Science Fair 2016 / Epidemiologic experiments (1) Abstract, Materials, Hypothesis

Science Fair 2016

Epidemiologic experiments

of probiotics’ mechanism

(direct antagonism) and

stability.

 

 

 

Kyu Been Lee

Irvine High School (10^th grade 2016)

Science Teacher: Mrs. Joan Elliott

Project Category: PHYSIOLOGY/MEDICAL BIOLOGY

               Title: Pharmacological effects: Epidemiologic experiment of

              probiotics’ mechanism (direct  antagonism) and stability.

 

 

 

Abstract

Objectives/Goals: There were a few purposes for this project one of which was to prove that one of the probiotics’ mechanisms was direct antagonism. Along with that, the project’s purpose was also to determine the probiotics’ stability in an intestine environment and also whether it is safe in the stomach or not. The final purpose was to study epidemiology to show the efficacy of dietary supplement probiotics.

 

Methods/Materials: To observe direct antagonism, I kept the probiotics’ tester tube with the body temperature(PH7.0) and combined it with cultured pathogens on microscope slides. Through the microscope, magnification by 400x and 1000x (with immersion oil for 1000x), showed real antagonistic action of the probiotics. In order to see the effect of the enteric coated capsule, many different stages were prepared: The first one was by putting a neutral pH(PH7.0) at room temperature and adding a neutral pH at body temperature to make an intestine environment for  the 5 and 90 billion probiotics capsules were observed to see their dissolving situation. Second was making a stomach environment with room temperature and body temperature along with vinegar (pH2.2), lemon extract (2.1), and apple vinegar (PH2.4). Finally, the stability experiments were tested through tester tubes: water at pH7.0 and dissolved probiotics in a body temperature environment. To observe the survival time of the probiotics, continued to observe for at least 24 hours.

 

Results: The experiment’s results showed that probiotics’ antagonism was revealed through observations from the microscope. Enteric coated capsule was not dissolved during those 3 hours where we put them in with pH2.1 and 2.2 liquids. However, when it was with pH 2.4 body temperature environment, the 5 billion probiotics  capsule dissolved in only an hour and 4 minutes.  In the pH7 body temperature environment experiment, capsule completely dissolved in 21 minutes(5 billions).  Also in the pH7 room temperature, it took around 45 minutes(5 billions) and 65 minutes(90 billions) to dissolve the capsule completely. Finally, the stability experiment continued until 30 hours passed and even then the probiotics were still alive and active.

 

Conclusion/Discussion: In the end, my hypothesis was proven correct. Since direct antagonism was shown throughout the experiments between probiotics and pathogens, it showed an efficacy of the probiotics’ important mechanism. The fact that the 90 billion probiotics capsule did not dissolved in a stomach environment during 3hours, but the 5 billion probiotics capsule dissolved completely in a stomach environment even before digestion time was up shows that perhaps Nature’s Way, the company, made an experimental problem with those capsules since through earlier experiments, it also showed that if probiotics were to be in acidic environments, they were to die in minutes. Finally, the stability experiment demonstrated the probiotics’ vitality and efficacy against bowel diseases in the end.

 

Summary Statement: My experiments were done to determine just how probiotics’ dietary supplement worked effectively in the small and large intestine.

 

Help was received: My father had purchased the microscope along with many instruments used throughout the experiments. My mother also supported me throughout the experimental processes.

 

 

Science Fair 2016 / Epidemiologic experiments (2) Procedures

Procedures

 

 

Experiment 1

1-1 Capsule melting time/ Room temp(81.6F/ph7.0 (water))

1)     Condition : room temperature-81.6F, tap water-PH 7.0, 6.73g in the plastic container

2)     Put 3 plastic containers with the same mass of water. Next, add one capsule to each container: 1 expired 5 billion probiotics, 1 new 5 billion probiotics , and 1 90 billion probiotics capsule.

