By Lizz Conover, Kimberly Flachs, Susan Frank, and Diane Tran
Fantastic Four Group
Abstract
The purpose of this investigation was to determine of rice, soy, and cow’s milk which would be easiest for a person with Type II Diabetes to digest. Analysis was based on carbohydrate, photosynthetic, and protein testing. In order to test the carbohydrate composition of each milk type, we used Barfoed’s, Iodine, Bial’s, and Selivanoff’s reagents in a series of tests. An absorption spectrum was determined for both the rice and soy plant chloroplasts. Via chromatography, the rate of flow was calculated in order to identify pigments found in soy and rice plants. The Bradford Assay was used to test the protein concentration of each milk. With previous research in mind, we predicted that the milk most suitable for a person with Type II Diabetes would be the milk with a higher ratio of complex to simple sugars. Based on this it was hypothesized that cow’s milk is the most beneficial for people with Type II Diabetes. The carbohydrate test implied that rice milk contains a higher ratio of complex sugars to simple sugars. The photosynthesis test suggested that soy plants will be able to absorb more blue and red light. Based on the protein test it was suggested that soy milk was best for a Type II Diabetic due to a high protein concentration and that is it a plant protein. Our investigation determined that soy milk was most likely the healthiest choice for a Type II Diabetic.
The purpose of this investigation was to determine of rice, soy, and cow’s milk which would be easiest for a person with Type II Diabetes to digest. Analysis was based on carbohydrate, photosynthetic, and protein testing. In order to test the carbohydrate composition of each milk type, we used Barfoed’s, Iodine, Bial’s, and Selivanoff’s reagents in a series of tests. An absorption spectrum was determined for both the rice and soy plant chloroplasts. Via chromatography, the rate of flow was calculated in order to identify pigments found in soy and rice plants. The Bradford Assay was used to test the protein concentration of each milk. With previous research in mind, we predicted that the milk most suitable for a person with Type II Diabetes would be the milk with a higher ratio of complex to simple sugars. Based on this it was hypothesized that cow’s milk is the most beneficial for people with Type II Diabetes. The carbohydrate test implied that rice milk contains a higher ratio of complex sugars to simple sugars. The photosynthesis test suggested that soy plants will be able to absorb more blue and red light. Based on the protein test it was suggested that soy milk was best for a Type II Diabetic due to a high protein concentration and that is it a plant protein. Our investigation determined that soy milk was most likely the healthiest choice for a Type II Diabetic.
Discussion
A person afflicted with Type II Diabetes is consistently presented with the issue of monitoring his or her diet, specifically sugar consumption. As a part of a healthy and balanced diet, the majority of people consume milk. In regards to the limitations of a healthy diabetic diet: of soy milk, rice milk and whole cow’s milk, which would be healthiest for a Type II Diabetic to drink? Based on prior knowledge and research it was predicted that cow’s milk would be best due to low simple sugar concentrations and the greater presence of protein.
One may assume that metabolism of simple sugars during digestion would be quickest and easiest for simple sugars, resulting in a lower blood glucose (a simple sugar) levels. However, the consumption of complex sugars and carbohydrates is considered to produce less extreme glucose levels in the blood stream than the consumption of simple sugars (Menedez and Stoecker, 1985).
Four different tests were conducted to analyze of the sugar composition of the whole cow’s milk, soy milk, and rice milk. In addition to the three milk samples, four sugar solutions were tested as positive controls. We analyzed two monosaccharide (glucose and fructose) solutions, one disaccharide (lactose) solution, and one polysaccharide (starch) solution. All solutions were diluted to 1% by volume.
The first test involved using Barfoed’s reagent to distinguish simple sugars (monosaccharides) from complex sugars (disaccharides and polysaccharides). Barfoed’s reagent contains compounds that would react with monosaccharides to form precipitate through the process of reduction of copper ions. The results of Barfoed’s test imply that the ratio of monosaccharides, to disaccharides or polysaccharides, is significantly low in all three milk samples during all three trials. Using the 1% solutions of glucose, fructose, lactose, and starch as positive controls, and water as a negative control, none of the milk samples, lactose, or starch reacted positively producing a rusty red color (Figure 1). Water as expected had no change. Only the glucose and fructose produced a rusty red color; which was to be expected due to the fact that both are monosaccharides.
