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Lab Section: 13156 Brett Spatola BISC 220 Lab The Effects of Lactase Solution on Glucose Concentrations within Different Types of Trader Joe’s Dairy, Plant-Based, and Non-Dairy Milk Introduction: Domesticated animals have proven themselves to be of great value to the human population over the past ten thousand years. A key feature of many of the domesticated animals is the ability to collect milk for consumption. Fast forward to today, and the dairy industry as a whole has become one of the largest parts of society’s modern food business (Silanikove et. al, 2015). Milk containing products are found in plentiful among grocery stores and supermarkets. However, within milk, there resides a special disaccharide called lactose. Lactose is a disaccharide molecule that is formed from a galactose molecule that bound to a glucose molecule. Lactose is an integral part in the importance of animal life, especially at the early years of development. Lactose serves as a main source of calories that can be collected from the milk in almost all living mammals. (Deng et. al, 2015). It is actually the main disaccharide found within milk. However, in order to break down lactose, the special enzyme called lactase is needed in order to break the bond. Lactase is the enzyme that has the ability to break down lactose, and lactose only. Lactase enzyme, also known as lactase-phlorizin hydrolase, is found within the small intestine of mammals, allowing the beta-glyosidic bond of D-lactose to be broken. This ultimately results in the formation of D-galactose and D-glucose. These broken-down molecules can then be processed by the body and used for energy for necessary tasks (Basso et. al, 2012). Lactase works best in an environment with a limited pH and temperature range. Due to its enzymatic

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structure, abnormal deviations from its optimal environment will result in the denaturation of the enzyme. An extreme change can result in permanent damage, whereas a small change can allow for the enzyme to renature. For example, higher temperatures can disrupt hydrogen bonds, which leads to enzyme denaturation, which cascades itself towards little to no affinity for a substrate and no catalytic activity. Likewise, lower temperatures can decrease the enzyme activity. The optimal environment for lactase is found within the small intestine of mammals. However, what is interesting, is that as a mammal develops into its mature physical form, the ability to process lactose decreases. At some point, the mammal is not able to process lactose whatsoever, forming what is known as a lactose intolerance. This can be characterized by diarrhea, gastrointestinal pain, vomiting, and gurgling noises due to the movement of fluids within the stomach (Deng et al., 2015). Interesting enough, some humans have developed a tolerance to lactose through mutations (Silanikove et al., 2015), in which their bodies continue to process lactose since their concentration of lactase is high enough. Since a significant population of humans cannot process lactose, many companies have been tasked to create lactose-free “milks.” These “milks” are sourced from other produce such as coconuts, soybeans, and almonds. Therefore, it is important to figure out if different milk types vary in lactose content so that lactose intolerant people can also enjoy the beverage without gastrointestinal flare-ups. The end goal of this experiment is to determine the glucose concentrations of these milks, both dairy and non-dairy, after adding the enzyme lactase to the milks. In our lab group, we hypothesized that lactose content would vary depending on the milk type being tested. Therefore, we can predict that non-dairy milks will have minimal if not any lactose in it for lactose intolerant people to enjoy. Materials and Methods:

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In our investigation, two milk samples were tested, one being a dairy sample, and one being a non-dairy or lactose-free sample. The group prepared a set of three replicates (of test tubes) for each condition. In total, eighteen tubes were created to accommodate for all samples and condition variations. Reagents were added to each tube carefully as described: Tube 1 receives 1mL of milk (Trader Joe’s brand) and 1mL of lactase solution from Lactaid tablets (enzyme solution), with no lactose solution (VWR branded), sucrose solution (Carolina Biologicals), or water. Tube 2 receives 1mL of milk and 1mL of water, but no enzyme solution, lactose solution, or sucrose solution. Tube 3 receives 1mL of enzyme solution and 1mL of lactose solution, but no milk, water, or sucrose solution. Finally, Tube 4 receives 1mL of enzyme solution and 1mL of sucrose solution, but no milk, water, or lactose solution. The tubes were set up one at a time, without adding the enzyme. Only after were all the tubes set up, was the enzyme added and a timer of ten minutes started. After ten minutes of waiting, the concentration of glucose in the milk type was tested for using a glucose test strip from Carolina Biologicals. Once the glucose strip was inserted into the sample, the strip was left to rest for about two to three seconds, and then pulled out and gently shook to remove excess fluid. Following the dip, the strip was laid out on a paper towel to dry for about three minutes. It is important to note here that labeling is very important as to avoid any confusion about which strips are paired with which milk type. After three minutes, the glucose strip was read according to its color change and the matching glucose concentration was recorded. This process was repeated for each of the trials. In this experiment, there were controls variables used to make sure all of the data was collected on an equal playing field and no data could be dismissed due to confounding variables. Firstly, the temperature was kept the same. This was kept the same in order to prevent any

