Late Elementary/Middle School #3

Got Lactose?

Grade 6

 
 

Abstract:

 

It is estimated that up to half of the population in the U.S. has some degree of lactose intolerance. This can lead to stomach pain and bloating. Some people with this condition avoid milk because of the pain and may not get enough calcium in their diet. Lactose intolerance is treated by taking a lactase enzyme supplement any time dairy is eaten. My study is to determine the best way of using the Lactose enzyme to convert the lactose in milk to a digestable sugar. Lactose intolerant people could use this information to best utilize the enzymes to treat dairy under individual circumstances. My study shows that the best method for treating milk is to use the liquid lactase and heat the milk to 115 degrees F if you are going to drink it immediately. If you can wait 24 hrs. the liquid lactase is just as effective in hot or cold milk (40 degrees F). The tablet form of lactase enzyme converted roughly half the amount of lactose to glucose per 3000 international lactase units. Overall, the liquid lactase was more effective at converting the lactose to glucose and I would recommend this form of the enzyme to highly intolerant people even though the cost is higher per treatment. I would recommend the tablet form for less lactose intolerant people because it is more cost effective.

 

Purpose:

 

Along with half the U.S. population, I am lactose intolerant. I wanted to know what the most effective way of treating dairy was because I noticed that my pain varied depending on how much dairy I ate and how much lactase enzyme I took. I designed a study to see if there was any difference in enzyme effectiveness between enzyme treated milk at 40 degrees F and 115 degrees F and if there was a difference between liquid and tablet forms of lactase. The reason that effectiveness matters is that symptoms are dependent on the amount of unconverted lactose is still left in the milk.

 

My hypothesis was that the tablet form would work better and that heating the milk would help. I thought the heat would help after I talked with my chemistry mentors and found that enzymes worked best near body temperatures and I have found in my chemistry experiments that heat often speeds up reactions. I hoped to learn the most effective way of treating milk to minimize the symptoms of lactose intolerance.

 

Materials:

 

Cups, micropipettes, digital scale, pill cutter, lab notebook, Good Neighbor Pharmacy Dairy Relief tablet lactase (3,000 lactase units in each ½ tablet), Pharmax liquid lactase (3,000 lactase units per 12 drops), weighing pans, microwave, spoons, toothpicks, test tube racks, test tubes, ParaFilm (for sealing the test tubes), 28 cell reaction plate, distilled water in a wash bottle, pen, masking tape, digital thermometer, milk for testing, Precision Xtra glucose meter and test strips, & waste container.

 

 

Procedure:

 

I bought a separate carton of milk for each trial. I did this because my research showed that there can be variations in lactose level between batches of cows milk. I  tested organic ultra pasturized fat free cows milk and non-organic ultra pasturized fat free cows milk. I choose fat free milk because my research showed that fat free had the highest lactose content. After the milk was bought, it was stored at 40 degrees F in my refrigerator. During testing, I took the milk out of the fridge and poured it into measuring cups that I  pre-marked with 94.6 milliliters (a fifth of a pint.). I wanted to use the smallest amount of materials possible. My scale could measure down to a ½ tablet (0.14 gms) of lactase. This represents 3,000 lactase units= LAU). I  poured 6 measuring cups of milk in this way. Before testing, I used a pill cutter to divide the solid lactase tablets in ½ and verified that they each weighed 0.14 gms (1/2 of an average 0.28 gm whole tablet). 3 of the cups remained at 40 degrees F and the other 3 were heated in the microwave to a goal temperature of 115 degrees F (+/- 2 degrees) as measured by a digital thermometer. I choose 115 degrees because my research and discussions with my mentors showed that, above 118 degrees, the lactase enzyme becomes deformed and is not as effective. I added 12 drops of liquid lactase (equal to 3000 LAUs) to one of the cold cups and one of the hot cups. I added ½ tablet (equal to 3000 LAUs) of powdered (I crushed this in the weighing pan) lactase to one of the hot and one of the cold cups. The other hot and cold cups had nothing added to them because they were my controls. All cups then were stirred for 10 minutes. I labeled 6 test tubes (Cold Control, Cold liquid lactase, Cold tablet lactase, Hot control, hot liquid lactase and hot tablet lactase) and poured them 3/4s full of the treated or control milk, covered the test tubes with Parafilm and placed them into a test tube rack labeled with the date the test was started. Into a 28 cell reaction plate I added 20 drops of room temperature (aver. 70 degrees F) distilled water from a micropipette into each of 6 cells of the plate. The 20 drop dilution rate was arrived at after trying many different dilution rates and seemed to be the best one that was not too high or low for my glucose meter to read. Before the sample drop was taken out, each test tube was shaken back and forth a few times to mix the milk in the tube uniformly. Using a separate micropipette for each milk sample and each test, I added one drop off a milk sample to a cell of the plate in the order specified above. I stirred each cell with a separate toothpick until it was uniformly cloudy. I tested each cell with the Precision Xtra meter according to the directions that came with the meter. I recorded the glucose level in a chart I made for each trial. Each milk sample was tested like this after the 10 min. stirring at the start of the experiment, 24 hrs. later, 48 hrs. later and 72 hrs. later. I arrived at these times by looking at the package directions for the lactase products and web sites and in consultation with my mentors and a physician (he said it might not be safe to drink milk treated like this after 3 days and did not think anyone would wait that long to drink their milk). During the 3 days of testing, the milk samples were kept at 40 degrees F in my refrigerator. I disposed of the milk samples after the last testing (72 hrs.) down the sink and washed all my equipment with soap and tap water then a final distilled water rinse. I analyzed my data by graphing the results using a spreadsheet program. There was no alternative to using cows milk for my experiment as the question I was trying to answer is how to make lactase work better on cows milk. Lactase is not a harmful substance and there was no alternative to using it.

