Enzyme Tests on Organic vs. Purchased “Fresh-squeezed” Citrus sinensis Indicates PPO Inhibition in Purchased Juice 

 

 

 

 

By: Shaun Wahab, Chad Zillich, Michelle Harvey, Zach Smith

Gelopees

LBS 145

W2

Dr. Luckie

10/16/02

 

ABSTRACT :

Using a series of experiments testing for sugars, pigments, and enzymes, concerning cell physiology, a comparison was made between the juices from freshly squeezed, organically produced oranges, and a particular brand of store bought juice (Tropicana Orange Juice - fresh squeezed).  The expectation was to find indications of a presence of additives in the brand name juice.   These differences would help support our hypothesis that store brand juice is not entirely pure.   The additives added would affect the characteristics of the juice, for example, the types and number of carbohydrates, types and number of pigments, and types and number of enzymes present.  There was speculation that the additives would have an effect on the enzymes already present in the orange. 

Experiments were done using common tests, Bial’s, Barfoeds’, and Selivanoff’s, searching for organic compounds, and found slight color changes in Bial’s indicating presence of five-ringed molecules.  Barfoed’s indicated the presence of reducing sugars that are monosaccharide along with Selivanoff’s test indicating ketoses in both of the juices. These results were compared to previously obtained knowledge in an effort to discover some characteristics of the juices’ carbohydrate compositions.  Paper chromatography tests and absorbance spectrums were used to compare pigment differences, and a series of enzyme experiments were done to examine the possible inhibition of enzymes by additives.

Our data suggested the possibility of additives, but showed no significant data to completely support out hypothesis. Further studies should be performed to better investigate the presence of additives in store bought juice.

 

DISCUSSION :

            In an effort to support our initial hypothesis that there are many additives to “freshly squeezed” store bought orange juice in comparison to the juice squeezed from an organically grown orange, we performed a series of experiments comparing them.  These experiments compared characteristics of present carbohydrates, differences in pigments, and varying enzyme levels.  Unfortunately, these experiments did not provide a significant amount of data to support our hypothesis.

            We expected that store bought juice would have a greater variance in present carbohydrates.  Our results unfortunately did not support our prediction, showing little variance in carbohydrate test results. Bial’s test, the test for the presence of a furanose ring, did not reveal any significant differences other than in concentration. When the resulting solutions turn an olive/brown color this indicates that a hexose furanose is present. If the color results in a greener color, the this indicates the presence of a pentose furanose. For all of the solutions that resulted in a color change, we found them all to look more olive than green, indicating the presence of a hexanose furanose. Both juices contained sugars with a furanose ring, however, in unequal proportions.  Selivanoff’s test, the test for a ketose or aldose, showed very similar results for both juices.  After the Selevanoff’s test was performed all solutions reacted with a sub-minute, red color change indicating that the sugars present were ketoses. The last carbohydrate test performed was Barfoeds’s test, which indicates whether monosaccharides are present versus di-/polysaccharides. A rusty brown precipitate indicates the presence of a monosaccharide, or positive reading. All tests resulted in positive readings, however, the 1% concentrations of both juices were only slightly positive, light rust. Therefore we noted that, both juices contain monosaccharides. This evidence did not support the prediction that manufactures add carbohydrates to the “fresh squeezed” juice during production because there were no significant differences in the sugar types or structures between the two juices.

              We anticipated that there would be natural pigments found in both juices.  We initially thought that carotene would obviously be found in both juices and that xanthophylls, which is a yellow pigment, would be found as well, probably more so in the store bought juice because of its pale yellow reflection. However, after performing the tests, the paper chromatography showed a presence of only carotene in both juices.  Neither of the juices we tested using chromatography showed a presence of chlorophyll a, chlorophyll b or xanthophylls.     It was not surprising to find the absence of chlorophylls, but to find that no xanthophylls were present whatsoever did not support our original hypothesis.  No bands of color showed on the chromatography paper until the carotene band, therefore giving us a rate of flow value of 1 for both juices (Figures 4 and 5). The rate of flow value indicates ratio of the distance traveled by the dissolved substances to the distance the solvent moved.  Next, we examined absorption values for supernatant solutions of both juices to see if any additives were present in the store bought juice.  As predicted, the absorption graph was significantly higher for the equivalently prepared solutions.  To compare these values we can look at the graph and see that at a wavelength of 400 nm, the Tropicana had an absorbance of .808 and the organic juice had a value of .108.  The greater absorbance of light shows that some additives were indeed added to the store bought juice. The two juices would likely have very similar absorbencies were there a lack of additives (Figure 6). 