The Iodine test further extended the investigation of the presence of polysaccharides. As stated in the results, the starch solution became a brown color when the iodine was added, the soy milk reacted and became pink, the rice milk changed to a dark purple appearance, and the other solutions were yellow (Figure 6). The yellow color is simply the diffusion of the iodine that did not react. The change observed in the starch solution was the basis of comparison in this experiment since starch is a polysaccharide. The color change seen in soy milk suggests it contains a significant amount of polysaccharides. Furthermore, the more intense color change seen in rice milk supports the idea of even larger amounts of polysaccharides compared with soy milk (Figure 6). These results suggest that of the three milk solutions, whole cow’s milk had the lowest amount of polysaccharides.
Based on the information obtained from Barfoed’s and the Iodine tests; rice milk, in comparison to soy and whole milk, has the greatest ratio of polysaccharides to monosacchardies. From the information found in Nutrition and Diabetes, rice milk can then be assumed to best for consumption by a Type II Diabetic.
Selivanoff’s test and Bial’s test focused on the structure of sugars present in the solutions. In an effort to validate the results of theses tests, absorbency values from 400 nm to 700 nm at 30 nm intervals, were found using a spectrophotometer. The absorption spectrum for each solution was then graphed (Figure 3, Figure 5). A higher absorption at a given wavelength would indicate a stronger, darker color, which would correlate to a stronger reaction. This allowed our testing to couple quantitative data with the otherwise only qualitative data. Specifically, Selivanoff’s was used to distinguish aldoses from ketoses. Both kinds of sugars react with Selivanoff’s reagent to produce a red solution (Figures 2). A solution of ketose sugars react (producing a red color) in under a minute, whereas aldose sugars take over a minute. The quick reaction times of fructose, soy, and rice milk provided strong support that these solutions have high ketose sugar concentrations. The rest of the solutions (whole milk, starch, lactose, glucose) tested took longer than one minute to react, indicating a higher concentration of aldose sugars.
Quantitatively we can observe the strength of the reaction from the absorption spectrum of the produced solutions (Figure 3). Since Selivanoff’s reagent produces a red color for both ketoses and aldoses, the strength of the reaction can be determined by the positioning of the peeks and valleys in the visual light spectrum. The quicker the reaction time, the longer the solution will be allowed to react, the darker the color. A darker color implies a quicker initial reaction time, indicating ketose sugars. This would suggest that the strength of a solution’s color directly correlates to the concentration of ketoses. A reaction later in the test produces a lighter colored solution, indicating aldoses.
Red light, as it seen by the human eye, is produced by an electromagnetic wave with a wavelength between 620 nm and 740 nm. Based on properties of electromagnetic waves, an object that the human eye views as red color, absorbs light least readily (reflects) at wavelengths between 620 nm and 740 nm, and most readily at other wavelengths. The absorbency of a given solution can be tested using a spectrophotometer, which will measure the absorbency at a given wavelength.
From the absorption spectrum (Figure 3) it can be observed that fructose, lactose, glucose, soy milk, and rice milk have the lowest absorbance for red light (620 nm to 740 nm). Whereas, whole milk and starch have comparatively high absorbance for red light, implying that fructose, lactose, glucose, soy milk, and rice milk had stronger reactions than whole milk and starch. It should also be noted that of the solutions with a strong reaction, only soy milk and fructose had high absorbance for light waves less than 620 nm, whereas rice milk and cow milk had moderate absorbance. Since lactose and glucose absorbed similar amounts of light over the entire visible light spectrum, it is possible that in regards to these solutions, we can not accurately infer that the reflection of red light corresponds to a strong reaction. Based on support of the absorption spectrum, soy milk likely has the highest concentration of ketoses to aldoses.
Bial’s test was set up not only to find furanoses, but also to make a distinction in the furan rings, five-membered versus six-membered. Solutions with five-carbon rings produce a green color when the reagent is added and solutions with six-carbon rings react to form an olive/brown solution. The observations made showed that the fructose, rice, and whole milk solutions became olive, and the soy milk turned brown, which suggested a higher concentration of six membered rings than five membered rings. The glucose, lactose, and starch solutions reacted to form green product solutions suggesting a higher concentration of five membered rings (Figures 4).
In the same way as Selivanoff’s test was analyzed by absorption spectrum, Bial’s test could be analyzed (Figure 5). A darker color corresponded to a higher concentration of hexose furanose. A lighter solution would indicate a higher concentration of pentose furanose. This only applies to positively reacting solutions. Fructose and soy milk had the greatest absorbency over the spectrum, indicating a higher concentration of hexoses furanoses. While whole milk and lactose had minimal absorbency across the spectrum, implying a higher concentration of pentoses furanoses. Rice milk, starch, and glucose fell in between the two groupings, implying a relatively more balanced distribution of hexose and pentose furanoses.
