Lactase Enzyme Lab

Kailen Roop
Biology A1
5/5/15

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Table of Contents Introduction……………………………………………………………………………………2­6
Methods…………………………………………………………………………………....…..7
Results………………………………………………………………………………………....8
Conclusion……………………………………………………………………………………..9
Citations…………………………………………………………………………………….10­11

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Introduction
This paper is about lactose intolerance, how it works and why it happens. This paper will discuss lactose intolerance form a molecular level to the evolutionary level. Also, this paper will outline an experiment designed to show the various levels of lactose and glucose in various samples of milk. First we must understand, what is lactose intolerance? To do that we must first understand what happens in the human body that defines lactose intolerance. The first step is learning what takes place in order to have a chemical reaction. Then, what happens during that reaction to make someone lactose intolerant or non­ lactose intolerant. Finally, what happened during evolution that makes some people lactose intolerant and some not.
Enzymes are proteins that make reactions happen faster. (National Enzyme Company
2015). Enzymes regulate chemical reactions. They do this by binding to molecules called substrates. Enzyme activators are molecules that start or speed up an enzyme’s activity.
(National Enzyme Company 2015). Like a baker getting an electric mixer instead of mixing by hand, the activators speed up the whole process.
Now, reactions can’t happen all the time or you’d have to much of whatever the reaction produced. Like if a baker made kept making bread nonstop, you’d have way too much bread.
Enzymes are controlled by inhibitors. An enzyme’s inhibitors are molecules that stop or slow down an enzyme’s activity (National Enzyme Company 2015). Inhibitors can be competitive or noncompetitive. Competitive Inhibition is the interruption of an enzyme’s activity to bind a substrate. This is achieved by an inhibitor binding to the active site. This is like if you put something else in the baker’s oven so he couldn’t bake. Noncompetitive Inhibition occurs when an inhibitor binds to the non active part of the enzyme. This causes the enzyme to change shape,

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not recognise a substrate and/or slow the enzyme down (National Enzyme Company 2015). This would be like if you moved all of the baker’s ingredients around and he couldn’t find them. It would either take him awhile to find them or he would stop baking (See ).
Fig 1
Enzymes rely heavily on their shape. An enzyme’s shape determines what substrate will fit with what enzyme. Enzymes function with substrates like a lock and key or a hand and glove.
(What are enzymes 2015). he substrate must fit perfectly with the enzyme or the reaction won’t
T
take place. What is an enzyme made of? Enzymes are proteins comprised of 100­1,000 amino acids.
Amino acids are organic compounds that bond together to make proteins. These amino acids then fold into a specific shape (How Cells Work 2015). Whatever shape the amino acids fold into determines the shape of the enzyme. Our cells and enzymes need energy to carry out these functions. But where do they get the energy? Plant cells get their energy from the food they make. Our cells cannot make their own food, so they rely on the foods we eat to provide them with energy. We eat energy rich molecules such as protein, carbohydrates, lipids and sugars everyday. Lactose is a milk sugar made of two smaller sugars. A molecule like lactose, a large sugar made up of two smaller sugars, is called a disaccharide. In your body, disaccharides cannot be absorbed into your bloodstream the way small sugars can. That’s where lactase comes in. Lactase is an enzyme that breaks down the substrate lactose into two smaller sugars called glucose and galactose. These sugars can now be absorbed into your body. This process is called lactose breakdown (See
Fig.
2)
.

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When you don’t have enough lactase, if any, to break down the lactose (like a lactose intolerant person) then a different process occurs. This process is called lactose fermentation.
The lactose in your small intestine cannot be broken down because of the lack of lactase, so it continues through the digestive system to the colon, where it is finally broken down by the bacteria that reside there. This causes gas, bloating and discomfort as the lactose travels undigested through the intestines (What is Lactose Fermentation 2015).
Fig 1
(IB biology syllabus 2015)
Not being lactose intolerant is called being lactase persistent. The lactose in your body has, for lack of a better word, persisted and continued to be produced. Lactase persistence is a genetic mutation, only about ⅓ of the world’s population is actually lactase persistent (Explore
Short Films 2015). Lactose intolerance is a dominant trait.
This means if one of your parents is lactose intolerant, you have a good chance of being lactose intolerant too.

It is important to understand the difference between lactose intolerance and a milk allergy. People with a milk or dairy allergy have much more severe symptoms and this condition can be life­threatening. This happens because your immune system thinks the lactose is a foreign invader and attacks it by releasing histones, causing your body to react violently. Some symptoms may include wheezing, vomiting or diarrhea (Food Allergy, or Something Else 2005).

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Lactose intolerance is mostly discomfort and gas and is not life­threatening (Food Allergy, or
Something Else 2005). Mainly because this is simply a case of lactose fermentation and involves your digestive system and not your immune system. Fig. 2
(Understanding Lactose Intolerance 2002).

Lactase persistence, was naturally selected for when passed down through the generations. What this means is that those who didn’t have the lactase persistence gene died.
This happened most likely because milk was the only safe thing ancient civilizations had to drink, as water was often contaminated, and alcohol was yet to be invented. If you couldn’t drink the milk, you would die. It has been discovered that people in northern Europe as well as certain part of Africa have a higher concentration of people with lactase persistence (Explore short Films
2015). The reason these places have a higher concentration of lactase persistent people is believed to be because the ancient tribes there drank milk a long time before anyone else and were able to make their bodies produce lactase again. In the lab, it was found that different types of milk have varying levels of glucose and lactose. This lab was researched and performed to validate the research done by Jeffrey D. Sack
(Explore short Films 2015). Our purpose was to understand lactose intolerance, lactase

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persistence and natural selection as well as evolution, the chemical reaction of lactose and lactase, and genetics.
The hypothesis was more of a prediction than a question as it was known what would happen because the lab was done to validate another’s work. It was predicted that lactase would break down the milk into glucose and galactose. The hypothesis was tested by testing the glucose levels of our samples, adding lactase and placing them in a water bath, then testing them again. If more glucose was there after the addition of lactase and the water bath, then the hypothesis can be accepted. The hypothesis can be accepted because the only way there could be more glucose after the addition of the lactase and the water bath would be if the lactase broke the lactose down.
Which was precisely what our hypothesis stated.

