Tuesday 1
Dr. Douglas Luckie
October 14, 2003
http://www.msu.edu/~kuelbsmi/cellular4
Abstract
Written By Jena Krueger
Revised By Mike Kuelbs
Revised By Lindsay May
Grapes and raisins are essentially the same fruit, as a raisin is simply a dehydrated grape. Our research attempted to discover if the dehydration process causes any differences to occur at the molecular level, concerning organic compound presence, pigment characterization, and enzyme presence. During the organic compound portion of our research, tests we utilized, included Barfoed’s test, the Iodine Test for Coiled Polysaccharides, Selivanoff’s test, and Bial’s test. Barfoed’s test came back positive for monosaccharides in both grapes and raisins. The Iodine Test for Coiled Polysaccharides showed negative results for the presence of starch in both grapes and raisins. Selivanoff’s showed the presence of ketoses in both grapes and raisins. Results of Bial’s Test detected hexose furanose in raisins and pentose furanose in grapes.
Concerning pigments, the paper chromatography test for pigment identification detected that the pigments found in grapes are chlorophyll a, chlorophyll b, xanthophyll, and carotene. We were not able to identify any pigments in the raisins.
The presence of Polyphenoloxidase (PPO) was detected in grapes, visibly resulting in a brownish coloration. However, no PPO was detected in the raisins. The Bradford Assay showed a higher total protein concentration in raisins than of that in grapes. Between the grapes and the raisins differences were found in pigmentation, presence of PPO, and protein concentration. The sugars present in each differed in that grapes contained pentose furanose while raisins contained hexose furanose.
Discussion
Written by Michael Kuelbs
Revised by Jessica Wolcott
Revised by Jena Krueger
Throughout our investigation our research compared the constitutional make-up of Thompson’s seedless grapes and Sun-maid raisins. Although raisins are made from grapes, we predicted there would be differences in the levels of carbohydrates, pigments and enzymes. Our research strived to answer the question as to whether grapes and raisins possess different structural qualities as an effect of the dehydration process. Our hypothesis is that grapes and raisins would be alike in sugar content, but may contain different pigments, and that raisins would have a higher concentration of protein.
We utilized the Barfoed’s, Iodine, Selivanoff’s, and Bial’s Tests to determine the different carbohydrates in each of our samples of grapes and raisins. For each of these tests, we carried out two trials on each of the three different concentrations (A, B, C) of both the grapes and raisins solution. The stock solution for the grapes contained 50.96 grams of grapes and 200 ml of deionized water. The raisin stock solution included 50.46 grams of raisins and 200 ml of deionized water. Since the stock solutions were too thick, we diluted the stock at three different concentrations to enable us to observe the color changes more accurately. Solution “A” consisted of 300 μl of either grape or raisin stock solution and 500 μl of deionized water. Solution “B” contained 300 μl of either grape or raisin stock solution and 1000 μl of deionized water. Solution “C” included 300 μl of either the grape or raisin stock solution and 1500 μl of deionized water.
Barfoed’s test can differentiate monosaccharides from di- and polysaccharides. The monosaccharides will react with copper ions, while the disaccharides will not. A positive reaction is recognized by the fabrication of a red precipitate. A monosaccharide is a carbohydrate that contains only one sugar molecule. Some examples of a monosaccharide are glucose, fructose, galactose, and xylose. A disaccharide is a carbohydrate that contains two sugar molecules, like lactose, maltose, and sucrose. A polysaccharide, for example starch, is a carbohydrate that contains more than two sugar molecules.
For Barfoed’s Test, our results for the grapes were the same for solutions A, B, and C in both trials (Figure 1). All grape solutions tested produced a red precipitate in a clear-blue liquid. The results of the raisins were consistent throughout both trials and for all three concentrations. When tested on the raisin solutions, the Barfoed’s test produced a teal/blue solution with a red precipitate. Our controls for this experiment, established in prior labs, were water and glucose (Table 1). Water produced negative results with a clear, bright blue solution. Glucose reacted positively with a teal/blue solution and a red precipitate. From our results we can infer both the grapes and the raisins contain monosaccharides (Table 3). We were unable to determine the quantity of the monosaccharides in each solution because the tests preformed were qualitative and only showed a presence or lack there of.