While furanoses does not directly affect the investigation of which milk is better, the information found in Selivanoff’s and Bial’s tests shows that fructose may possibly be found in rice milk or soy milk. In both tests and all trials the rice milk, soy milk, and fructose reacted similarly. Being that fructose is the only naturally occurring ketose sugar (tested in Selivanoff’s test), it is implied that rice and soy milk contain amounts of significant fructose. The possibility that fructose may be evident in rice milk leads to more support that rice milk is best for a person with Type II Diabetes. However, based solely this idea stems from further research that states “low-dose fructose improves the glycemic response to an oral glucose load in adults with type 2 diabetes” (Cherrington, Davis, Mann, and Moore, 2001). Fructose raises blood glucose levels less than glucose and many starches (Anonymous, unknown 3). Fructose takes longer than glucose for the body to absorb. This additional time allows the liver to first process the glucose consumed with the fructose, more evenly distributing total absorption of sugars (Nuttall and Gannon, 1999).
The analysis of the milks continued with a look at the photosynthetic qualities of the original plants. Because whole milk originates from an animal it does not have any chloroplasts that carry out the light reactions of photosynthesis, it was impossible to analyze the photosynthetic qualities of the origins of cow’s milk. However, in the case of rice milk and soy milk, both are derived from plants. Comparing the absorption spectrum of chloroplasts found in rice plants and soy plants suggested the potential sugar contents of each plant before processing. Photosynthesis is a series of reactions utilizing light energy, CO2, and water to produce sugar, among other things. The likely correlation between light absorbency and sugar production will deal greatly with the light wavelengths of 680 nm and 700nm. This is because the pigments predominantly responsible for carrying out photosynthesis, chlorophyll α and chlorophyll β, absorb most readily between 680 nm and 700 nm. A high absorbency at these wavelengths would indicate a darker pigment and a greater ability for higher rates of photosynthesis. It should be noted however, that high potential does not always induce high rates of photosynthesis, as black colored inanimate objects absorb all visual light waves, yet are incapable of photosynthesis. We can assume that since we are dealing with two plants, the relationship between absorbance and higher ability for photosynthesis is plausible.
Furthermore, the efficiency of photosynthesis determines the type of sugars that the resulting glucose is stored in. A plant typically will use glucose to make sucrose (a combination of fructose and glucose), however if the plant is acting at a high rate of photosynthesis, and so producing glucoses more quickly, it will simply string the glucoses together to form starch (Freeman, 2002). Logically, a plant that is capable of a higher rate of photosynthesis will likely contain a higher concentration of starch. It should be further noted that rates of photosynthesis can not be directly obtained from the absorption spectrum, they can only be inferred. Those interested in further investigation may desire to perform an action spectrum for soy and rice chloroplast solutions.
By the absorption spectrum, the relative absorption of different wavelengths of light by pigments in soy and rice chloroplasts is shown (Figure 8). In rice milk, there were distinct peaks at 445 nm, 490 nm, and 670 nm. This indicates that the colors blue, blue-green and red are absorbed by rice chloroplasts. In soy milk, there were distinct peaks at 400 nm and 670 nm. This indicates that the colors violet and red are absorbed by soy chloroplasts. The rice chloroplasts solution had a higher absorbency in the range of 680 nm to 700 nm, than soy chloroplasts. This would suggest that rice plants have a higher potential for greater photosynthetic rates and likely a higher concentration of starch.
Paper chromatography determined the pigments present in the soy and rice plants. Pigments present can lend further insight regarding the original sugar contents in the plants. Different pigments have different absorption qualities. Soy and rice plant chloroplasts both contained four pigments: carotene, xanthophyll, and as discussed above, chlorophylls α and β (Figure 7). These were determined through the pigment chromatography procedure and the rate of flow of each pigment compared to that of the solvent used, referred to as solvent front (Table 3). While chlorophyll pigments are most abundant and so strongly influence absorption rates, the other pigments allow the plant to absorb and so utilize a greater range of light waves. Both plants contained the same pigments, which negates any comparison between the two. However, it does suggest an increased efficiency of photosynthesis in both plants, supporting the likelihood of more complex sugars being formed. From this it would suggest that rice milk would be the healthier choice because it contained more complex sugars.
