Enzyme, carbohydrate and photosynthetic analysis show frozen green beans (Paseolus vulgaris) as best preparation

 

The Chloroplasts

 Tom Barkham, Jennifer Lindley, Laura Neuder, and Julie Nap

LBS 145

 b

 

Abstract

Several tests were used in order to differentiate between the various preparation methods of frozen, cooked, and canned beans with respect to carbohydrates, pigments, and the presence of enzymes.  Raw green beans were used as a control in this analysis.  We predict that canned green beans would have the greatest similarities to raw green beans in comparison to cooked and frozen. To test the presence of carbohydrates the Barfoed’s, Selivanoff’s, and Iodine tests were used.  The Barfoed’s test exhibited a presence of monosaccharides in cooked green beans while frozen, canned and raw green beans showed a presence of polysaccharides.  The Selivanoff’s test showed all beans reacting within one minute indicating a presence of ketoses.  The Iodine test indicated the presence of starch in all beans. Pigment absorption was tested using paper chromatography and the absorption spectrum test.  Our data from the paper chromatography was inconclusive. The absorption spectrum showed the greatest absorbance in descending order: frozen, cooked, canned, and raw. The presence of enzymes was tested using the presence of polyphenol oxidase (PPO), and the presence of heat and pH on enzyme activity diagnostic tests.  PPO was found in frozen and canned green beans while raw and cooked did not exhibit the enzyme.  There was no significant difference in the presence of PPO when heat was added.  Frozen and raw green beans had the highest presence of PPO when the pH of the solution was altered. Therefore, the preparation of frozen green beans closely resembles the characteristics of raw.

 