The Iodine Test for coiled polysaccharides tests for the presence of starch. If starch is present, the solution will turn a blue/black color with the addition IKI. All trials and concentrations for both the grapes and raisins remained a yellowish gold color when we added IKI (Figure 2). The results suggest that grapes and raisins do not contain starch (Table 4). However, it is possible grapes and raisins do contain starch, just not enough to react in our test. In addition, our samples were blended and diluted, making it even harder for any substance to show up.
Selivanoff’s Test is a way to confirm if ketoses or aldoses are present. A ketose is a carbohydrate, in which the carbonyl group (carbon double bonded to oxygen) is attached neither at the top nor the bottom of the chain. An aldose is a carbohydrate in which the carbonyl group is attached to either the top or the bottom of the chain. When heated, the reagent forces the ketose to transform the clear liquid to one red in color. Our results of the Raisin solutions for the three different concentrations imply that monosaccharide ketoses are present. Our results of the grape solution for both trials of concentrations “A” and “B” show that monosaccharide ketoses are present. However, the grape solution for both trials of concentration “C” suggested an aldose (Table 5). We believe the reason for this difference is because both fructose and glucose are involved in the organic composition of grapes and raisins. Fructose is a monosaccharide ketose and glucose is a monosaccharide aldose. In Selivanoff’s Test, if ketoses are present, they will be detected first because they react before aldoses. So the only way for aldoses to be detected is if there were not any ketoses present. Our trials suggest either there was an error in the process, making trial “C” produce different results, or that the more diluted concentration in trial “C”, causing and aldose to show up.
The results given by Bial’s Test allows for the determination of furanoses, which are five-membered rings. Pentose furanoses are five-membered rings with four carbons in the ring and the other carbon is a substituent. They are present when the solution turns a green/olive color. Hexose furanoses are five-membered rings with four carbons in the ring and two other carbons are substituents. They are present when the solution turns a muddy brown color. Pyranoses are six-membered rings, and are present when the color remains the same. Our results show that grapes contain pentose furanoses because all three concentrations in both trials were an olive/green color. In both trials for the raisins, the three concentrations were a dark brown color. This indicates that raisins contain hexose furanoses (Table 6).
The data collected in our Carbohydrate tests suggest a consistency in monosaccharides, ketoses and furanoses. The sugars in each are consistence and stay the same, falsifying our hypothesis.
To look at identify the different pigments in each solution we preformed a paper chromotography. We predicted the grape solution would show cholorphyll a and b, carotene and xanthophyll. We predicted the raisin solution would not provide any distinguishable pigments. During the dehydration process the raisins obtain their dark brown color through polyphenol oxidation (Mullins, et al 1992). This brown color prevents the observation of other pigments in the solution.
We performed the paper chromatography twice. Our first attempt provided no results so we used a higher concentration on the second trial. The chromatography strips used to test the raisin solution showed no visible separation of pigments, leading us to believe the solution did not contain any of the pigments chlorophyll a, chlorophyll b, carotene or xanthophylls (Figure 6). The grape chromatography strips showed a light run of dark green and a light strip of orange-yellow (Figure 6). From this we inferred the solution contained chlorophyll b and carotene (Table 7).
To look at the enzymes found in each of our two samples we tested for the presence of polyphenoloxidase (PPO) and preformed a Bradford Assay. The PPO test specifically detects whether or not this enzyme is present in grapes and raisins using litmus paper and pH (Table 2). We predicted that PPO is present in both grapes and raisins (Table 8). The Bradford Assay is a dye-binding analysis. Based on the various protein concentrations, the dye (CBBG) will respond with different colors to show this variation. This variation can be detected by performing an absorbance reading at 595nm. It is read at 595 nm because this is the absorbance maximum of CBBG when it binds to amino acids. This must first be performed using bovine serum albumin (BSA) to create a protein standard curve on which to compare the absorbance values of our grape and raisin solutions. This will determine the protein value, and dividing the protein value over the amount of grape and raisin solution we used will yield the protein concentration in µg/µl. Our predicted result for the Bradford Assay is that raisins will have a higher protein concentration than grapes. We predicted this because raisins have a smaller volume than grapes; therefore, all of the proteins that are found in grapes are compacted into a smaller volume though dehydration.