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denaturation of enzymes or change the catalytic activity. Secondly, the volume of any milk type was kept the same to prevent any trial from having more or less activity than the rest of the trials. Obviously, if there is more enzyme concentration, then there would be a higher concentration of glucose. Likewise, if there was much more lactose than lactase, then glucose concentrations would reflect this disproportionality, so the volume of each milk type was kept the same to prevent either of these instances from occurring. The amount of time the enzyme was allowed to act was also kept constant. By doing this, then no enzyme could complete more lactose breakdown than another trial. Sucrose acted as the negative control of the experiment as lactase enzyme would not be able to bind to the substrate. Lactose with lactase acted as the positive control of this experiment as lactase can bind to the enzyme to split lactose into galactose and glucose. Water was used in the experiment to determine the base level of pre-existing glucose within a certain milk type. Data was collected by viewing the color of the glucose strip from Carolina Biologicals and matching the subsequent color to that on the labeling. The corresponding glucose concentrations were then recorded into a table. After collecting the three trials of data, the trials were averaged, and the average calculated became the glucose concentration of the milk product being tested.

Results: The data depicted in Table 1 shows all the measured values of glucose concentrations in all types of dairy, non-dairy, and plant-based milk. Using the data in Table 1, Table 2 can be constructed for the calculated average of the three trials, as well as the standard deviation between the three trial measurements. We can note that there is no linear trend between the

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different types of milks, and there is no evident pattern between glucose concentrations. Even between different tests of the same combinations of solutions (i.e. lactose + lactase), the values of glucose concentration are not even the same. Almost all the data seemed to be uncorrelated between each other. Figure 1 provides a graphical depiction of the data collected from Table 2.

Table 1: Measured Values of Glucose Concentrations (mg/dl) Over Three Repeated Trials Within Different Types of Dairy, Non-Dairy, and Plant Based Milks Solution Type Whole Milk + Lactase Whole Milk + Water Soymilk + Lactase Soymilk + Water Lactose + Lactase Sucrose + Lactase 2% Milk + Lactase 2% Milk + Water Almond Milk + Lactase Almond Milk + Water Lactose + Lactase Sucrose + Lactase 1% Milk + Lactase 1% Milk + Water Coconut Milk + Lactase Coconut Milk + Water Lactose + Lactase Sucrose + Lactase No-Fat Milk + Lactase No-Fat Milk + Water No-Lactose + Lactase No-Lactose + Water Lactose + Lactase Sucrose + Lactase

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Trial 1 (mg/dl) Trial 2 (mg/dl) Trial 3 (mg/dl) 300 300 300 0 0 0 0 0 0 0 0 0 3000 3000 3000 0 0 0 300 300 300 0 0 0 0 0 0 0 0 0 100 100 100 0 0 0 1000 1000 1000 0 0 0 0 0 0 0 0 0 1000 1000 1000 0 0 0 1000 1000 1000 0 0 0 3000 1000 3000 1000 1000 1000 3000 3000 3000 0 0 0

Table 2: Measurements of the Average Glucose Concentrations (mg/dl) and the Respective Standard Deviation Values of the Repeated Trials Within Different Types of Dairy, Non-Dairy, and Plant Based Milks

Solution Type Whole Milk + Lactase Whole Milk + Water Soymilk + Lactase Soymilk + Water Lactose + Lactase Sucrose + Lactase 2% Milk + Lactase 2% Milk + Water Almond Milk + Lactase Almond Milk + Water Lactose + Lactase Sucrose + Lactase 1% Milk + Lactase 1% Milk + Water Coconut Milk + Lactase Coconut Milk + Water Lactose + Lactase Sucrose + Lactase No-Fat Milk + Lactase No-Fat Milk + Water No-Lactose + Lactase No-Lactose + Water Lactose + Lactase Sucrose + Lactase

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Average Glucose Concentration (mg/dl) Standard Deviation (mg/dl) 300 0 0 0 0 0 0 0 3000 0 0 0 300 0 0 0 0 0 100 0 1000 0

0 0 0 0 0 0

0 0 1000 0 1000 0 2333.33 1000 3000 0

0 0 0 0 0 0 1154.70 0 0 0

Average glucose concentration (mg/dl) of 18 different milk types and solutions after ten minutes of lactase enzyme exposure 3500 3000 2500

GlucoseConcentration (mg/dl)

2000 1500 1000 500 0

Name of Tested Solution

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Figure 1: Average glucose concentration (mg/dl) of eighteen different milk types and solutions after ten minutes of lactase enzyme and water exposure. The error bars represent the standard deviation between the trials used to calculate the average concentration.