Discussion

            There are various ways to prepare green beans. In this laboratory investigation, we decided to determine which preparation method (cooked, canned or frozen) most closely resembles the characteristics of raw green beans (control). Our initial hypothesis stated canned green beans most closely reflected raw green beans. We preformed a series of different tests to look for carbohydrate, enzyme and pigment variations in the four samples of green beans. Our results did not confirm our hypothesis, instead, we found that frozen prepared green beans are closely comparable to raw.
In our first experiment, tests were completed in order to determine the different types of carbohydrates. Plants synthesize starch and then stockpile it in the form of glucose, which is a major cellular fuel. Since starch is stored energy in the plant, all preparation types of beans should contain starch. Previous experiments have shown that that heating process undergone by prepared vegetables depletes the vegetable of starch. Therefore, we predicted that frozen, canned and cooked samples would all have starch present, but the amount would be less compared to raw (Nyman, Svanberg and Asp, 1994). Using Iodine staining, we tested for the presence of starch. Each type of bean (raw, cooked, canned and frozen) varied in the color change, but no assumptions as to the amount of starch could be drawn from the variations in color. As expected, due to the importance of starch to a plant; raw, cooked, canned and frozen beans all contained starch.
The Selivanoff's test determined whether any carbohydrates present were ketoses or aldoses. Ketoses are sugars, which contain a carbonyl group within a carbon chain. An aldose is a sugar, which contains a carbonyl group on the terminal end of the carbon chain. As stated above, galactose is a common sugar found in green beans, which consist of aldoses. When green beans are heated, galactose is diminished into the solution that the green beans are cooked or canned in. Therefore, we predicted that prepared green beans would have more ketoses than raw green beans, which would consist mainly of aldoses (Nyman, Svanberg and Asp, 1994). Within one minute, each of the green bean solutions formed a red/brown precipitate, indicating the presence of ketoses. Our results showed that raw green beans do not have a differing dominant functional group carbohydrate (aldose) than the prepared methods of green beans.. Instead frozen, cooked, canned and raw all contained ketoses. This does not mean that aldoses are nonexistent in all green beans. Instead, the concentration of ketoses is higher than the concentration of aldoses.
The Barfoed's test distinguished the presence of monosaccharides (single sugars) versus polysaccharides (many sugars) within the green bean samples. Once again, we predicted that the raw beans would contain monosaccharides as oppose to polysaccharides as with the processed beans because cooking and canning processes deplete the beans of carbohydrates. "During wet heat treatment, as in blanching, boiling and canning of vegetables and fruits, there is a considerable loss of low molecular weight carbohydrates (i.e. mono- and disaccharides)" (Effects of Food Processing on Dietary Carbohydrates: Carbohydrates in Human Nutrition , 1997). The observation of a precipitate among the green bean solutions indicated that each sample contained monosaccharides sugars. The results did not confirm our prediction that fresh beans do contain monosaccharides whereas the other types did not contain them. Instead, our results show that raw, cooked, canned and frozen green beans all contain monosaccharides. Again, this does not mean that polysaccharides are not present within the sample, instead, their concentrations are lower than monosaccharides.
Our second experiment involved pigments and absorption rates within the chloroplast. The chloroplast is the component of a plant cell, which helps to produce chemical energy in the process of photosynthesis. The pigments that are released from the chloroplast include carotene, xanthophyll, chlorophyll a and chlorophyll b. Using chromatography, the samples of green beans were tested to see how much of each pigment was present. Our group predicted that canned and cooked green beans would have smaller Rf values for chlorophylls than raw because chlorophylls are depleted when heat is added. Caretenoids would remain the same in comparison to raw green beans (de la Cruz-Garcia, Gonzalez-Castro, Oruna-Concha, Lopez-Hernandez, Simal-Lozano and Simal-Gandara, 1999). In contrast, frozen green beans should have smaller Rf values than raw green beans for chlorophylls and caretenoids because a decrease in temperature depletes the presence of pigments (Monreal, de Ancos, Cano, 1999). In this experiment, none of the pigments were observed using paper chromatography. This is because the samples of green beans are not soluble in the solutions that were provided in the laboratory. This does not mean that carotene, xanthophyll, chlorophyll a and chlorophyll b are not present in the bean. Instead, the methods provided in the laboratory were not capable of expressing these pigments. Therefore, our results are inconclusive.
The absorption spectrum analysis test was performed as another way to look at the pigments in each type of green bean. A solution was prepared using each sample of green bean and placed in a spectrometer in which the absorbance was determined. We predicted that cooked and canned would have the closest absorption rate to that of raw green beans. This is because caretenoids stay intact when the green beans are heated during preparation. Therefore, the spectrum would resemble the caretenoids still present within the bean. Chlorophyll on the other hand is depleted when green beans are heated and therefore would not be representitive in the spectrum (de la Cruz-Garcia, Gonzalez-Castro, Oruna-Concha, Lopez-Hernandez, Simal-Lozano, and Simal-Gandara, 1999). We also predicted that frozen green beans would have a spectrum quite different than raw. This is because the freezing process depletes the green bean of both chlorophyll and caretenoids (Monreal, de Ancos, Cano, 1999). After experimentation, we actually found frozen green beans to have the highest absorption rate followed by cooked, canned and raw. This could be because of the chlorophyll degradation of pheophytin is favored in acidic conditions. Frozen green beans were found to be the most acidic in the enzyme portion of this lab, therefore confirming these results (Cruz-Garcia, Gonzalez-Castro, Oruna-Concha, Lopez-Hernandez, Simal-Lozano, Simal-Gandara, 1999). The freezing process actually increases the absorption of pigments within green beans because the absorption spectrum of frozen samples are significantly higher than the absorption spectrum of raw green beans. Considering that at 400nm, both had relatively the same absorption, frozen surpassed raw green beans at 430nm. Therefore, the acidic conditions as well as the process that the frozen green beans are prepared allow for a greater absorption of pigments.
For the third experiment, the activity of the enzyme polyphenoloxidase (PPO) was determined using the effects of heat and pH. First, we determined if PPO was present in green beans. We could not find any previous documentation to whether PPO is present in green beans. Instead we hypothesized that it did contain the enzyme because often green beans will discolor after harvesting. We found that frozen and canned green beans had PPO because there was a slight color change when 0.1% catechol was added. Next, we tested the effect of heat on enzyme activity. Again the enzyme of study was PPO. We predicted that cooked and canned green beans would have the highest enzyme activity because heating increases the catalyzation of enzymes (Kaack, 1995). Instead, we found that only cooked green beans had a change in the catalyzation due to heat. The color change was very slight indicating that a slight change in enzyme catalyzation was undergone. When testing for the effects of pH, we predicted that canned green beans would have the greatest presence of enzymes. This is because we assumed that canned green beans are in acidic solutions, as well as since they are heated during preparation. Enzyme activity increases if an acidic pH is at higher temperatures. On the other hand, a basic pH will decrease the presence of enzymes when the green beans are heated, therefore implying that cooked and canned green beans will have significantly less presence (Delincee and Radola, 1974). Our results contradicted our predictions, instead frozen green beans have the highest presence of enzymes due to the effect of pH. When we applied litmus paper to the green beans during the presence of PPO tests, we noticed that frozen green beans had the most acidic pH. Therefore, this information would correspond with the assumption that frozen green beans have the highest enzyme presence due to pH.
Overall, due to carbohydrates, photosynthetic potential and enzyme activity, frozen green beans most closely resemble raw green beans. The carbohydrate diagnostic tests and the pigment identification tests were inconclusive in this analysis. The absorption spectrum of pigments as well as the effects of heat and pH on enzyme activity lead us to conclude that the preparation of frozen green beans closely resembles raw. This does not mean that canned and cooked green beans are not comparable to raw green beans. Although the conclusion was drawn that frozen beans are most like the control, there was not an abundance of evidence pointing in the direction that preparation highly affects green beans content. Instead, frozen green beans are most comparable to raw in general with the experiments done on sugars, photosynthesis and enzymes. Testing different aspects of green beans, such as vitamins or any other type of nutritional content may show another type of processing most effective at preserving the values of a raw green bean. Also, the fact that we used the same samples of green beans each week, while refrigerating the samples when not in use, was done to retain consistency. Forms of contamination as well as spoiling could have altered our results. The time in which the green beans were removed from their preparation method and were ground down to a liquid like form could have altered the content of the bean. Although the brand of beans used and the methods to obtain analyzed materials was only one way to gather results, it was the only way in which we were able to use within the laboratory.
Therefore, in this analysis of green beans, we conclude that the preparation of frozen green beans closely resembles raw green beans. This conclusion was made due to the data that we collected while analyzing carbohydrate, photosynthetic and enzyme content. We do not condone the consumption of cooked and canned green beans. They also offer their consumer excellent tasted and nutrients. But ultimately because of this analysis, we propose that frozen green beans are the best substitute for raw.