When tested, we found that the grapes had levels of PPO and it was not possible to establish if the raisins did or not (Figure 7). Using a whole grape and a whole raisin, we put drops of catechol on each sample. The grape turned brown, indicating PPO. The raisin, however, was already brown and this test was inconclusive. For the Bradford Assay we made a standard curve and compared each sample to it (Figure 8). Our absorbance value found from the grape was 0.018. When compared to our curve, this is a protein value of 0.37µg, which yields a concentration of 0.0148 µg/µl. The absorbance value associated with the raisin solution was .074. When compared to the standard curve, this is a protein value of 3.76 µg, which yields a concentration of 0.1504 µg/µl. This data suggests that raisins do have a higher protein concentration than grapes (Table 10). We predicted this to occur due to the condensed nature of the raisin, compared to the grape.
The basis of our study was to determine how the structural components of grapes and raisins compare. Overall, we concluded that our data from the various tests supports our hypothesis. The sugar make-up of grapes and raisins are very similar, as found in the Carbohydrate tests. The pigments that are found in grapes are different than those that are found in raisins, as tested by paper chromatography. We found PPO in the grape samples, but could not conclusively verify the presence of PPO through our trials. However, we did find a higher concentration of protein in raisins than in grapes.
We found our data to be beneficial because raisins are more convenient, as they fit into the fast paced lives of college students. We were able to show the different molecular components of both grapes and raisins. However, our experiment had a few aspects that could be altered, having an effect on the results.
For starters, we used the wrong kind of grapes. We should have used Thompson’s seedless green grapes instead of red grapes. Our results from the various tests may have shown a closer relationship of the structural qualities of grapes and raisins. Another error is perhaps that because we diluted our stock solutions may be we did not get a good representation of grapes and raisins as a whole. In Selivanoff’s Test, we might have observed and recorded the incorrect times that the solution started to turn red. This test reacts fast and it is therefore difficult to record all of the times accurately. For Bial’s Test, our results might have been thrown off because the raisin solution was somewhat brown to begin with. So maybe raisins would have pentose furanoses present, like grapes, instead of hexose furanoses. These colors were very similar and hard to decipher between the slight shade differences.
Through our investigation we learned valued information about the similarities and differences in the grape and the raisin. Although they both contain monosaccharides and ketoses, their structure and taste are very different. Our research intrigued us, and in the future we would be interested to follow up on our experiment, and possibly look for quantitative results rather then the qualitative we found.
Photo A Photo B
Figure 7: The Presence of PPO. The photographs above compare the results of testing for PPO in grapes versus the results in raisins. Polyphenoloxidase (PPO) is an enzyme that catalyzes the oxidation of catechol, a colorless substrate, resulting in a brownish color change. Photo A shows trials one and two of the reaction of after catechol and water were each added to a grape slice. The addition of catechol resulted in a brown color seen in the middle of the grape, while the addition of water did nothing, suggesting the presence of PPO. In both of two trials, when catechol and water were added to a slice of raisin, no visible reaction occurred, suggesting there is no PPO present in the raisins.
References
Written by Mike Kuelbs
Revised by Jessica Wolcott
Revised by Jena Krueger
Bakhru, H.K. Food that Heal. Delhi: Orient Paperbacks. IndianGyan.com, 2000 <http://www.indiangyan.com/books/healthbooks/food_that_heal/raisins.shtml>
Cowan, M. M., E.A. Horst, S. Luengpailin, R. J. Doyle. Inhibitory effects of Plant Polyphenoloxidase on Colonization Factors of Streptococcus sobrinus. Antimicrobial Agents and Chemotherapy 44: 2578-2580, 2000.
Eisenman, Lum. The Home Winemakers Manual. (online book), 1999.
<http://home.att.net/~lumeisenman/contents.html>
Krha et al. LBS-145 Cellular and Molecular Biology. MSU Printing Services. 2003.
Mullins, Michael G., Bouquet, Alain., Williams, Larry E. Biology of the Grapevine. Cambridge University Press, 1992.
Parsons, H. Grapes Under Glass. Transatlantic Arts, Inc., 1955.
Petucci, Vincent E., Carter D. Clary. A Treatise on Raisin Production, Processing, and Marketing. Malcom Media Press, 2001.
Weiss, Kathryn, Linda Bisson. Optimisation of the Amido Black assay for the Determination of the Protein Content of Grape Juices and Wines. Journal of the Science of Food and Agriculture 81: 583-589, 2001.
