In the Laboratory
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Carbohydrate Analysis: Can We Control the Ripening of Bananas? S. Todd Deal,* Catherine E. Farmer, and Paul F. Cerpovicz Department of Chemistry, Georgia Southern University, Statesboro, GA 30460; *
[email protected] Background Laboratory exercises involving carbohydrates tend to be based on classic qualitative analyses employing Benedict’s reagent, Fehling’s solution, polarimetry, etc. (1). Recent exceptions are the synthetic experiments published by Lowray (2) and Norris (3) in this Journal. While the latter are excellent examples of nontraditional carbohydrate investigations, we wanted to develop an introductory-level experiment that focused on quantitative carbohydrate analysis and maintained the theme of our laboratory program, which is the utilization of common foodstuffs to investigate biochemical principles relevant to nutrition.1 The result is an experiment with a threefold purpose: (i) determination of the carbohydrate composition of a banana, (ii) analysis of the changes in carbohydrate composition as the banana ripens, and (iii) investigation of the control of the ripening process. In a 1986 study, Englyst and Cummings demonstrated that while the total carbohydrate content of a banana remains relatively constant (~28% of dry matter) during ripening, the free sugar content increases sharply while the starch content shows a similar decrease (4). Using this study as our basis, we developed a carbohydrate analysis experiment entitled “Ripening Bananas: What’s Happening and Can We Control It?” In it, students determine the carbohydrate composition of bananas at various stages of ripeness. Using these data as a benchmark, they then test various storage methods to ascertain which method best preserves the desirable qualities (taste, color, etc.) of bananas for the longest period. Description of the Experiment
Determining the Carbohydrate Content of Bananas In the first part of the experiment, each pair of students analyzes the starch and free reducing sugar (glucose and fructose) content of a green, ripe, or overripe banana. For the free reducing sugar analysis, we chose the 3,5-dinitrosalicyclic acid–potassium sodium tartrate (DNS) assay originally developed by Sumner for reducing sugars (5). We decided to analyze only for reducing sugars because, as demonstrated by Englyst et al. (4), the change in free sugar content of a banana as it ripens is due mainly to an increase in the amount of reducing sugars. Additionally, the chosen method has been shown to be an accurate quantitative measure of reducing sugar content in solutions containing both unhydrolyzed starch and free reducing sugars (6 ). The DNS assay is a colorimetric assay based upon the redox reaction between a reducing sugar such as glucose or †
Initially presented at the 52nd Southeast/56th Southwest Combined Regional Meeting of the American Chemical Society, Dec 6–8, 2000, New Orleans, LA.
Table 1. Student Data on Carbohydrate Composition of Bananas Stage of Ripeness (Appearance) Green with some yellow
Reducing Sugarsa Starcha (% of Total Sample) (% of Total Sample) 12.9
4.0
Yellow (ripe)
6.2
4.4
Yellow with black spots (overripe)
2.0
11.6
aBased on wet weight of original sample. Note that the data in ref 5 are based on dry matter.
fructose and 3,5-dinitrosalicyclic acid. The reaction results in the formation of a reddish brown reduction product, 3amino-5-nitrosalicyclic acid, from the colorless 3,5-dintrosalicyclic acid. The inclusion of potassium sodium tartrate (Rochelle’s salt) as a component of the reagent was a later modification designed to limit the solubility of oxygen in the reaction mixture, since dissolved oxygen can interfere with the oxidation of glucose. To begin the analysis, the students homogenize a 1-g sample of banana in cold distilled water using a hand-held glass homogenizer. The resulting suspension is centrifuged and the pellet is isolated, dried, and weighed to assess the starch content of the banana. (The dry pellet is composed mostly of starch.) Then, a sample of the supernatant is analyzed using the DNS assay to determine the banana’s reducing sugar content. Each pair of students completes duplicate analyses of starch and reducing sugar content and records the averages of the analyses on the chalkboard or on a pre-prepared class data sheet. Using the collective class data, the students can observe the differences in the carbohydrate composition of bananas at various stages of ripeness. Table 1 shows some representative student data.
