Miles Hirata FST101A Thurs 9-12 SecA05 11/18/13 HP Lab 6: Measuring Enzyme Inactivation by Absorbance I.

Purpose/Objective: The purpose of the experiment is to observe the enzyme denaturing effect that blanching has on enzymes, specifically in turnips. Equal-sized turnip portions were divided and blanched for various lengths of time before measuring their subsequent enzyme activity. II. Introduction: Blanching is a procedure in which food is placed in boiling water for a short period of time. Blanching inactivates the enzymes found within the food and is usually done before the food is frozen to better preserve it. Determining the amount of time the food is blanched is essential to preserving the food. If the blanching time is too short, the enzymes will not be completely inactivated, leading to further maturation and degradation of the food. If the blanching time is too long, the food tissues will degrade and nutrients will be lost. Enzymes are denatured when the thermal energy available is greater than the attractive forces that bond together the higher levels of structure. This energy makes the enzymes unfold from their original structure. While the enzyme shape changes leading to the denaturing effect, the amino acid sequence stays intact. The amino acid sequence is held together by strong covalent bonds, which requires a great amount of heat in order to break these bonds. Understanding the blanching process on enzyme activity can be important in the food and nutrition industry. When processing food, such as preparing frozen meals, blanching for the correct range of time that denatures enzymes while preserving the food tissue is essential for preserving and delivering the food. Properly blanching food will preserve the color, texture and nutrients of the food and also prevent microbial growth from occurring. Thus, blanching is a key procedure in processing safe to eat meals. III. Procedure: The procedure followed for the experiment is found in Principles of Food Composition Laboratory Manual (2013) Experiment 5, Thermal Inactivation of Enzymes,

pages 53-54. Modifications included using 100 microliters of turnip extract rather than 3 drops. Spectrophotometer readings were also done in 5 second increments until 1 minute. At which point, recordings were done every 30 seconds for 1 minute. IV. Data: Table 1. Absorbance and times at which they were recorded for each of the turnip blanched samples. Time (sec) 5 10 15 20 25 30 35 40 45 50 55 60 90 120 Abs 120s .006 .004 .003 .004 .005 .007 .009 .010 .012 .013 .014 .015 .021 .023 Abs 90s .008 .011 .013 .014 .015 .015 .016 .016 .017 .018 .019 .020 .023 .030 Abs 60s .027 .012 .017 .022 .029 .043 .062 .076 .093 .110 .129 .140 .228 .268 Abs 30s .044 .088 .150 .220 .280 .333 .404 .466 .510 .546 .574 .588 .690 .965 Abs 15s .090 .170 .263 .246 .418 .500 .636 .728 .820 .890 .920 .943 1.33 1.58 Abs 0s .160 .270 .390 .470 .700 .820 .975 1.110 1.240 1.310 1.370 1.420 1.700 1.850

Absorbance vs Time
2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 50 Time (sec) 100 150 Abs 120s Abs 90s Abs 60s Abs 30s Abs 15s Abs 0s Linear (Abs 90s) Linear (Abs 60s) Linear (Abs 30s)

Graph 1. Absorbance vs time graph of data from blanched samples.

Table 2. Slope, enzyme activity unit, and ln enzyme activity unit of each blanch sample. Blanch Sample Slope (A.U./sec) Slope (A.U./min) Enzyme Activity Unit 120 sec 90 sec 60 sec 30 sec 15 sec 0 sec 0.0002 0.0002 0.0025 0.0078 0.0136 0.0158 0.012 0.012 0.15 0.468 0.816 0.948 12 12 150 468 816 948 2.48 2.48 5.01 6.15 6.70 6.85 Ln E.A.U

Absorbance

Rate of Inactivation
1000 900 800 700 600 500 400 300 200 100 0 0 50 100 150 Blanching Time (sec) Enzyme Activity

Enzyme Activity Unit

Graph 2. Rate of inactivation of enzyme vs time.

Enzyme Inactivation
8 7 Ln (Enzyme Activity) 6 5 4 3 2 1 0 0 50 100 150 Blanch Time (sec) Ln E.A.U Linear (Ln E.A.U) y = -0.0422x + 7.1618

Graph 3. Ln of the EAU versus blanch time.