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Methods
First, test tubes are labeled
A, B, C, D, E and is Half and Half milk, is Whole milk,
. A B
C is 2% milk, is Skim milk, and is Lactaid®. 5 ml of milk samples added to respective D E tubes. One indicator strip is dipped into tube one in tube etc. Strips are removed and
A
,
B
, compared to the color chart on the indicator bottle. The approximate concentration of glucose in the milk sample is noted and recorded in Table 1. 5 drops of lactase is added to each sample of milk. Tubes are placed in a warm water bath for 10 minutes. It is important to note that the warm water bath was 37 degrees celsius, about the temperature of the stomach, where this reaction occurs. The tubes are then tested again as earlier. The test tubes are cleaned and put away
(Explore short Films 2015).
My group consisted of Aleta Prysock, Brenner Beck, and myself. For this lab, we used
Lactaid® as our control. Which means it was the trial when we knew what would happen. We also controlled the other milk samples, by using the same amount and maintaining the same temperature. You will notice glucose indicator strips were used not lactose indicator strips.
Glucose indicator strips were used because, after lactose is broken down, glucose and galactose are formed. We tested the amount of glucose in the milk to test how much glucose was in each type of milk. We also tested all of the samples before and after the lactase was added. This was to understand that most samples are not broken down before being ingested.

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Results
It was found that prior to adding the 5 drops of lactase, there was little to no glucose found in the milk samples. After adding the 5 drops of lactase, there was more glucose found than the initial reading in all samples. In sample Lactaid®, there was glucose prior to adding
E
, the lactase.

Table 1
Sample:
A: ½ and ½ B: Whole C: 2%

D: Skim E: Lactaid
®

before after

before

after

before after

before

after

before

after

Color of

light

dark

sea

dark

sea

dark

sea

dark

light

dark

glucose

green

green foam

green

foam

green

foam

green brown

brown

200

1000

2000

mg/dl

mg/dl

mg/dl

strip

green

Amt. of

100

200

0

glucose

mg/dl

mg/dl mg/dl

green

green

200

0

200

0

mg/dl

mg/dl

mg/dl mg/dl

The only data trend was that all our samples had more glucose when the lactase was added. No statistical run was done on this data because we were only validating another persons research. Only one trial was done because this lab was done to validate another’s work.

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Conclusion
The purpose of this lab was to better understand lactose intolerance, lactase persistence, genetics, natural selection and evolution. Our hypothesis was our prediction that our samples would have more glucose after the addition of lactase and a water bath. Our outcome was that our results proved our hypothesis.
The only trend was that we had more glucose after the lactase was added to the samples.
The experiment was controlled by using the same amount of each sample, and keeping temperature constant.
The scientific reasoning for the results in this lab are simple. Milk will not have much glucose until it is broken down into glucose and galactose. Lactase breaks it down. Therefore, it follows that our samples of milk won’t have a glucose reading prior to adding lactase. If this experiment was repeated, another trial would be added, and more lactase would be added to find the amount of lactase needed to break down the entire sample of milk. Other samples may also be added, such as evaporated milk or heavy cream. Lipids, or fats, from the samples may be covering our strips causing error in reading, so for another trial I would try taking out the lipids first, then running the trials. This lab is useful for discovering lactose intolerance by substituting blood for milk. This can also be used for a company to test if their supposedly lactose free milk is actually lactose free. Other questions include: why does ½ and ½ milk have more glucose than the other samples? Would more lactase result in more glucose?

Citations

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● What are enzymes? (2015, January 1). Retrieved April 22, 2015, from http://www.novozymes.com/en/about­us/our­business/what­are­enzymes/Pages/default.as px#set ● National Enzyme Company. (2015, January 1). Retrieved April 22, 2015, from http://nationalenzyme.com/education/what­are­enzymes/ ● Enzymatic Reactions: Inhibition and Regulation. (2003, January 1). Retrieved April 22,
2015, from http://study.com/academy/lesson/enzymatic­reactions­inhibition­and­regulation.html ● What Is Lactose Fermentation? (2015, February 27). Retrieved April 23, 2015, from http://www.livestrong.com/article/443997­what­is­lactose­fermenting/ ● Food Allergy, or Something Else? (2005, January 1). Retrieved April 23, 2015, from http://www.webmd.com/allerghttp://www.webmd.com/allergies/foods­allergy­intoleranc eies/foods­allergy­intolerance ● Explore Short Films | HHMI's BioInteractive. (2015, January 1). Retrieved April 23,
2015, from http://www.hhmi.org/biointeractive/explore­short­films Roop 11

● Brian, M. (2015, January 1). How Cells Work. Retrieved May 3, 2015, from http://science.howstuffworks.com/life/cellular­microscopic/cell2.htm ● I Love Ice Cream, But It Doesn't Love Me: Understanding Lactose Intolerance. (2002,
January 1). Retrieved April 28, 2015, from http://www.sciencebuddies.org/science­fair­projects/project_ideas/BioChem_p031.shtml ?from=Blog ● IB Biology Syllabus. (n.d.). Retrieved May 3, 2015, from http://ibbiology.wikifoundry.com/page/Explain the difference between competitive and non­competitive inhibition ●