Determining the Best Method for Storing Bananas At the beginning of the first laboratory period, each pair of students is given a yellow-green banana (in addition to their green, ripe, or overripe banana) taken from the same bunch and told to store this banana (see below). During the following laboratory period, the students analyze their stored banana to determine the best method of storage to control the ripening process. To design the “storage” part of the experiment, we turned to popular culture. Many readers will recall the jingle that was used long ago by one of the major banana producers, which implored consumers not to put their bananas in the refrigerator. Why shouldn’t we store bananas in the refrigerator, we wondered? Thus, the refrigerator became one of the test storage methods. Next, we noted that it is nearly impossible to walk into the produce section of any grocery store without seeing a display of white plastic or wooden “ba-
JChemEd.chem.wisc.edu • Vol. 79 No. 4 April 2002 • Journal of Chemical Education
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In the Laboratory
nana hangers” with their attendant claim to keep bananas fresher, longer. The “banana hanger” became another test storage method. Then, one of us (PFC) recalled the instructions of his wise grandmother to “always store bananas in a brown paper bag.” So, the brown paper bag became yet another test storage method. Finally, we threw in the “conventional wisdom” method of fruit storage and chose the lab drawer as a cool, dark place to serve as the final test storage method. In this portion of the experiment, the students are assigned one of the test storage methods and instructed to store their bananas for the week. During the following lab period, they repeat the analyses from the first week, taking samples from their stored banana. We also ask them to record the appearance of the skin of the banana when they remove it from storage. The students enjoy this second part of the lab exercise, and the collective results are always eagerly anticipated. Hazards 3,5-Dinitrosalicylic acid is toxic and an irritant. Potassium sodium tartrate is an irritant. Always wear gloves and appropriate eye protection when handling these compounds. Discussion As any trained chemist might expect, the refrigerated banana “ripens” the least during the week of storage. Typically, students find that the composition of the refrigerated banana has changed very little, whereas the composition of each of the other three bananas is similar to that of the ripe banana from the first part of the experiment. After all the data are collected, we pose the question, “Which of the storage methods would you recommend to consumers?” While this seems like a straightforward question, it turns out to be far more complex than it first appears. Since most of our students are nutrition majors, they understand that food is more than taste and smell. We’ve often overheard them in class discussing mouth feel, consistency, and appearance as important facets of food and eating. It is the last characteristic—appearance—that presents the dilemma. At the end of a week, a banana stored in the refrigerator is not a pretty sight! Typically, a refrigerated banana has a mottled green-brown color that makes it appear almost bronze—not at all an appetizing appearance. The banana stored in the brown paper bag, on the other hand, has a rich, bright yellow color with very few brown spots, while the other two are yellow but have many patches of brown. Thus, the students are forced to make a tough decision. Refrigeration is
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obviously the best storage method to slow the ripening process, but many consumers would refuse to eat a refrigerated banana because of its appearance. The brown paper bag method yields the most appealing fruit, but the outward appearance belies the almost overripe flesh that lies beneath the skin. Summary While we utilize this experiment in our Nutritional Biochemistry course, we believe that it is broadly applicable. We feel confident that this experiment is appropriate for courses such as introductory chemistry and organic chemistry in which the basic principles of carbohydrate analysis are introduced. Techniques taught in this experiment include construction and use of standard curves, spectrophotometric analysis, and centrifugation. Additionally, interpreting the results of the experiment requires both critical-thinking and decision-making skills. W
Supplemental Material
Notes for the instructor and a handout for students with detailed instructions, a data sheet, and questions are available in this issue of JCE Online. Note 1. This is the fourth report in a series on the development of our nutritional biochemistry laboratory program. For the first three parts see ref 7.
Literature Cited 1. See for example: Williamson, K. L. Macroscale and Microscale Organic Experiments; Heath: Lexington, MA, 1989; pp 499–506. Boyer, R. F. Modern Experimental Biochemistry, 2nd ed.; Benjamin/Cummings: Redwood City, CA, 1993; pp 347–362. 2. Callam, C. S.; Lowray, T. L. J. Chem. Educ. 2001, 78, 73–74. 3. Norris, P.; Freeze, S.; Gabriel, C. J. J. Chem. Educ. 2001, 78, 75–76. 4. Englyst, H. N.; Cummings, J. H. Am. J. Clin. Nutr. 1986, 44, 42–50. 5. Sumner, J. B. J. Biol. Chem. 1921, 47, 5–9. 6. Bernfeld, P. Methods Enzymol. 1955, 1, 149–158. 7. Deal, S. T.; Pope, S. R. J. Chem. Educ. 1996, 73, 547. Pope, S. R.; Tolleson, T. D.; Williams, R. J.; Underhill, R. D.; Deal, S. T. J. Chem. Educ. 1998, 65, 761. Mullis, T. C.; Winge, J. T.; Deal, S. T. J. Chem. Educ. 1999, 76, 1711.
Journal of Chemical Education • Vol. 79 No. 4 April 2002 • JChemEd.chem.wisc.edu