3)     Observe each for 5 minutes to observe any transitions of capsules. (enteric coated capsule has two layers: white part and a  clear inner part)

4)     Write down the changing room temperature for each stage

5)     Cracking time is more important than when it completely dissolves because when the capsule cracks, probiotics spurt out into the liquid.

6)     Continue until the capsules dissolve completely in the water

7)     Take photos each stage for recording

 

 1-2 Capsule Melting/ Body temperature(Average 98.4F/ph7.0)

1)     Condition: tap water-PH7.0, 6.73g in the plastic container, 5billion probiotics capsule, and boiled water to refill the water in order to maintain the body temperature for each stage.

2)     Keep in mind! Measuring the temperature is important. Start the temperature at 98.6 °F. This experiment is an independent variable of the stomach’s environment (the average internal temperature is 37.0 °C (98.6 °F)). An error value of 0.1~0.5 will be allowed because it can be assumed the body temperature is different for each person.

3)     Observe for 3minutes for the transition of the capsule (in this case, condition 3minutes gap because it expects fast dissolving time).

4)     Before observing transition of the capsule for each stage, measure the temperature of water and add water with the eyedropper and keep refilling warm water until body temperature.

5)     Mark the breaking capsule time. Continue to observe transition until dissolved completely.

 


 

1-3  5 billion Capsule Melting Time in ph2.1/ph2.2(room temperature81.5F)

1)     Condition: room temperature-81.6F, lemon extract(PH2.1) 6.73g, vinegar(PH2.2) 6.73g , and 2, 5billion enteric coated capsule

2)     Measure the PH with a PH tester for the lemon extract and vinegar.

3)     Put one container with vinegar and the other with lemon extract, put them at the same time.

4)     Observe the stage every 30 minutes during 3 hours.

5)     Check what phase the capsule is in and write down.
 

1-4 Capsule Melting Time Experiment (Body Temperature Average 98.6F Apple Vinegar PH 2.4) 5billion Probiotics

1)     Condition: body temperature 98.6F(±0.4F), apple vinegar (PH2.4) 6.73g, heated vinegar 100g, 1- 5billion enteric coated capsule, eye dropper, digital thermometer, 1 small plastic bowl, clock

2)     Note: It is very important to keep the body temperature the same. If you are able to keep the same temperature (98.6F), it will have a perfect result. If you don’t have special machine like me, use an eye dropper and add more heated vinegar whenever the temperature falls below 98.4F (nearly every 10-15 seconds).

3)     Prepare 6.73g apple vinegar in the plastic container.

4)     Add 1 5billion probiotics enteric coated capsule.

5)     Record every temperature, and observe phase of the capsule

6)     Cover the container with warm cloth.

7)     Check and write down during those 3hours.

 

 

 

 

1-5 Capsule Melting Time Experiment (body temperature 98.6F, Apple Vinegar PH2.4) 90 billion probiotics

1)     Keep temperature at 98.6F with heated vinegar, apple vinegar PH2.4  500g, one plastic container, thermometer, clock,

90   billion probiotics capsule, eye dropper, clock

2)  Prepare 6.73g apple vinegar in the container. Add 1 90 billion probiotics capsule.

3) Heat apple vinegar on the stove top. Pour the container, and measure the temperature until it is 98.6F.

4) Every 20-30 seconds, observe the temperature, and add some heated vinegar with eye dropper.

5) Watch the transition of the capsule.

6) Record every stage that change.

 

 

Experiment2

Probiotics’ stability in a body temperature environment

4 tester tubes, 4-6.4g water with expired 5 billion probiotics capsule, new 5 billion probiotics capsule, new 90 billion probiotics capsule, Kimchi juice, microscope slide, cover glass, eye dropper, 1 bottle Immersion oil type A, 40swabs, alcohol for cleansing the slides

1)     Fill 6.4g of water in 3 tester tubes, fill 6.4g Kimchi juice in the tester tube.

2)     Break 3 capsules gather only powder of probiotics, Add different subjects in the tester tubes.