 

Data:
 
*********several charts have been omitted here**********
 
    Results and discussion:

In my data, the controls were the only ones that showed no detectible glucose throughout the three days of testing and temperature variations. This was expected because I did not put any lactase enzyme in the controls, therefore the lactose would not have broken down into a level of glucose my meter could read.

 

Adding the tablet lactase to heated milk showed an overall increase in the amount of glucose at the end of three days of testing. I think the amount of glucose in the milk increased because enzymes, like lactase, work better at body temperatures. Therefore, the lactase would work better at higher temperature. Heat may also have helped because the lactase is a catalyst. If the molecules of lactose are moving around faster because of heat, they will have more of a chance to encounter a lactase enzyme molecule, bind to the active sites on it and get broken into the digestible sugars of glucose and galactose.

 

The behavior of the liquid lactase is a bit harder to explain than the tablet. Heat did not make much of a difference (approximately 5mg/dl glucose difference between hot and cold on average). The liquid lactase, performed almost identically at 24 hrs. no matter what the temperature. A possible reason for the higher glucose level in the liquid enzyme trials is that the liquid lactase may have dispersed itself better despite the fact that I crushed the tablet as much as I could and stirred it for 10 minutes to help it dissolve. Better dispersal means more enzyme can contact more lactose, therefore breaking apart more of the sugar.

 

The highest readings in the liquid enzyme trials came at twenty four hours. After this point, the liquid enzyme tests showed a decrease in the amount of glucose. I do not really know why this happened but I have some theories:

  1. There was some kind microorganism contamination in the samples. The organisms could have been eating the glucose for energy thus lowering the amount of glucose in the samples. The counter argument to this theory is that if there was contamination why would it have only affected the liquid lactase and not the tablet? Perhaps the liquid lactase itself was contaminated before it was added to the samples.
  2. Possibly the glucose and galactose were reforming into lactose but this is unlikely because this enzyme only breaks apart lactose, it does not catalyse its reformation.
  3. Perhaps the glucose became bound to the inside of the test tube and no matter how hard I shook the test tube before taking the reading, it did not go back into solution.
  4. Maybe it was just a human or meter error. This is not likely because it happened in numerous trials.  I don’t think the meter or I could have made such a consistent mistake in only the liquid enzyme trials.

 

If I repeated this project, I would like to have equipment that could test for both glucose and galactose. I would have liked to test more kinds of milk such as whole milk, 2% and raw milk. Unfortunately, the raw milk was not allowed by the science fair rules. It would have been fun to see if pasteurization had any effect on lactose concentration. It might have been interesting to test the milk for a longer period of time but since milk spoils, this was probably not a good idea.

 

Conclusion:

My data shows that liquid lactase is more effective than tablet form. Heating the milk to 115 F allowed more lactose to be converted to glucose. Waiting 24 hours after treating your milk gives you the highest conversion rate for liquid enzyme. If you are using tablets, heating will help the conversion rate slightly as will waiting 72 hrs. to drink the milk. Not treating your milk, not matter how long you wait, will not increase the amount of glucose in the milk. My orginal hypothesis was partially correct. The heat did help the conversion rate but the tablet did not work as well as the liquid lactase.

 

Acknowledgements:

I would like to thank my chemistry mentors ******personal info removed*****for answering all my questions and helping me work through my dilution rate dilemma. Thanks to *****personal info removed***** for helping me with the Excel spreadsheet charts. Thanks to *****personal info removed***** for help in understanding enzymes and catalysts.

 

 

 

 

Bibliography:

 

Interviews and emails with *****personal info removed***** Chemistry teacher, *****personal info removed*****, and *****personal info removed***** throughout my experiment.

 

Website: http://www.lactaid.com

 

Website: http://en.wikipedia.org/wiki/Lactose_intolerance

 

Understanding Normal and Clinical Nutrition 2E by Whitney, Cataldo and Rolfes pp. 62-63 and 732-734. 1987

 

Biochemistry by Cambell p.112 copyright 1991

 

Website: http://digestive.niddk.nih.gov/ddiseases/pubs/lactoseintolerance/