Grapes. <http://www.innvista.com/health/foods/fruits/grape.htm>
Written By Jena Krueger
Revised By Mike Kuelbs
Revised By Lindsay May
Grapes and raisins are essentially the same fruit, as a raisin is simply a dehydrated grape. Our research attempted to discover if the dehydration process causes any differences to occur at the molecular level, concerning organic compound presence, pigment characterization, and enzyme presence. During the organic compound portion of our research, tests we utilized, included Barfoed’s test, the Iodine Test for Coiled Polysaccharides, Selivanoff’s test, and Bial’s test. Barfoed’s test came back positive for monosaccharides in both grapes and raisins. The Iodine Test for Coiled Polysaccharides showed negative results for the presence of starch in both grapes and raisins. Selivanoff’s showed the presence of ketoses in both grapes and raisins. Results of Bial’s Test detected hexose furanose in raisins and pentose furanose in grapes.
Concerning pigments, the paper chromatography test for pigment identification detected that the pigments found in grapes are chlorophyll a, chlorophyll b, xanthophyll, and carotene. We were not able to identify any pigments in the raisins.
The presence of Polyphenoloxidase (PPO) was detected in grapes, visibly resulting in a brownish coloration. However, no PPO was detected in the raisins. The Bradford Assay showed a higher total protein concentration in raisins than of that in grapes. Between the grapes and the raisins differences were found in pigmentation, presence of PPO, and protein concentration. The sugars present in each differed in that grapes contained pentose furanose while raisins contained hexose furanose.
Discussion
Written by Michael Kuelbs
Revised by Jessica Wolcott
Revised by Jena Krueger
Throughout our investigation our research compared the constitutional make-up of Thompson’s seedless grapes and Sun-maid raisins. Although raisins are made from grapes, we predicted there would be differences in the levels of carbohydrates, pigments and enzymes. Our research strived to answer the question as to whether grapes and raisins possess different structural qualities as an effect of the dehydration process. Our hypothesis is that grapes and raisins would be alike in sugar content, but may contain different pigments, and that raisins would have a higher concentration of protein.
We utilized the Barfoed’s, Iodine, Selivanoff’s, and Bial’s Tests to determine the different carbohydrates in each of our samples of grapes and raisins. For each of these tests, we carried out two trials on each of the three different concentrations (A, B, C) of both the grapes and raisins solution. The stock solution for the grapes contained 50.96 grams of grapes and 200 ml of deionized water. The raisin stock solution included 50.46 grams of raisins and 200 ml of deionized water. Since the stock solutions were too thick, we diluted the stock at three different concentrations to enable us to observe the color changes more accurately. Solution “A” consisted of 300 μl of either grape or raisin stock solution and 500 μl of deionized water. Solution “B” contained 300 μl of either grape or raisin stock solution and 1000 μl of deionized water. Solution “C” included 300 μl of either the grape or raisin stock solution and 1500 μl of deionized water.
Barfoed’s test can differentiate monosaccharides from di- and polysaccharides. The monosaccharides will react with copper ions, while the disaccharides will not. A positive reaction is recognized by the fabrication of a red precipitate. A monosaccharide is a carbohydrate that contains only one sugar molecule. Some examples of a monosaccharide are glucose, fructose, galactose, and xylose. A disaccharide is a carbohydrate that contains two sugar molecules, like lactose, maltose, and sucrose. A polysaccharide, for example starch, is a carbohydrate that contains more than two sugar molecules.
For Barfoed’s Test, our results for the grapes were the same for solutions A, B, and C in both trials (Figure 1). All grape solutions tested produced a red precipitate in a clear-blue liquid. The results of the raisins were consistent throughout both trials and for all three concentrations. When tested on the raisin solutions, the Barfoed’s test produced a teal/blue solution with a red precipitate. Our controls for this experiment, established in prior labs, were water and glucose (Table 1). Water produced negative results with a clear, bright blue solution. Glucose reacted positively with a teal/blue solution and a red precipitate. From our results we can infer both the grapes and the raisins contain monosaccharides (Table 3). We were unable to determine the quantity of the monosaccharides in each solution because the tests preformed were qualitative and only showed a presence or lack there of.
