Carbohydrate and Enzyme Analysis Reveal Better Nutritional Value Within Homemade Vegetable Juice as Opposed to V8

 

By: Team S.A.E.

Elizabeth Firchau, Stephanie Nelson, Sarah Janisse and Andrea Hawk

 

 

Abstract

First Draft By: Andrea Hawk

Revised By: Sarah Janisse, Elizabeth Firchau, and Stephanie Nelson

 

            When performing carbohydrate, photosynthetic and enzyme analysis our group was able to show that a homemade vegetable juice contains more complex sugars and a higher protein concentration. When compared to commercially canned V8 vegetable juice, a homemade vegetable juice displays a higher nutritional value. Our most notable results came directly from carbohydrate and enzyme analyses of both juice varieties.

            Looking specifically at carbohydrates, a dark red precipitate that formed from Benedict’s test indicate that both juices contained free aldehyde and ketone groups where-as only a dark red precipitate was produced in the trials containing V8 juice, indicating the presence of monosaccharides and suggesting the presence of more complex sugars within the homemade blend. Results from Selivanoff’s and Bials test showed similarities between the two juices in which both blends tested positive for ketoses and hexose furanoses. No reaction occurred in either juice variety when mixed with iodine indicating the absence of starch molecules.

            Paper chromatography allowed us to separate photosynthetic pigments such as: carotene, chlorophyll a, chlorophyll b, and xanthophylls. The canned V8 juice only produced a deep orange band, suggesting carotene is present within the juice and producing an average Rf factor of 1. Photosynthetic pigments could not be separated out of the homemade juice. Because the vegetables used within both juices are all ‘products’ of photosynthesis rather than photosynthetic sites (i.e. leaves) the absence of xanthophylls, chlorophyll a and b was not an unexpected result. The presence of carotene within the V8 suggests a higher carrot concentration within this juice blend.

            Examining absorbance versus pH showed a common trend of increasing absorbance to an increase in alkalinity within both juices. The Bradford assay showed that homemade vegetable juice contains an average protein concentration of 1.31mg whereas the V8 averages a protein concentration of 0.78mg. Comparing the two numbers shows that, on average, the homemade juice contains 67.7% more protein than its commercial counterpart. In conclusion, containing complex sugars and a higher protein concentration, homemade vegetable juice can be said to have a higher nutritional value.

 

Discussion

 

First Draft By: Sarah Janisse

Revised By: Andrea Hawk, Elizabeth Firchau, and Stephanie Nelson

 

Our experiment compared commercially bottle V8 juice and a homemade solution of V8 at a molecular level, by analyzing carbohydrates, enzymes, and photosynthetic pigments.  We did this in order to test whether or not pasteurization (of the V8 juice) reduces the nutritional value of the product, and if so, what molecules are affected.  Pasteurization entails the heating of a specific food to a high enough temperature in which disease-causing microorganisms would be killed (Anonymous, 2001), and it is known that molecules such as enzymes (proteins) will become denatured at certain high temperatures (Freeman, 2002). This would, in turn, reduce the amount of protein in the juice affecting the juice’s overall nutritional value. Carbohydrates (sugars) are also known to act differently under certain conditions based on their structure (Krha et al., 2002), which could also affect the nutritional content of a certain product.  Based on this information we performed a series of tests analyzing the carbohydrate structures, enzyme concentrations, and photosynthetic pigments in order to support our hypothesis that the pasteurization of the commercially bottled V8 juice will lessen its nutritional value because it will break down the protein lowering its concentration, and also will change the composition of the sugars by evoking hydrolysis, which breaks hydrogen bonds creating water molecules; reducing them in complexity. 

Carbohydrates vary in complexity. They may contain one sugar molecule, monosaccharides, two sugar molecules, disaccharides, or many sugar units, polysaccharides (Krha et al., 2002). In order to determine the chemical makeup and structures of the different types of sugars present within homemade vegetable juice and V8, we performed a conclusive carbohydrate analysis, which also showed many subtle differences between the two.

