The effects of table salt (Sodium chloride)

The most frequently used table salt for food seasoning is sodium chloride. Normally, it is very common for one to add a pinch of table salt to their cooked dishes. As an Asian, we love dishes such as Chinese-style fried rice, fried noodles with pork in soy sauce and Hakka style salt-baked chicken, and we love ordering this dishes often when we are eating out in a restaurant. However, these dishes are high in salt content. Sodium is important in our diet to balance blood pressure and nerve signalling. But, too much consumption of salt can result in serious health complications such as high blood pressure and heart disease. I would consider investigating how the concentration of salt affects the rate of breaking down food.

By observing the rate of enzymatic breakdown of starch to glucose in different salt concentrations, I can know the rate of digesting food when consuming a different amount of salt in the diet, and not involving any human experiments.

Amylase is an enzyme that functions to breaking down starch into sugars. It is an enzyme that takes part in the digestion process in human. Amylases are found in almost all plants, animals and microorganisms. Significant amounts of amylase are found in the pancreas and saliva in people (Birch et al. 45–49). Like all enzymes, amylase has to be kept in a particular condition to function at its optimum level. This internal assessment will explore the effect of different concentrations of sodium chloride (0.5%, 1%, 5%, 10%, 20%) on the functioning of amylase by using starch collected by the grinding of rice and iodine.

When ground rice is mixed with iodine, the mixture will turn to blue-black due to the presence of starch. However, when added amylase to the mix, the mixture will become colorless since the starch molecules will be broken down into glucose molecules by amylase. Thus, the rate of enzymatic reaction can be calculated by the speed of changing in color of the mixture. Since the rate of change in color is equal to the rate of change (time used to change color) in absorbance of the blue-black solution. The rate of enzymatic reaction of amylase can be calculated by the following formula (Rate of Reaction =absorbance/Time) (Krishna 22–32). The aim of this experiment is to find out the rate of reaction between amylase and starch in a range of concentrations of sodium chloride concentrations.

Hypothesis:

A faster rate of decreasing in absorbance of the blue-black color solution means there is a higher rate of amylase reaction, whereas a slower decrease in absorbance of the blue-black color solution equals a lower rate of reaction. Sodium chloride concentration will be manipulated into the solution to examine the effect of different concentrations on the rate of amylase reaction. A higher sodium chloride concentration might cause a conformational change in the enzyme and can no longer bind to its active site. Therefore, the hypothesis is that a high concentration of sodium chloride will denature amylase thus not being able to break down starch into glucose, which will result in a slow rate of color change of the solution. When sodium chloride concentration increased, the rate of amylase reaction decreased.

Research Question:

Does sodium chloride concentration affect the rate of enzymatic breakdown of starch to glucose in Oryza sativa, and if so how?

Variables:

Materials and Apparatus:

1. Apparatus

• Test tubes
• Test tube rack
• Labels
• 50mL Beaker
• Stopwatch
• Colorimeter

2. Materials

• Rice (with juicing materials)
• Sodium chloride solutions (0.5%, 1%, 5%, 10%, 20%)
• 0.01M Iodine solution
• 1% Amylase concentration

Safety:

• Wear safety goggles, lab coat, and appropriate laboratory shoes.
• Avoid the ingesting of chemicals.
• Make sure the experiment is conducted in a well-ventilated area.
• Avoid direct contact with chemicals

Procedures:

Preparation of starch mixed with iodine

1. The starch solution: Oryza sativa will be weighted (appropriate amount is 0.5g to 1.0g) and then added to water that has been heated in order to make a paste. It will then be diluted to about 50 to 100 cm³.
2. 300μl of iodine is put into the starch solution. It is stirred several times using a plastic stirrer until a blue-black solution is made.

Conducting the experiment:

1. The experimenter will make 50cm³ or 100cm³ of 1% of the soluble starch in the distilled water. Quantities of 0.5 cm³, 1cm³, 2cm³, 3cm³, 5cm³, and 10cm³ will be measured using a graduated pipette and the solution will be placed in different test tubes.
2. The solution in each tube will then be filled to 10cm³ using distilled water.
3. There will also be another tube that will have 10cm³ of distilled water (control purposes)
4. In each tube, add a drop of iodine solution and then mix thoroughly.
5. From each solution, transfer enough quantities from the solution in order to fill the cuvette of a clean colorimeter. Take the reading of the absorbance at ‘orange’ wavelengths (610nm).
6. Plot the graph of absorbance against the concentration.

Results:

Observation

Even with the naked eye, one could observe the disappearance of color inside the cuvette, from a dark, blue-black coloration to a clear, colorless state.

