Question:

Which natural antibacterial solution made by either under ripe pomegranate peels, under ripe mango peels, or under ripe banana peels will inhibit the most bacteria growth?

Hypothesis:

If I make three antibacterial solutions, one made with pomegranate peels, one made with mango peels, and one made with banana peels, then the one made with pomegranate peels will inhibit the growth of bacteria the best because of the higher tannin content in pomegranate peels.

(Tannins can pass through the bacteria cell wall and interfere with the metabolism of the bacteria cell, which causes the destruction of the bacteria cell. Tannins also cause bacteria to not be able to attach to a surface, and a lack of adhesion to a surface causes bacteria cells to die.)

What are tannins?

Tannins, sometimes called tannic acid, (which is a specific type of tannin) are a type of polyphenol (a group of compounds that are naturally found in plants) that are found in many plants and fruits. When they are present in fruits, they usually cause an astringent or bitter taste. Under ripe fruits are high in tannin content, which makes them unappealing to eat. As the fruit ripens, the tannin content decreases, but doesn’t disappear completely, and the sugar content increases. Therefore, more under ripe fruits would be better to use for my experiment so I can extract more tannins from the fruit peels. 

How do you effectively extract tannins from fruits?

Most of the experiments and methods of extracting tannins I found involve having the plant that you want to extract tannins from in a hot solvent for extended periods of time, and the way that the tannins get extracted is similar to how tea is made using tea leaves. It's important that the solvent is hot because a study involving extracting tannins from spruce tree bark (https://www.sciencedirect.com/science/article/pii/S138589472203296X) reported that lower levels of tannins were extracted when the solvent (water at 21°C) was cold, and higher levels of tannins were extracted when the solvent (water at 85°C) was hot. However, temperatures higher than 70-85°C can cause a minimal increase in the amount of tannins that are extracted and a greater amount of bound sugars in the extract. Higher temperatures like that can also cause tannin degradation, so it’s important that I choose a temperature that isn’t too hot or too cold for the solvent (water) in my experiment.

https://web.archive.org/web/20190812161851/https://www.googlesciencefair.com/projects/2018/056075c3255ccc9d9ef035618eadbef4690dd38aac815e4a10419b1c4bfcfbc0 This experiment also found that higher extraction temperatures of 100°C, 150°C, and 200°C did not inhibit the growth of bacteria. The same experiment also found that the most bacteria growth was inhibited when tannins were extracted from a persimmon calyx that was in water heated at 50°C for 20 minutes. Because of this, I’ll probably use a similar amount of time that the water and fruit peels are heated at for my experiment.

Also, adding sodium sulfite to the solvent can increase the contact surface between tannins and the solvent and this increases the extraction efficiency. However, very high levels of sodium sulfite can decrease the amount of tannins extracted.

How do tannins inhibit the growth of bacteria?

Tannins are very effective as an antibacterial substance because they have the ability to pass through the bacterial cell wall, reach the internal membrane of the bacteria cell, and interfere with the metabolism of the bacteria cell, which leads to the destruction of the bacteria cell. In addition to this, tannic acid causes bacteria to be unable to attach to a surface, and a lack of adhesion to a surface causes bacteria to die. 

Weatherspoon, D., & Cherney, K. (2022, April 7). What Is Bacteria? Good vs. Bad, Benefits, and Common Types. Healthline. https://www.healthline.com/health/bacteria (Bacteria diagram image)

Can tannins destroy a certain type of bacteria faster?

Most bacteria can be categorised as either Gram-positive or Gram-negative. The cell walls of Gram-positive bacteria are composed of thick layers of peptidoglycan, which is a substance that forms the cell walls of many bacteria. Gram-negative bacteria have a thinner layer of peptidoglycan in their cell walls compared to the Gram-positive bacteria, but their cell wall is also surrounded by an outer membrane which the Gram-positive bacteria don’t have. This outer membrane causes the antibacterial effects of tannins to be slower on Gram-negative bacteria compared to Gram-positive bacteria. 

Along with this, Gram-negative bacteria are becoming increasingly resistant to multiple drugs and antibiotics. This makes finding a new and natural way like using tannic acid for the destruction of Gram-negative bacteria important. Even though the effects of tannins is slower on Gram-negative bacteria, it can still destroy them and the bacteria isn’t resistant to it.

Tannic acid has been tested and shown antibacterial effects against these bacteria:

•  Staphylococcus aureus
•  Escherichia coli
•  Streptococcus pyogenes
•  Enterococcus faecalis
•  Pseudomonas aeruginosa
•  Yersinia enterocolitica
•  Listeria innocua 
•  Bacillus cereus

How do tannins inhibit the growth of viruses?

Tannic acid has effective antiviral properties, and it has shown antiviral effects against these viruses:

• Influenza A virus
• Papilloma virus
• Noroviruses
• Herpes simplex virus type 1
• Herpes simplex virus type 2
• Human immunodeficiency virus (HIV)

Tannins inhibit the growth of viruses by binding to the cell receptor of a virus and preventing viruses from attaching to different types of surfaces. Tannins also stop proteins from attaching to a virus.