While sugar intake is important in this exploration, one has to consider this in context of tradeoffs of other beneficial milk components, such as protein. The Bradford Assay gave quantitative results of protein concentration. The highest levels of protein were found in the whole milk. Soy milk contained the next highest concentration, and rice milk contained the least.
Protein content is particularly pertinent for persons with Type II Diabetes. Recent studies have found that when simple sugars, such as fructose and glucose, are consumed with protein rich foods, blood glucose levels rise less than if consumed alone. This is due to protein stimulating insulin secretion (Gannon and Nuttall, 1999). Moreover, for persons at risk of kidney disease due to diabetes, studies have suggested that substituting plant protein for animal protein in their regular diet can aid in the prevention and treatment of kidney disease. The plant proteins appear to reduce hyperfiltration of the kidneys (Strain, 2002).
As a means to check our data, we compared our protein concentrations from the Bradford Assay to the information given on the nutritional label of each milk (Figure 10, Figure 11, Figure 12). Our data fell significantly short of the predicted levels from the nutritional labels. The Bradford Assay would suggest that we had 1.26 g of protein per 8 oz serving for whole milk, 1.10 g for soy milk, and 0.090 g for rice milk. This is much lower than the 8 g for whole milk, 11 g for soy milk, and 1 g for rice milk per 8 oz serving as stated on the nutritional labels. While discrepancies such as this were hoped to be avoided by multiple repetitions, our data was consistent across all trials. Due to the amount of testing done by both manufacturers and the Food and Drug Administration, it is likely that the nutritional labels are accurate. This would imply that error during the Bradford Assay was the cause of the difference in findings. This would be useful for those interested in further study into our hypothesis to note, and take into special account our potential sources of error. Error for the protein concentration for whole, soy, and rice were 84.25%, 90%, and 91% respectively.
Based strictly from our experimental data, we can infer that either whole or soy milk would be best for a person with Type II Diabetes, since they each contain similar protein levels. Based on the types of protein, soy milk would likely be the better of the two. Soy milk is a plant protein, so along with a beneficial amount of protein to reduce blood glucose levels, the type of protein can aid in combating kidney disease.
Judging from the information collected during these experiments soy milk would be best for a person with Type II Diabetes. While rice milk was indicated as the healthiest choice by two of our investigations, in both investigations soy milk behaved similarly. Whereas, in regards to protein concentration, soy was significantly different than rice milk, prompting the conclusion that soy milk would be the healthiest choice. Cow milk has been a long standing favorite among milk drinkers, only recently have soy milk and rice milk become widely available. The results of the tests show that there is a higher ratio of complex sugars in reference to simple sugars in soy milk than in cow milk. In addition the evidence of what is possibly fructose (from Selivanoff’s and Bial’s tests), potentially high rates of starch (photosynthesis testing), and high protein concentration (the Bradford’s Assay) in soy milk further supports the idea that soy milk is the most suitable and beneficial for Type II Diabetics.
It is safe to assume that for this, as with any laboratory investigation that there are many possible sources of error. Human error is always a potential source, such as error in timing or incorrect reading of a value. One specific area highly susceptible to errors was the absorption spectrums of Selivanoff’s and Bial’s tests, of chloroplast solutions, and the Bradford Assay. Before this could be preformed, each solution had to be diluted in order to be read by the spectrophotometer at all light wavelengths. While dilution insured complete readings of an initially dark solution, it caused solutions that were already light to become even lighter and harder to read. During the photosynthetic analysis it would have been helpful to test the action spectrum of the soy and rice chloroplasts, but due to difficulties and inconsistencies of the experiment, it was omitted.
Other sources of error include the materials used in the experiments. New packages of milk had to be purchased every few weeks, this produced variation in our test subjects. We attempted to decrease this variation by using the same brand of milk each time. Also, in regards to materials; since we shared materials with other persons within the lab, it is impossible to completely know about the prior treatment of materials. Specifically light sensitive reagents such as Bradford’s.
Time constraints of growing and harvesting seasons forced us to obtain soy plants nearly 3 weeks before they we actually used. Further failures in initial photosynthetic testing, further increased the time between harvesting and the chloroplast extraction. The soy leaves were stored in the refrigerator to prevent potential damage due to freezer-burn, however the refrigerator did not completely prevent decay. Moreover, due to limitations of time, climate, and availability of specimens and product information: the rice and soy leaves used were not known to be of the same species used in each of the milks.
Links for additional information
American Diabetes Association
http://www.diabetes.org/type2/type2.jsp
The Endocrine System
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookENDOCR.html