Bial's Test detects Pentoses in Original style and Hexoses in Hamburger style Gardenburgers®

By: Kathryn Wearsch, Amber Staudacher, Shannon Meissner, and Jessica Kaunelis



Companies such as Nabisco process vegetables to taste amazingly similar to beef patties and other meats, but the same companies still produce and sell other varieties of vegetable meats that taste nothing like the meats they substitute. This study compares two such kinds of Gardenburgers® "on the molecular level", original and hamburger style, and the chemical changes they undergo upon cooking.

Raw and heated patties of both varieties were analyzed in order to compare three major components: carbohydrate, photosynthetic material, and enzyme composition. Carbohydrate properties of the two styles of Gardenburgers®, both raw and cooked, were found to be identical, using Benedict's, Barfoed's, and Iodine tests. Selivanoff's showed that 'original' style contained aldoses, while 'hamburger' style contained ketoses. Bial's Test showed structural differences - 'original' style had pentoses and 'hamburger' style had hexoses. We determined through the use of spectrophotography and paper chromatography that neither style of Gardenburgers® possessed carotene, xanthophyll, chlorophyll a, or chlorophyll b. Cooking them did not change the presence of these pigments. Finally, we tested for active enzymes by applying a strip of litmus paper to the Gardenburgers®, and found that none were present in any of our samples.



The purpose of the experiments carried out on different varieties of cooked and uncooked Gardenburgers® was to find some of the major differences of the macromolecules they contained. Tests on the structure of the sugars and other carbohydrates were the only ones that gave useful results. Because the patties did not really contain vegetables, tests designed to look at pigments and enzymes found in plants only confirmed that the vegetables were not in the samples. If time and resources allowed, further testing on the pigments and enzymes that they do contain would have been carried out.

The team analyzed the types of carbohydrates, searched for photosynthetic pigments, and tested for the enzyme polyphenoloxidase (PPO) in the two styles of Gardenburgers®, Original and Hamburger Style. This information was used to answer the question of how these properties differ between the styles and also to test if heat would affect them.
Before forming a hypothesis, a taste test was held. Each member of the group observed the taste of a piece of the Original and Hamburger Style Gardenburgers® to compare how sweet they tasted, how similar they tasted to real hamburgers, and how much vegetables contributed to the taste.
Four different test samples were obtained by grouping the Original and Hamburger Style into cooked and raw samples, resulting in Original raw, Original cooked, Hamburger raw, and Hamburger cooked. A series of preparations performed throughout the entire research project was to conduct control experiments of each section in order to correctly interpret the results of the actual experiment. For example, for the carbohydrate section, all of the six carbohydrate tests found in the fall 2002 LBS145 lab manual on different sugar samples were ran in the first week. The following week the team ran selected tests on the actual samples and compared them with the control results. Similar comparisons were done for the photosynthesis and enzyme sections.

In discussing the research results of these experiments, the carbohydrate section of the project will be considered first. After performing the control experiment, we decided to perform five of the six carbohydrate tests, believing that a test specifically for galactose would not provide useful information considering the large number of sugars found in foods. Benedict's Test indicated all four samples lacked free aldehyde and ketone groups. Barfoed's Test showed all samples contained di- or polysaccharides, but not monosaccharides.The Iodine Test indicated all samples contained starch. The last two tests revealed some differences between the Original and Hamburger styles regardless if they were cooked or not. In Selivanoff's Test, the Original style reacted with the reagent to show that it contained aldoses, while Hamburger style did not react with the reagent, which means they did not contain aldoses. Bial's Test revealed that Original style contained hexose furanose rings while Hamburger style contained pentose furanose rings (Figure 1.)

The predictions were that there would be no difference between the carbohydrates in the two styles. However, we did find structural differences of the carbohydrates within the Gardenburgers® patties. Because they were different, this could explain a fraction of the taste difference in the two patties. It could also explain, if studied further, the differences in how each patty is digested, for example one kind of carbohydrate might take longer or require different enzymes to break down than a differently structured carbohydrate.

Our prediction was that since the Original Style appeared to have more pigments and was supposedly the more vegetable-like patty compared to the Hamburger Style that it would contain chlorophyll a and b, xanthophyll, and carotenoids. We did not expect to find the green chlorophyll pigments in the Hamburger Style. Spectrophotography of the samples gave no similarities to the action spectrum for photosynthetic pigments in plants denying that they contained the pigments for photosynthesis. The results of the chromatogram completely failed to show the presence of any pigments. This seemed quite an odd result for a product called "the Original Gardenburgers® veggie patty," since either they contained no colored vegetables or too few to be observed. According to the box, this conclusion is correct; the only vegetable was onions. Original does, however, contain significant amounts (over 2%) of Annatto, or vegetable color. Since the patties are not a homogeneous color, pigmented parts should have been cut out for the photosynthesis experiments in addition to the experiment we did do to obtain a chromatogram that was not blank. The observation that no plant pigments are in the others makes sense for Hamburger Style, though, after learning that they are actually soy patties and not veggie ones. It would have been a good idea to read the nutrition facts and ingredients list beforehand. In fact, not doing so was the most significant mistake of our series of experiments. Garden Fiesta would have probably been a better sample to test than Hamburger Style since we now know it really has vegetables in it.

We expected that the enzyme section of the research would reveal differences between all four samples, Hamburger raw, Hamburger cooked, Original raw, and Original cooked. We thought that Hamburger Style would contain less PPO than Original because PPO is found in most plants (McHale,) and we believed original contained more vegetables. The cooked samples were expected to contain no active PPO. This was based on the knowledge that proteins often denature when brought to high temperatures. For example, when an egg is cooked this can be observed by comparing the stringiness and color of the egg white before to after. Since enzymes are part of the protein class of macromolecules (Bruice p916,) this seemed like a reasonable prediction. These hypotheses were shown to be incorrect by the results. Although the samples had a few plant products in them (rolled oats, rice, and soy) they had been processed so much that no PPO could be detected by adding catechol to the sample solutions if there was any to begin with. Without an enzyme that we could test for, further planned experiments on how cooking and pH of the digestive system affected their activity could not be carried out.

Original did, however, contain several milk products. Further, more useful testing could combine lactase and an indicator with the sample to test for the presence of or concentration of lactose, a sugar that 10% of Caucasians stop producing digestive enzymes for, leading to lactose intolerance (Bruice p902.) According to the box, the mozzarella and cheddar cheeses in Original Style do include some enzymes whose activity could possibly be tested. Another test on Hamburger Style could explore the denaturation of soy protein under the stresses of cooking temperature and stomach acidity.

In sum, the only difference in macromolecules of Original and Hamburger Style Gardenburgers® the team was able to find in the structure of the carbohydrates. Original style had hexose furanose rings, whereas Hamburger Style instead contained five-membered furanose rings. Hamburger Style also lacked the aldehyde sugars found in Original Style Gardenburgers®.


Figure 1. These are the results from Bial's test in our gardenburger samples. A greenish-brown color change indicated the presence of hexose furan ring sugars, while a green color change indicated the presence of pentose furan ring sugars.

Raw samples of Original and Hamburger style Gardenburger® are on the left. Cooked samples of Original and Hamburger style Gardenburger® are on the right.



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