3)      To keep the body temperature(98.6F), grab 4tester tubes with left hands. Our body temperature is 98.6F, but the body skin temperature is about 93.2F, but this temperature is also the probiotics’ growth temperature. When you have to use left hand you can keep the tubes between belt of your pants and skin of waist.

4)     Observe the status of probiotics sample from tube with swab. Rub with a swab some probiotics with water on the surface of  the microscope slide, and cover with cover glass.(write down their name tag on the slides)

5)     After watching the microscope, clean up the slides and microscope lenses of every stage.

6)     Use your microscope. From 40x lens to 1000X observe the activities of every subject. (1000X lens need immersion oil)

7)     Note: For metric transfer, according to the degree you can make score 1-10.

8)     During your sleep time, you can cover the tubes by throwing several layers of clothing.

9)     Observe Until stage12.(30hours)

Experiment 3

Survival of Probiotics in the pH2(vinegar)

1)     In the middle of stage 4or 5, collect probiotics(90billion) with moisture from the tube.

2)     Drop the probiotics on the microscope slide.

3)     Add one drop of vinegar(use eye dropper)

4)     Observe the slide from 40X to 1000X

5)     Record the probiotics’ activity and disappearing time.

Note: This experiment can analogize the stomach environment.

 

Experiment 4

Observation of Direct Antagonism  

1)     During  experiment 2 stage 4, fill one drop of 90 billion probiotics onto another microscope slide.

2)     Add a sample of moldy bread, orange, and/or onions.

3)     Observe the reaction and activities of probiotics through the microscope.

 

 

Observation of Direct Antagonism (Experiment 4)

(probiotics, moldy bread, moldy orange, onion black fungi : Aspergillus niger)


 

Science Fair 2016 / Epidemiologic experiments (3) conclusions

 Conclusions  

 

 

    While in the beginning of the experiment, I looked under the microscope to see probiotics fighting pathogens, it was merely because of my fascination and wonder at seeing microorganisms not only move but also attack other threatening bacteria as well. However, the reason I had done this experiment was for the two purposes: to first have some kind of epidemiologic study about the dietary supplements my father eats since he was a consumer and also because my father suffered from IBS (Irritable Bowel Syndrome) and I wanted to find out what the best way was to cure him from it. Through research, I realized right off that what the researchers had stated about probiotics being able to battle pathogens was correct as I had observed the probiotics fight against molds and other kinds of pathogens. What really amazed me about the probiotics was that even when they were going to die, they stuck onto the pathogens and refused to let go, showing their dedication towards fulfilling their job even to the very end. Following direct antagonism, in order to see whether or not probiotics would really be able to survive in a small intestine’s environment (pH level 7 and body temperature of 98.6 degrees F) and be able to enter the colon active and well, I carried around 4 test tubes containing kimchi, new 5 and 90 billion probiotic capsules, and one expired capsule with 5 billion.

     The results for this was very successful since all except the expired probiotics managed to maintain stability even after 30 hours in the intestine environment. Finally, while earlier on one of my first experiments I tested to see if the enteric coating was efficient at resisting the environment of the stomach at room temperature (which succeeded as the capsules remained the same even after hours), also I added vinegar with the temperature of my body, then added a 5 and 90 billion probiotics capsule and waited the results. The results showed that while the 90 billion remained sturdy even after three hours, the 5 billion capsule dissolved in about 1 hour and 4 minutes showing that the probiotics of that capsule would die even before reaching the small intestine (since my third experiment showed that when probiotics were put in acid or a pH level of 2.2, they would die in 15 minutes).

     While the entire experiment was successful in general, the dissolving of the 5 billion capsule at body temperature meant that there was an error made by the company Nature’s Way which made many people in a way, waste their money since the results showed even if we consumed those capsules, the probiotics there wouldn’t even be able to aid one’s body, showing that there wouldn’t be much point in buying those capsules. In the end, the experiments showed that the enteric coatings of the capsules were really stimulated by the temperature and PH level. Most importantly, it showed that the reason why my father’s IBS gradually subsided was because of these fierce micro organisms that fought pathogens even to the end, showing that my thesis was thus proven.