The Iodine Test for coiled polysaccharides tests for the presence of starch. If starch is present, the solution will turn a blue/black color with the addition IKI. All trials and concentrations for both the grapes and raisins remained a yellowish gold color when we added IKI (Figure 2). The results suggest that grapes and raisins do not contain starch (Table 4). However, it is possible grapes and raisins do contain starch, just not enough to react in our test. In addition, our samples were blended and diluted, making it even harder for any substance to show up.
Selivanoff’s Test is a way to confirm if ketoses or aldoses are present. A ketose is a carbohydrate, in which the carbonyl group (carbon double bonded to oxygen) is attached neither at the top nor the bottom of the chain. An aldose is a carbohydrate in which the carbonyl group is attached to either the top or the bottom of the chain. When heated, the reagent forces the ketose to transform the clear liquid to one red in color. Our results of the Raisin solutions for the three different concentrations imply that monosaccharide ketoses are present. Our results of the grape solution for both trials of concentrations “A” and “B” show that monosaccharide ketoses are present. However, the grape solution for both trials of concentration “C” suggested an aldose (Table 5). We believe the reason for this difference is because both fructose and glucose are involved in the organic composition of grapes and raisins. Fructose is a monosaccharide ketose and glucose is a monosaccharide aldose. In Selivanoff’s Test, if ketoses are present, they will be detected first because they react before aldoses. So the only way for aldoses to be detected is if there were not any ketoses present. Our trials suggest either there was an error in the process, making trial “C” produce different results, or that the more diluted concentration in trial “C”, causing and aldose to show up.
The results given by Bial’s Test allows for the determination of furanoses, which are five-membered rings. Pentose furanoses are five-membered rings with four carbons in the ring and the other carbon is a substituent. They are present when the solution turns a green/olive color. Hexose furanoses are five-membered rings with four carbons in the ring and two other carbons are substituents. They are present when the solution turns a muddy brown color. Pyranoses are six-membered rings, and are present when the color remains the same. Our results show that grapes contain pentose furanoses because all three concentrations in both trials were an olive/green color. In both trials for the raisins, the three concentrations were a dark brown color. This indicates that raisins contain hexose furanoses (Table 6).
The data collected in our Carbohydrate tests suggest a consistency in monosaccharides, ketoses and furanoses. The sugars in each are consistence and stay the same, falsifying our hypothesis.
To look at identify the different pigments in each solution we preformed a paper chromotography. We predicted the grape solution would show cholorphyll a and b, carotene and xanthophyll. We predicted the raisin solution would not provide any distinguishable pigments. During the dehydration process the raisins obtain their dark brown color through polyphenol oxidation (Mullins, et al 1992). This brown color prevents the observation of other pigments in the solution.
We performed the paper chromatography twice. Our first attempt provided no results so we used a higher concentration on the second trial. The chromatography strips used to test the raisin solution showed no visible separation of pigments, leading us to believe the solution did not contain any of the pigments chlorophyll a, chlorophyll b, carotene or xanthophylls (Figure 6). The grape chromatography strips showed a light run of dark green and a light strip of orange-yellow (Figure 6). From this we inferred the solution contained chlorophyll b and carotene (Table 7).
To look at the enzymes found in each of our two samples we tested for the presence of polyphenoloxidase (PPO) and preformed a Bradford Assay. The PPO test specifically detects whether or not this enzyme is present in grapes and raisins using litmus paper and pH (Table 2). We predicted that PPO is present in both grapes and raisins (Table 8). The Bradford Assay is a dye-binding analysis. Based on the various protein concentrations, the dye (CBBG) will respond with different colors to show this variation. This variation can be detected by performing an absorbance reading at 595nm. It is read at 595 nm because this is the absorbance maximum of CBBG when it binds to amino acids. This must first be performed using bovine serum albumin (BSA) to create a protein standard curve on which to compare the absorbance values of our grape and raisin solutions. This will determine the protein value, and dividing the protein value over the amount of grape and raisin solution we used will yield the protein concentration in µg/µl. Our predicted result for the Bradford Assay is that raisins will have a higher protein concentration than grapes. We predicted this because raisins have a smaller volume than grapes; therefore, all of the proteins that are found in grapes are compacted into a smaller volume though dehydration.