Benedict's test is a test that allows one to determine if the sugars present have free or potentially free aldehyde or ketone groups in a solution. Because Benedict’s reagent is composed of sodium bicarbonate, sodium citrate and copper sulfate, the aldehyde or ketone group will become oxidized, which in turn reduces the copper ions, creating a red precipitate (Krha et al., 2002).  In our trials, we found that both the commercially bottled V8 and the homemade solution produced, in similar levels, a red precipitate in solution, which suggested that both solutions were composed of carbohydrates comprising of free aldehyde and or ketone groups.

The next test performed was Barfoed's test, which enables one to identify monosaccharides from more complex sugars such as disaccharides and polysaccharides. Barfoed’s reagent is much like that in Benedict’s test with slight variance in pH, for which Barfoed’s reagent reads approximately 4.5. With a lower pH and a shorter incubation time only monsaccharides can react fast enough to produce reduce copper ions once again resulting in a red precipitate (Krha et al., 2002). No reaction or a reaction occurring after two minutes is an indication of disaccharides and polysaccharides contained in the solution. Some of our most notable results came directly from this test. Our group hypothesized that the homemade juice would contain the more complex sugars, leaving only the commercial product to react with the reagent. Because of the extreme temperatures we thought the more complex sugars that naturally occur within vegetables would be prematurely broken down within the pasteurization process.  In our results, all four trials of the canned V8 vegetable juice tested positive for monosaccharides. The remaining four trials of the homemade vegetable juice didn’t react with the reagent at all, leading us to believe there were more complex sugars contained in this juice blend. With these results we could correlate carbohydrate breakdown within the commercial product as a direct result of the pasteurization process. 

To gather more information about the structure of the sugars present within the juices, Selivanoff's test was performed. Selivanoff’s reagent is made up of resorcinol in concentrated HCl, and when heated along with a sugar will produce furfural or hydroxymethylfurfural, which in turn produces a deep red hue (Krha et al, 2003). Based on reaction times, one can distinguish ketoses that react in a minute or less from aldehydes, which take longer to react. All four trials of both the homemade and V8 juices reacted between forty-five and fifty seconds allowing us to conclude that present carbohydrates were ketoses. Ketoses differ from aldoses structurally, in which the carbonyl group of a ketose is located centrally within the carbohydrate chain where as the aldehyde carbonyl group is situated on the end of the chain.

Another test performed that provided us with insight on carbohydrate structure was Bial's test, which is used to identify different furanoses present in the carbohydrate chain. Bial’s reagent contains orcinol in concentrated HCl, which reacts to distinguish between hexose furanoses and pentose furanoses. Hexose furanoses are six-membered carbon rings and if present, Bial’s reagent will react to produce an olive-green color. Pentose furanoses are five-membered carbon rings that will react with Bial’s reagent to produce a green color (Krha et al., 2002).  In both the homemade and V8 juices all trials reacted with Bial’s reagent to produce an olive-green color suggesting the presence of hexose furanose rings.

The final carbohydrate test performed was one that utilized IKI (iodine-potassium iodide) to confirm the presence of starch. The significance of this tests lies within the fact that iodine interacts with the coils of glucose that make up starch. If present, the IKI reagent will turn from its original color of a yellow brown color to a bluish black (Krha et al, 2003). No trials of either the homemade or the V8 juices reacted with the iodine, therefore it can be concluded that starch was not present in either of the juices.

The only carbohydrate test that was available to us that we did not perform was the mucic acid test which tests specifically for galactose. After careful examination of the ingredients of both juices it was believed that galactose would not be present and therefore unnecessary to perform.