            For tests done on enzymes, we initially tested both juices for the presence of PPO by taking absorption graphs of solutions of catechol and supernatant juices.  The solution of the organic orange juice increased slowly, but steadily for the entire fifteen minutes tested.  The absorption increased because PPO was enabled by the catechol, resulting in a slightly darker solution.  The darker color occurs when PPO reacts with catechol producing o-benzoquinone, which is a brown color.  This indicated a small but noticeable amount of PPO.  As for the store bought juice, no PPO was found, as the absorption of the solution decreased over the fifteen minute period.  The absorption fell because the catechol diluted the juice solutions.  The absence of the effects of PPO was found to be a result of pasteurization.  Pasteurization is the heating of a substance to cause a loss of enzyme activity.  In doing so, enzymes such as PPO are inhibited; resulting in a much longer shelf life for store bought orange juice (Pasteurization, 2000).

After discovering that the store bought juice was pasteurized, we performed our own pasteurization on the organic juice.  We used methods of direct heating and heating via water bath.  Both heating methods proved to pasteurize successfully as the absorptions for both cases decreased steadily over the ten minute time period.  This showed that PPO was inhibited in the reaction, as it caused no increase in absorption. 

We also performed another pasteurization process on the store bought juice to see if there would be any difference in the rate of change between the original and re-pasteurized store juices.  After exposing the juice to both direct and water bath forms of heating, no significant change was found as to how fast the absorption decreased.  This shows that the PPO was inhibited or denatured in the initial pasteurization of the juice by the company.  Our additional heating did not produce any reduction of PPO enzyme presence.

            More concrete evidence could have been obtained with a few improvements in our methods. First, more extensive research on the molecular properties of oranges should have been obtained to better understand our subjects and focus our research on relevant characteristics or properties of those subjects. Second, with more time we could have performed a greater number of trials, which would ensure more accurate results.. Lastly, additional subjects should have been tested such as different brands of juice as well as non-organically grown oranges. This would increase our understanding of additives found in store bought juices and/or the characteristics of organic vs. inorganically grown oranges.  

            Further research could be done concerning additives in orange juice. Suggested research includes investigating more enzymes other than PPO, additives commonly added to juice and how they affect the enzymes. Other research could include the investigation of added pigments to the orange fruit and how or if at all they affect human beings.       

            In sincere efforts to rigorously investigate the effects of additives in store bought and organic “fresh-squeezed” orange juices there was no significant support for our hypothesis. We expected to find noteworthy differences on the molecular level due to additives in the store bought juice. With improvements in protocol and additional research we believe that a more supportive conclusion could have been obtained.

           

 

Table 3. The effect of time, and heat on polyphenyloxidase (PPO) in Orange Juice solutions. Absorbancies were taken of organic and store bought fresh squeezed juice solutions, after the addition of catechol (1.5:1.5ml) which increases the reaction rate of the PPO, and enzyme in oranges.  The change in absorbancies indicate a presence, non-presence, or inhibition of the PPO in the juices. After heating in a water bath for five minutes and adding direct heat absorbancies were taken again for comparison.

Non-Heated

 

Water Bath (5 min)

 

Direct Heat (5 min)

Organic

 

 

Organic

 

 

Organic

 

Absorbance

Time (min)

Absorbance

Time (min)

Absorbance

Time (min)

1.054

0

 

1.104

1

 

1.05

1

1.056

1

 

1.08

2

 

1.24

2

1.058

2

 

1.082

3

 

1.014

3

1.052

3

 

1.08

4

 

11

4

1.058

4

 

1.068

5

 

1.01

5

1.058

5

 

1.066

6

 

1.01

6

1.06

6

 

1.064

7

 

1.008

7

1.06

7

 

1.068

8

 

1.004

8

1.062

8

 

1.062

9

 

1

9

1.062

9

 

1.062

10

 

0.999

10

1.062

10

 

 

 

 

 

 

1.062

11

 

 

 

 

 

 

1.062

12

 

Tropicana

 

 

Tropicana

 

1.064

13

 

Absorbance

Time (min)

Absorbance

Time (min)

1.068

14

 

1.356

1

 

1.246

1

 

 

 

1.276

2

 

1.232

2

Tropicana

 

 

1.276

3

 

1.224

3

Absorbance

Time (min)

1.266

4

 

1.22

4

1.932

0

 

1.58

5

 

1.214

5

1.93

1

 

1.248

6

 

1.216

6

1.926

2

 

1.242

7

 

1.206

7

1.92

3

 

1.222

8

 

1.2

8

1916

4

 

1.214

9

 

1.196

9

1.914

5

 

1.212

10

 

1.186

10

1.912

6

 

 

 

 

 

 

1.91

7

 

 

 

 

 

 

1.908

8

 

 

 

 

 

 

1.98

9

 

 

 

 

 

 

1.908

10

 

 

 

 

 

 

1.908

11

 

 

 

 

 

 

1.906

12

 

 

 

 

 

 

1.904

13

 

 

 

 

 

 

1.9002

14