 

Table 1. Carbohydrate Test Results.  Each of the following tests have a specific characteristic that help to determine which carbohydrate is exhibited in cooked, raw canned and frozen samples.  The Barfoed’s test distinguishes between monosaccharides (reduced red cupric ions) and polysaccharides (blue).  Selivanoff’s test distinguishes between aldoses and ketoses (ketoses will react red within one minute while aldoses will react after several).  The Iodine test identifies starches; it will turn a substance a blue-black color when starches are present. (three test tubes refers to each of three trials done)

Selivanoff’s

Barfoed’s

Iodine

Cooked             25%

3 test tubes reacted within 1 minute, with a red/brown precipitate.

3 test tubes reacted which showed a slight red precipitate.

3 test tubes resulted in a light gray color.

                         50%

3 test tubes reacted within 1 minute, with a red/brown precipitate.

3 test tubes reacted which showed a slight red precipitate.

3 test tubes resulted in a light gray color.

Raw                  25%

3 test tubes reacted within 1 minute, with a red/orange precipitate.

3 test tubes reacted staying a blue color with a very small red precipitate.

3 test tubes resulted in an orange color.

                         50%

3 test tubes reacted within 1 minute, with a red/orange precipitate.

3 test tubes reacted which showed red precipitate.

3 test tubes resulted in a light gray color.

Frozen              25%

3 test tubes reacted within 1 minute, with a dark red/brown precipitate.

3 test tubes reacted which showed a red precipitate.

3 test tubes resulted in a dark purple color.

                         50%

3 test tubes reacted within 1 minute, with a dark red/brown precipitate.

3 test tubes reacted which showed a red precipitate.

3 test tubes resulted in a dark purple color.

Canned             25%

3 test tubes reacted within 1 minute, with a light red/brown precipitate.

3 test tubes reacted which showed no change staying a blue color.

3 test tubes resulted in a darker purple color.

                         50%

3 test tubes reacted within 1 minute, with a light red/brown precipitate.

3 test tubes reacted which showed no change staying a blue color.

3 test tubes resulted in a darker purple color.

Control             (+/-)

(Deionized-Water) showed no change.

(Sucrose) reacted to show no color change (blue)

(Starch) resulted in a dark purple color