During manufacture of food products, manufacturers must put different factors into consideration. The health of consumers becomes a paramount factor of consideration during food manufacturing processes . Consumers present a very sensitive and complex group. People have different medical conditions and as such, manufacturers have to put this into consideration. One common health condition that has been on the rise among consumers is diabetes . Diabetes is caused by foods with high glucose levels. Therefore, this is an aspect that food manufacturers have devised techniques to deal with during food preparation, storage and packaging. Foods with high glucose levels pose a health challenge to patients with glucose intolerance.
For instance for consumers with lactose intolerance, lactase supplements are added to the food products to reduce the amount of lactose present in the food. Enzymes are substrate specific and as such, lactase added to food products is breaks down lactose in food and that way reducing the concentration of the sugar in foods to levels for consumption by consumers with lactose intolerance.
One symptom of diabetes is the presence of glucose in urine. In fact, this is the test for diabetes. Diastix strips are used to test for the presence of glucose in urine and thus diabetes. For consumers with diabetes, food products with high glucose levels lead to high glucose levels in the body that is excreted out of the body through urine. Therefore urine tests using Diastix is used to determine whether someone is suffering from diabetes.
Lactose is contained in many food products that contain milk as one of its ingredients. Milk products are added lactase supplements to counter high levels of lactose during storage and packaging of the products.
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Therefore the objective of this experiment is to determine the effects of varying temperature, PH, substrate concentration (kinetics) and to test for specificity of lactaid (lactase) on substrates. In this experiment Diastix will be use to test and measure the presence of glucose.
Results and Discussion
Effect of Substrate specificity
1. Clean, dry and label six test tubes from 1 to 6
2. In each test tube place, dextrose, lactose, sucrose, maltose, galactose and fructose respectively.
3. To every test tube add 1 ml of lactase and wait for 30 seconds
4. Dip a Diastix strip to test the presence of glucose
5. Record the observation
Enzymes are substrate specific. This is one among the many characteristics of enzymes. A seen with the above results, this trend is apparent. In the first test-tube the Diastix strip tested positive for glucose. Dextrose is glucose and lactase had no impact on the substrate.
Similarly, in the second test tube Diastix strip shows a positive test. Lactase breaks down lactose into glucose within the 30 seconds and this explains why the substrate tested positive for glucose.
In test tube 3, 4, 5 and 6, Diastix detected no glucose. The enzyme used in this experiment is lactase. Lactase cannot break down other substrates apart from lactose.
Enzymes have got special sites known as the active sites. The active sites are the sites that bind with the substrate to catalyze an enzymatic reaction. Active sites have unique shapes that are only particular for a particular type of a substrate. Due to the unique shape of the active site, an enzyme can only bind with only one kind of substrate. Lactase is an enzyme that is only specific to one substrate (lactose). This explains why all other sugars except lactose tested positive for glucose. Dextrose did not require any enzymatic break down as it is in itself a glucose.
Lactase breaks down lactose according to the following biochemical reaction
Lactose + H2O ——(lactase)——galactose +glucose
C12H22O11 + H2O → (lactase) C6H12O6 + C6H12O6 + heat.
The effect of temperature
– Wash and dry three test tubes.
– In each test tube put 10ml of lactose.
– Label the test tubes as A, B, and C.
– For each test tube, add 1ml of lactase and shake the mixture well.
– Take test tube labeled A , and place it in ice at -10c.
– Take the second test tube labeled B and place it in a warm water bath at 370C.
– Take the last test tube labeled C and place it in a hot water bath at 600C
NB: for steps 6 and 7 make sure the temperature is maintained at the specified level to achieve consistent results. A source of heat is recommended in this case.
– Allow the solutions to remain in the water baths and ice respectively for fifteen minutes and 30 seconds each.
– After the specified time elapses, remove each test tube from the water bath or ice respectively and add 1ml of diastix strip to the content of each test tube.
– Observe the results and analyze them.
Temperature at -10C
When the diastix is added to the solution, a light green color is observed. This indicates the absence of glucose in the solution. This is because at very low temperatures, enzymes are deactivated and can therefore not catalyze a reaction. In this case, the low temperatures to which the solution in the test tube was subjected means that the lactase enzyme was deactivated by the surrounding low temperatures of ice at -10C. This deactivation implies that the catalytic breakdown of lactose (substrate) to glucose could not take place. Thus the lactose remained in its original state. Diastix was used to test for glucose in the solution. The glucose meter indicated a negative response to imply that there were no traces of glucose in the mixture.
Temperature at 370C
The solution in test tube turned to a brown color when the diastix solution was added. The brown color implies presence of glucose in the solution. This in essence means that the lactase enzyme was subjected to suitable temperatures that could enable the catalytic conversion of the lactose to glucose. It therefore implies that lactase enzyme is active at room temperature. The glucose meter on the other hand recorded 250mg/dL (1/4). This implied that the total amount of glucose converted constituted a quarter of the total content of the solution.