When tested, we found that the grapes had levels of PPO and it was not possible to establish if the raisins did or not (Figure 7). Using a whole grape and a whole raisin, we put drops of catechol on each sample. The grape turned brown, indicating PPO. The raisin, however, was already brown and this test was inconclusive. For the Bradford Assay we made a standard curve and compared each sample to it (Figure 8). Our absorbance value found from the grape was 0.018. When compared to our curve, this is a protein value of 0.37µg, which yields a concentration of 0.0148 µg/µl. The absorbance value associated with the raisin solution was .074. When compared to the standard curve, this is a protein value of 3.76 µg, which yields a concentration of 0.1504 µg/µl. This data suggests that raisins do have a higher protein concentration than grapes (Table 10). We predicted this to occur due to the condensed nature of the raisin, compared to the grape.
The basis of our study was to determine how the structural components of grapes and raisins compare. Overall, we concluded that our data from the various tests supports our hypothesis. The sugar make-up of grapes and raisins are very similar, as found in the Carbohydrate tests. The pigments that are found in grapes are different than those that are found in raisins, as tested by paper chromatography. We found PPO in the grape samples, but could not conclusively verify the presence of PPO through our trials. However, we did find a higher concentration of protein in raisins than in grapes.
We found our data to be beneficial because raisins are more convenient, as they fit into the fast paced lives of college students. We were able to show the different molecular components of both grapes and raisins. However, our experiment had a few aspects that could be altered, having an effect on the results.
For starters, we used the wrong kind of grapes. We should have used Thompson’s seedless green grapes instead of red grapes. Our results from the various tests may have shown a closer relationship of the structural qualities of grapes and raisins. Another error is perhaps that because we diluted our stock solutions may be we did not get a good representation of grapes and raisins as a whole. In Selivanoff’s Test, we might have observed and recorded the incorrect times that the solution started to turn red. This test reacts fast and it is therefore difficult to record all of the times accurately. For Bial’s Test, our results might have been thrown off because the raisin solution was somewhat brown to begin with. So maybe raisins would have pentose furanoses present, like grapes, instead of hexose furanoses. These colors were very similar and hard to decipher between the slight shade differences.
Through our investigation we learned valued information about the similarities and differences in the grape and the raisin. Although they both contain monosaccharides and ketoses, their structure and taste are very different. Our research intrigued us, and in the future we would be interested to follow up on our experiment, and possibly look for quantitative results rather then the qualitative we found.
Photo A Photo B
Figure 7: The Presence of PPO. The photographs above compare the results of testing for PPO in grapes versus the results in raisins. Polyphenoloxidase (PPO) is an enzyme that catalyzes the oxidation of catechol, a colorless substrate, resulting in a brownish color change. Photo A shows trials one and two of the reaction of after catechol and water were each added to a grape slice. The addition of catechol resulted in a brown color seen in the middle of the grape, while the addition of water did nothing, suggesting the presence of PPO. In both of two trials, when catechol and water were added to a slice of raisin, no visible reaction occurred, suggesting there is no PPO present in the raisins.
References
Written by Mike Kuelbs
Revised by Jessica Wolcott
Revised by Jena Krueger
Bakhru, H.K. Food that Heal. Delhi: Orient Paperbacks. IndianGyan.com, 2000 <http://www.indiangyan.com/books/healthbooks/food_that_heal/raisins.shtml>
Cowan, M. M., E.A. Horst, S. Luengpailin, R. J. Doyle. Inhibitory effects of Plant Polyphenoloxidase on Colonization Factors of Streptococcus sobrinus. Antimicrobial Agents and Chemotherapy 44: 2578-2580, 2000.
Eisenman, Lum. The Home Winemakers Manual. (online book), 1999.
<http://home.att.net/~lumeisenman/contents.html>
Krha et al. LBS-145 Cellular and Molecular Biology. MSU Printing Services. 2003.
Mullins, Michael G., Bouquet, Alain., Williams, Larry E. Biology of the Grapevine. Cambridge University Press, 1992.
Parsons, H. Grapes Under Glass. Transatlantic Arts, Inc., 1955.
Petucci, Vincent E., Carter D. Clary. A Treatise on Raisin Production, Processing, and Marketing. Malcom Media Press, 2001.
Weiss, Kathryn, Linda Bisson. Optimisation of the Amido Black assay for the Determination of the Protein Content of Grape Juices and Wines. Journal of the Science of Food and Agriculture 81: 583-589, 2001.
Grapes. <http://www.innvista.com/health/foods/fruits/grape.htm>