V.

Calculations: -Enzyme Activity of 120 second blanch sample: Slope = 0.0002 a.u./sec 0.0002a.u./sec x 60 sec/1min = 0.012 a.u./min

0.012a.u./min x 1E.A.U./0.001a.u./min = 12 E.A.U. -Half Life Half life = 0.693/k -k = 0.0422 0.693/0.0422 = 16.42 seconds VI. Discussion: The data seen on graph 1 indicates the absorbance of each blanch sample. There is a negative correlation seen between the amount of blanch time and the amount of absorbance. This is expected as the absorbance or color change is indicative of enzyme activity. Peroxidase found within the turnip, is responsible for converting guaiacol to tetraguaiacol. This conversion is also the reason for browning of the turnips as it matures. Thus, the 0 second blanch sample had the highest peroxidase enzyme activity, which is exhibited from the brown color change. The 120 second blanch sample exhibited the least amount of color change due to the inactivation of peroxidase. The thermal energy from the boiling water denatured the peroxidase enzyme. Once the enzyme unfolds and denatures, it loses its function. In this case, peroxidase lost the ability to convert guaiacol to tetraguaiacol. After the 60 second blanch time, absorbance declines extremely, indicating the enzyme activity is almost completely deactivated. Graph 3 represents the inactivation of peroxidase over time. The slope indicates the rate at which the peroxidase enzyme is becoming inactivated over time. The calculated half life was determined at 16.42 seconds. Meaning the peroxidase enzyme activity is reduced by half every 16.42 seconds. Since the water used to blanch each sample was held at a constant temperature, the samples are comparable. Graph 2 indicates the rate of enzyme inactivation over time. After the 60 second blanch sample, the enzyme activity begins to stop completely. At the 90 second blanch sample, there is no recorded enzyme activity. This represents the optimal blanch time for turnips as there is no enzyme activity to further mature the turnip and cause browning. It is also the least amount of time to reduce the enzyme activity to 0, thereby preserving the nutrients found within the turnip. This data can be used by food manufacturers to properly preserve foods to increase shelf life. While this optimal blanch time might not be the same in other vegetables, it can be used as a starting point. Vegetables that are smaller and more permeable, such as spinach, would most likely have a shorter blanch time for optimal preservation. The reason for cutting the turnips into small uniform pieces was to create equal size units in each blanch sample. If each sample

had different sized turnip pieces, then some pieces would require longer blanch times to inactivate their enzymes, while enzymes in smaller pieces would already be denatured. While each piece was not exactly the same size as others, they were similar in size enough for the experiment to provide accurate results. VI. Conclusion: Overall, the experiment was acceptable in showing the difference in peroxidase enzyme activity by measuring absorbance. Although this method does not specifically measure the enzyme activity, it is a viable method as long as each sample is prepared and handled correctly. While finding the optimal blanch time for a specific food item may require testing, I have learned that blanching is an effective procedure to preserve and store food. The experiment could be improved by using more samples to blanch, specifically between 60 and 90 seconds. VII. Questions: 1. What does the calculated half life represent? The calculated half life represents the amount of time needed for the concentration of peroxidase to decrease by half. As the calculated half life was 16.42 seconds, every 16.42 seconds the peroxidase concentration was reduced by half. 2. During the blanching, we have assumed that the enzyme was continuously exposed to 100C. Was this the case? If not, how would you redesign this experiment to improve it? Although we tried to create equally sized turnip pieces, making each one exactly the same is near impossible. Thus, while some pieces were completely exposed to the boiling water, some larger pieces were not. I would suggest the use of some type of machine which would cut pieces of exactly the same size. Possible machines that could be used are potato slicers for French fries or a cheese grater. 3. Why is the most blanched sample always the first to be measured, and the least blanched sample last? The reason for this is to prevent any peroxidase from the previous sample getting into another sample. The most blanched sample is measured first since it theoretically has the least amount of peroxidase enzyme.