Data Processing

Calculations (Average Rates of Reaction)

20% Concentration

(-0.00124+-0.00127+-0.00123+-0.00122+-0.00125)/5= -0.00124

10% Concentration

(-0.00141+-0.00135+-0.00141+-0.00134+-0.00137)/5= -0.00138

5% Concentration

(-0.00163+-0.00162+-0.00164+-0.00161)/4= -0.00163

1% Concentration

(-0.00163+-0.00175+-0.00173+-0.00174+-0.00167)/5= -0.00170

0.1% Concentration

(-0.00183+-0.00197+-0.00199+-0.00187+-0.00179)/5= -0.00189

Effect of Sodium Chloride Concentration on the Rate of Reaction of Amylase

Uncertainties:

Example of a Calculation for Standard Deviation:

NaCl Concentration at 20%

σ =square-root [(-0.00124) -(-0.00124)] ² + [(-0.00124) -(-0.00127)] ²+ [(-0.00124) -(-0.00123)] + [(-0.00124) -(-0.00122)] + [(-0.00124) -(-0.00125)] ²=0.00002

Similar calculations were conducted for the other NaCl concentrations (10%, 5%, 1% and 0.1%)

Uncertainty due to the Dilution of Glucose Solutions:

The uncertainty is caused through the use of a 10cm³ pipette and it will result in an uncertainty value of +/-0.02cm³

Conclusion:

The results supported the hypothesis to the extent that an increase in sodium chloride concentration decreased the rate of reaction of enzyme amylase. However, the decline in the rate of reaction was not exponential; rather, the relationship between NaCl concentration and the average rate of reaction was kind of linear (Purich 443–448). As sodium chloride concentration grew, the average rate of reaction declined at a relatively constant rate. After extrapolating the figures on the graph, it demonstrates that if one were to conduct the reaction further, the rate of reaction would be 0, when the NaCl concentration is at 60% (Rejzek et al. 718–730). Based on the data collected from the test, one can state that the enzyme amylase will be denatured at a 60% NaCl concentration.

In the hypothesis of this experiment, it says that a high concentration of NaCl will denature amylase, and therefore there will be no breakdown of starch into glucose. The result of this will be; a slow rate concerning the color change of the solution (Scholarly Educations 21–35). The results collected from the experiment provide an agreement with the hypothesis. The reason for this is that; an increase in sodium chloride ions that have a different charge group with the enzyme protein amylase will result in increasing protein hydration and denature the enzyme. The graph extrapolated shows that even a concentration of 0.1% of NaCl is enough, to reduce the rate of reaction of amylase by a large proportion.

Evaluation:

The results that were achievable for this experiment are relatively accurate and reliable. During the experiment, there were no striking outliers, and even the standard deviations calculated are not significant enough to contradict the conclusion drawn from the analysis. The relationship between the NaCl concentration and the rate of reaction of the amylase is a negative correlation (University Tun Hussein Onn Malaysia 45–48). The results that were derived from this experiment are proof that even a small concentration of NaCl (0.1%) is enough to have an effect on the electrostatic bonds of the enzyme amylase. In future experiments, for the experimenters to be able to observe the effect of NaCl concentration on the rate of reaction of the enzyme, it will be advisable to use even more minute levels of the sodium chloride.

Human error more likely caused the standard deviation ranges that were calculated in the experiment. The error may result from time delay that took place between when the enzyme amylase was placed in a cuvette, and the moment it took for it to be put in the starch solution (Wingard et al. 128–132). Also, another problem could have been brought about during the process of mixing the amylase with sodium chloride. Ten separate micro tubes had to be filled, one by one. They then had to be mixed through a micro-centrifuge, and in some cases, the amylase solutions in the micro-tubes had taken a longer time to interact with the sodium chloride. Therefore, it meant that in some cases, the sodium chloride took a long time to denature the enzyme, and therefore, it lowered the rate of reaction (Austin Peay State University Department of Chemistry 1–5). However, it is hard to prove the statement, and therefore it can be termed to be a form of speculation.

One can state that the experiment was successful because of the accuracy of the results that were achievable. It was noteworthy that the presence of sodium chloride led to the lowering of the enzyme activity of the amylase. However, there is the need to improve on the errors that were in the experiment through the production of consistent and reliable data. It will facilitate in bringing about a solid conclusion for the experiment. The experimenter felt that the amylase could have been provided with enough time to break down the starch in some of the trials than in others. Therefore, it caused differences in the rate of reaction from one trial to another and also increased the standard deviation as was calculated in the different concentrations.

Limitation and Improvements:

Problem: Calibration errors

Improvement: The error can be improved using different methods. One can conduct the primary test. In this test, it is important to allow the colorimeter to warm enough (recommended time length is 5 minutes). One should then place the cuvette that has ¾ full of distilled water in the colorimeter, close the lid, and then select an appropriate wavelength. One should also press and hold the CAL button that is on the Colorimeter, and when the red LED begins to flash, one should then release the CAL button. After the LED stops flashing, the Colorimeter will have been calibrated and then one should check the absorbance readings which should be close to zero.