Temperature at 600C
The solution turned to a dark brown solution. This observation meant that there was presence of glucose. At 600C, lactase enzyme was able to facilitate the catalytic conversion of lactose to glucose and galactose. The glucose meter recorded 400mg/dL (1/3). This value indicated that the total amount of glucose content in the mixture constituted approximately one third of the total solution content. This high rate of conversion implies the optimum temperature for lactase.
Low temperatures inhibit the action of enzymes. This means the rate of reaction is slowed to very insignificant levels. At an optimum temperature, the rate of reaction is very high as long as all other conditions are favorable for the action of the enzyme. Beyond this temperature, the enzymes are completely denatured.
Their ability to catalyze the conversion of substrates is thus lost. Thus the range of temperatures between which an enzyme can actively support catalytic reactions is limited and specific to each enzyme.
Effect of Lactase Kinetics
– A 1ml stock solution with an initial lactose concentration of zero was the starting point.
– Lactose enzyme solution (2ml) was added to the stock solution and a Diastrix strip was introduced. The strip was tested for glucose concentration was measured and 30 seconds after the introduction of the Diastrix
– The experiment was repeated successively but with some specific alterations with alterations. The enzyme concentration remained the same. But the substrate concentration was altered whereby the lactose concentration was increased by 1ml and the water concentration was decreased by 1ml in each successive experiment. In each of the experiments, a Diastrix strip was introduced to measure the substrate concentration. The results were recorded 30 seconds after the introduction of the strip and the resultant color change was compared with the provided scale to show the substrate concentration.
– All the results from the above experiments were recorded and tabled.
Below is a table showing the recorded results from the experiment above.
After inserting a Diastrix strip into the solution, the strip’s test area changes color in accordance to the amount of glucose in the sample. This color change corresponds to a specific glucose amount.
In our experiment, we started with a solution containing no lactose. The result was ultimately negative because there was no substrate for the enzyme to act on. However, as the concentration of the lactose is increased, there is an observable color change in the Diastrix strip. As mentioned above the increase in the amount of lactose translates into an increase in the action of the enzyme lactase which converts each lactose molecule into two glucose molecules.
One thing that should however be noted is that the enzyme concentration remains the same throughout the entire experiment.
The results from the above experiment are consistent the findings from many researchers that the amount of the desires test specimen (glucose) increases with the increase of the analytes (the lactose solution). It is therefore safe to conclude that our experiment was a success and indeed yielded the expected results.
Effect of PH on enzyme action
– Three test tubes each containing 10ml of lactose solution were prepared
– The solutions in the test tubes were placed under three different Ph mediums. The first test tube was placed under a basic medium of ph 9. The second test tube was placed in a neutral medium of around ph 7. The third sample was placed in an acidic medium of ph 2.
– The enzyme was left to act on the substrates for about 15 minutes after which a Diastix strip was introduced into the solution. The strip was tested for color change 30 seconds after insertion of the strip.
– The results were recorded
– In the test tube containing lactose solution in a basic media of Ph 9, the result was negative. This means that there was no enzyme action on the lactose solution. The lactose was not converted to glucose for a positive test to be yielded.
– In the test tube containing lactose solution lactose solution in an acidic media, the results were also negative. This means that there was no lactose breakdown into glucose for a color change and hence a positive result to happen.
– In the test tube containing lactose solution in a neutral media, there was a positive result in that there was a color change in the Diastix strip. This means that there was enzymatic breakdown of the lactose into glucose.
During preparation of food substances, the impact of enzymes on food quality is enormous. Food substances contain enzymes and as such enzyme, catalyzed reactions continue to take place even after foods have been released to the market for consumption. For that reason, it is worth to assess the various enzyme properties and set conditions right to prevent possible compromise of the consumer health.
In this experiment, the aim was to determine the various characteristics of lactase under different temperature, pH conditions and at different concentrations of the substrate. Moreover, the experiment sought to understand the substrate specificity of lactase enzyme.
As seen in the above experiment, lactase is substrate specific as it did not break down all other sugars into their simple sugars. Enzymatic reactions are influenced by Ph. Some enzymes work best in basic conditions, others in neutral and others in acidic medium. However, for the case of lactase, the enzyme attains its optimum working in neutral conditions. Lactase at very low temperatures is deactivated and this hinders enzymatic break down of the substrate. As seen in the experiment, Diastix strip tested positive for glucose at 600C. As substrate, concentration increases the rate of enzymatic break down of lactose into its simple sugars increases.
Processing of foods containing lactose and lactose supplements (lactaid) such as in most milk products, the above discussed aspects should be put into consideration in order to achieve the best set of conditions to attain the safety and health of the foods.
The experiment was successful in terms of unveiling the enzymatic properties of lactase. However, improvements should include the action of other enzymes that are used in food processing and packaging in order to get a wider view of how various enzymes behave in different conditions.
Bettelheim, Frederick A, and Joseph M. Landesberg. Laboratory Experiments for Introduction to General, Organic, and Biochemistry. Belmont, CA: Brooks/Cole, Cengage Learning, 2010. Print.
Tamanoi, Fuyuhiko, Ross E. Dalbey, and Carla M. Koehler. The Enzymes: Volume 25. Amsterdam: Elsevier/Academic Press, 2007. Print.