Discussion and Conclusion: Given the data we collected, we should be able to accept our hypothesis. However, this hypothesis would be accepted with much skepticism. When looking at Table 2, in trying to find a correlation between the data and potentially milk fat concentration, nothing existed. In fact, no linear or consistent correlation existed at all between lactose content of milks. For there to be a trend, glucose concentrations between milk types would have to be equivalent or changing at a consistent scale, due to the fact that glucose is a product of lactose decomposition. Whole milk with lactase and 2% milk with lactase had the same glucose concentrations, however 1% milk and non-fat milk had far greater glucose concentrations than did the latter pair (Table 2, Fig. 1). We can also see that in every non-dairy milk tested, no lactose was present. (Table 1, Table 2, Fig. 1). An offsetting point top mention is that a supposed equal volume of lactose and lactase when mixed resulted in different glucose concentrations (Table 1, Table 2, Fig.1). With conflicting data, and potentially incorrect data, we cannot definitely accept the hypothesis. We can only accept it with a grain of salt. It is of utmost importance, however, that correct labeling be done to these milk products in order to offer them to corresponding audiences. It has been researched of the importance of milks nutritional components in the daily diets of people. Milk, and dairy products like cheeses,

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yogurts, and butters, are an important source of calcium for many individuals. On top of calcium, milk also serves as a great supplier of healthy fats (approximately 35g/L) for brain development and protein (32g/L) for muscle repair and growth (Erich et al., 2012). For lactose intolerant people, non-dairy or lactose free milks are the only option. Yet, it would be remiss to say that the non-dairy or lactose free milks lack sufficient nutrients, and also hold some benefits. Lactosefree milks are filled with sufficient glucose and galactose, which ultimately enhances the sweetness of the drink. Because of this, no additional sugar needs to be added, which reduces the number of excess calories being put into the beverage. (Gerbault et al., 2011). Also, to mention, in recent studies, negligent differences were discovered in gastric emptying in rats in comparison to lactose and a glucose-galactose consumption (Dekker et al., 2019). Furthermore, in a study done on baby cattle, blood sugar levels were very similar in those who drank milk and those who drank lactose free milk (Dekker et al., 2019). Limitations in the lab were due to human error. In delivering a 1mL dosage of solution or milk type to the tubes, not exactly 1mL was transferred. This little error could have resulted in either an increase or a decrease in glucose concentration depending on if the volume was less than or greater than 1mL. Secondly, in adding the enzyme to the solution, some solutions got a few seconds longer to have enzymatic activity. Obviously, this would create massive error in the data, but it is still important to note. To fix these two errors in the data, using more precise instruments would allow the user to get as close as possible to 1mL; having multiple people insert glucose strips at the same time would eliminate the time difference between test tubes in the trial. A future experiment that relates to the topic of lactase enzyme would be determining the implications that lactose milk has in comparison to lactose free milk on people with type II diabetes. This could be accomplished by testing blood sugar levels after a certain amount of time

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after drinking the milk. Our research did not necessarily create any new avenues to draw new conclusions. However, the inability to draw a solid conclusion allows for more research to be put into the lactose content of different types of milk.

References: Basso, M., Luciano, R., Ferretti, F., Muraca, M., Panetta, F., Bracci, F. (2012). Association between celiac disease and primary lactase deficiency. European Journal of Clinical Nutrition, 66(12): 1364–1365. Dekker, P., Koenders, D., & Bruins, M. J. (2019). Lactose-Free Dairy Products: Market Developments, Production, Nutrition and Health Benefits. Nutrients, 11(3), 551. Deng, Y., Misselwitz, B., Dai, N., Fox, M. (2015). Lactose Intolerance in Adults: Biological Mechanism and Dietary Management. Nutrients, 7(9): 8020–8035. Erich, S., Anzmann, T., Fischer, L. (2012). Quantification of lactose using ion-pair RP-HPLC during enzymatic lactose hydrolysis of skim milk. Food Chemistry, 135(4): 2393–2396. Gerbault, P., Liebert, A., Itan, Y., Powell, A., Currat, M., Burger, J., Swallow, D. M., & Thomas, M. G. (2011). Evolution of lactase persistence: an example of human niche construction. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 366(1566): 863–877. Silanikove N, Leitner G, Merin U. (2015). The Interrelationships between Lactose Intolerance and the Modern Dairy Industry: Global Perspectives in Evolutional and Historical Backgrounds. Nutrients, 7(9): 7312–7331.

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