In the secondary test, one will prepare a solution that contains color. After a person has calibrated the Colorimeter, one should fill the cuvette with three-quarters of the test solution. One should then place the cuvette in the Colorimeter and then close the lid. The absorbance readings that are noted in this test are between 0.8-1.5. It is important to note that the ideal or normal transmittance is normally between 10-90% transmittance.

Problem: In the procedure, there was a 450µl of sodium chloride solution that was inputted in the micro-tube. On the other hand, only 50µl of amylase solution was inputted, and it resulted in an imbalance. As was shown in the results, and noted in the conclusion, it showed that; even a small concentration of sodium chloride had an impact on the rate of reaction. Therefore, it shows that too much sodium chloride was used in the experiment, as was compared to the amount of amylase. The abundance of the sodium chloride could have had an immense impact on the enzyme activity.

Improvement: In future experiments, it will be wise to balance the amount of sodium chloride solution to that of the enzyme amylase. The appropriate quantity that will be used will be 250µl for every trial in the experiment. The reason for this is that; it is more likely to produce results that are closer to the ones that were hypothesized.

Problem: The 10cm³ that was used in the serial dilution of the sodium chloride decreased the precision and also increased the range of uncertainty in the experiment.

Improvement: In future experiments, it will be preferable to use a micropipette in the serial dilution process. It will most likely reduce or lower the range of uncertainty in the experiment.

Works Cited

Austin Peay State University Department of Chemistry. “BREAKING DOWN STARCH USING SALIVARY AMYLASE.” APSU, 2011, https://www.apsu.edu/sites/apsu.edu/files/chemistry/SP11_1021_BREAKING_DOWN_STARCH_USING_SALIVARY_ENZYMES.pdf. Accessed 2 Jan. 2017.

Birch, G. G., et al. Enzymes and Food Processing. Springer Science & Business Media, 6 Dec. 2012, https://books.google.co.ke/books?id=32Z-BgAAQBAJ&pg=PA45&dq=enzyme+amylase&hl=en&sa=X&redir_esc=y#v=onepage&q=enzyme%20amylase&f=false. Accessed 2 Jan. 2017.

Krishna, Prasad Nooralabettu. Enzyme Technology: Pacemaker of Biotechnology. PHI Learning Pvt., 2011, https://books.google.co.ke/books?id=E6A1aqqLqRIC&pg=PA335&dq=enzyme+amylase&hl=en&sa=X&redir_esc=y#v=onepage&q=enzyme%20amylase&f=false. Accessed 2 Jan. 2017.

Naik, Pankaja. Biochemistry. JP Medical, 30 Nov. 2015, https://books.google.co.ke/books?id=YsMqCwAAQBAJ&pg=PA110&dq=how+external+factors+affect+enzymes&hl=en&sa=X&redir_esc=y#v=onepage&q&f=false. Accessed 2 Jan. 2017.

Perry, George H, et al. “Diet and the Evolution of Human Amylase Gene Copy Number Variation.” Nature Genetics, vol. 39, no. 10, 9 Sept. 2007, pp. 1256–1260, 10.1038/ng2123.

Purich, Daniel L. Enzyme Kinetics: Catalysis and Control: A Reference of Theory and Best .. Elsevier, 16 June 2010, https://books.google.co.ke/books?id=SkSQNNACcrYC&pg=PA443&dq=how+external+factors+affect+enzymes&hl=en&sa=X&redir_esc=y#v=onepage&q=how%20external%20factors%20affect%20enzymes&f=false. Accessed 2 Jan. 2017.

Rejzek, Martin, et al. “Chemical Genetics and Cereal Starch Metabolism: Structural Basis of the Non-Covalent and Covalent Inhibition of Barley β-Amylase.” Mol. BioSyst., vol. 7, no. 3, 2011, pp. 718–730, 10.1039/c0mb00204f.

Scholarly Educations. Amylases: Advances in Research and Application: 2011 Edition. Scholarly Editions, 9 Jan. 2012, https://books.google.co.ke/books?id=k2sLSlx1e70C&pg=PA1&dq=enzyme+amylase&hl=en&sa=X&redir_esc=y#v=onepage&q=enzyme%20amylase&f=false. Accessed 2 Jan. 2017.

University Tun Hussein Onn Malaysia. Amylase Enzyme Detection System Using Image Processing Technique. University Tun Hussein Onn Malaysia, 2010, https://books.google.co.ke/books?id=3eS2DAEACAAJ&dq=enzyme+amylase&hl=en&sa=X&redir_esc=y. Accessed 2 Jan. 2017.

Wingard, Lemuel B., et al. Immobilized Enzyme Principles: Applied Biochemistry and Bioengineering. Elsevier, 28 June 2014, https://books.google.co.ke/books?id=HTiaBQAAQBAJ&pg=PA127&dq=how+external+factors+affect+enzymes&hl=en&sa=X&redir_esc=y#v=onepage&q=how%20external%20factors%20affect%20enzymes&f=false. Accessed 2 Jan. 2017.