To follow up our carbohydrate analysis, we performed a paper chromatography test, which allowed us to separate any photosynthetic pigments present within the juices. The specific pigments that we were interested in were carotene, xanthophyll, chlorophyll a and chlorophyll b. By comparing the distance in which the pigment travels up the chromatogram to the solvent front, an Rf factor can be calculated, allowing for easier identification of the pigment. In all three trials of the V8 juice, a dark orange band was produced in which the pigment distance was equal to that of the solvent front producing an Rf factor of one. These results indicate a strong presence of carotene within the commercially bottled product. The homemade juice, however, did not show any sort of pigment separation; therefore an Rf could not be calculated. A possible explanation for this could be that pigments are a result of photosynthetic activity, however the vegetables that were used were all products of photosynthesis, rather than photosynthetic sites, such as the leaves of a plant. For this very same reason, the action and absorbance spectrum tests were not carried out because they provide a way to measure photosynthetic activity within the chloroplasts, which in turn are located within the leaves of a plant.

To conclude our research our group conducted enzyme analysis in which we looked specifically and the effectiveness of pH as well as protein concentration by means of the Bradford assay. When looking at the effects pH has on absorbance we mixed a constant concentration of juice and varied the pH by using a phosphate buffer. After running two trials on both the homemade and canned V8 juices the general trend showed that as we increased the alkalinity of the solution, the absorbance was also increased. These results suggest that pH and absorbance are directly proportional to each other.

When comparing protein concentrations of both vegetable juice blends, concentrations were varied; absorbance was taken at a wavelength of 595nm, and then compared to a bovine serum albumin (BSA) standard curve. The main reason why protein concentration can be measured has to deal specifically with the reagent being used. Bradford’s reagent is composed of a Coomassie brilliant blue G-250 dye which binds to specific amino acids (arginine, tryptophan, tyrosine, histadine and phenylalanine) with in the polypeptide protein chain (Krha et al, 2003). Once this dye binds to these specific amino acids, the protein is pulled out into a precipitate turning the dye from a dull gray to a blue, which coincides with the name of the dye. After performing two trials of the Bradford assay on the homemade vegetable juice, an average absorbance could be calculated and then using the equation: y = 0.0069x + 0.1215, produced from the linear relationship of the BSA standard one can solve for the x value and then divide by the juice volume used to calculate a protein concentration. The average protein concentration of the homemade juice was 1.31. Two trials of the Bradford assay were also performed on the canned V8 juice as well. Using the same equation: y = 0.0069x + 0.1215, produced from the BSA standard, using the absorbance to calculate the x value and then once again dividing by the juice volume protein concentration can be calculated. The average protein concentration for commercially, canned V8 vegetable juice was 0.78. When comparing the two average protein concentrations, it can be calculated that homemade vegetable juice contains 67.7% more protein.

With the conclusion of our research one can believe that pasteurization has a significant affect on the nutritional value of commercially canned vegetable juices. The higher protein concentrations and more complex sugars found within the homemade vegetable juice suggest that the extreme heat that the V8 product endures during pasteurization is a key reason for lesser amounts of protein an abundance of simple, monosaccharide sugars. Although the commercial nutritional values are significantly lower than those of a homemade juice, does not neglect the fact that V8 juice is still a healthy product to consume. But to gain the most energy for your body, it is better to consume foods with higher protein concentrations and more complex sugars, so they aren’t metabolized so readily and stick with your body for longer periods of time.

 

 

 

 

A.

B.

C.

 

Barfoed’s Test Results. Barfoed’s test is used to distinguish between monosaccharides, disaccharides and polysaccharides within sugar solutions. No reaction suggests the presence of more complex sugars. In this figure picture A depicts our positive control, galactose, and our negative control, lactose. Picture B represents trials one through four for the canned V8 juice, and picture C represents trials one through four for the homemade vegetable juice. Only the canned V8 reacts with Barfoed’s reagent to produce a rusty brown precipitate indicating the presence of monosaccharides within the juice. The homemade trials retained the original blue color of the reagent indicating the presence of more complex sugars.