What are the types of tannins?

Hydrolyzable tannins and condensed tannins are the two major types of tannins. Hydrolyzable tannins are composed of ellagic and gallic acid (antioxidants found in plants or combined with tannins) and they have a sugar core which is made out of primarily glucose. Condensed tannins are composed of flavonoids (biologically active compounds found in many plants) and they don’t have a sugar core, unlike hydrolyzable tannins. Condensed tannins have many more sources in nature compared to hydrolyzable tannins.

Is it possible to confirm whether or not something has tannins in it?

An effective way of testing a solution to see if it has tannins in it is by adding FeCl3. (Iron III chloride or ferric chloride) FeCl3 usually has a yellow-orange colour, and in the presence of tannins, it turns black or very dark green. This could be used to confirm whether or not the antibacterial solutions have extracted tannins from the fruit peels in them.

What kind of properties do foods that contain tannins have?

Foods that contain tannins usually have a bitter or astringent taste. The three fruit peels that I’m using for my experiment (pomegranate peels, mango peels, and banana peels) are described to have a bitter taste, with pomegranate peels also having an astringent taste. Because of this, all of the fruit peels that I’m using for my project most likely contain tannins. In addition to this, pomegranate peels are described by multiple websites to contain high amounts of tannins.

Manipulated variable:

• The fruit that the antibacterial solution is made with (pomegranate peels, mango peels, or under ripe banana peels)

Responding variable:

• The amount of bacteria colonies that grew in the petri dishes where the antibacterial solutions were added. (the amount of squares on grid paper that the bacteria colonies touch in the petri dishes where the antibacterial solutions were added)

Controlled variables:

• The amount of tap water in the antibacterial solutions (60 mL)
• The amount of time that the antibacterial solutions are heated for (20 minutes)
• The setting on the electric burner that each antibacterial solution is heated at (1)
• The amount of antibacterial solution that is added to each petri dish (2mL)
• The amount of each dried fruit peel that is added to the tap water (20g)
• The amount of sodium sulfite added to the antibacterial solutions when they're being made (1 teaspoon)

Procedure:

(To make the disinfectants)

1. On a cutting board, use a knife to cut the pomegranate peel into small pieces. (to expose the most surface area to the water which increases the tannin extraction efficiency)

2. Put 20g of the pomegranate peel into 60mL of tap water in a beaker.

3. Heat the tap water and pomegranate peel mixture on a portable electric burner on the “1” setting for 20 minutes, adding 1 teaspoon of sodium sulfite to the mixture after 1 minute of being heated. (adding sodium sulfite also increases contact surface between tannins and the water, which increases the extraction efficiency)

4. Let the mixture cool until it’s at room temperature.

5. Repeat steps 1-4 using dried mango peels.

6. Repeat steps 1-4 using dried banana peels.

(To prepare the petri dishes)

1. Get all materials and put plastic gloves on.

2. Make the nutrient agar according to the instructions on the package.

3. Pour the nutrient agar equally into 10 petri dishes.

4. Leave the nutrient agar to harden in the petri dishes with lids on for an hour.

(To test the disinfectants in the petri dishes)

1. Once the nutrient agar has hardened, drag a sterile swab over a light switch.

2. Gently drag the swab in a zigzag pattern over one of the petri dishes.

3. Repeat steps 1 and 2 for all the petri dishes, using a new sterile swab for each one.

4. Pour 5 teaspoons of the antibacterial solution made with pomegranate peels onto one of the petri dishes.

5. Repeat step 4 for two more petri dishes.

6. Repeat steps 4 and 5 for the antibacterial solution made with mango peels.

7. Repeat steps 4 and 5 for the antibacterial solution made with banana peels.

8. Leave the last petri dish without any antibacterial solution in it to be the control.

9. Close and tape all the lids to the petri dishes.

10. Add 1 teaspoon of the pomegranate antibacterial solution into 20mL of ferric chloride/iron III chloride to confirm if the antibacterial solution contains tannins. (iron III chloride will turn black or very dark green in the presence of tannins.)

11. Repeat step 10 for the mango antibacterial solution and the banana antibacterial solution.

12. Put all the petri dishes into a box and leave them in a dark area for 4 days.

13. After 4 days, put grid paper under each petri dish and count the number of squares that the bacteria colonies in each petri dish touch.

Number of Grid Paper Squares that Bacteria and Fungi Colonies Touch in Petri Dishes with Different Types of Fruit Peel Antibacterial Solutions Added

Number of grid paper squares that bacteria colonies touch in a petri dish with no antibacterial solutions added: 247 squares

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The banana antibacterial solution left a bit of a residue on the petri dishes since it was left on them for multiple days. I knew the bacteria colonies I circled were bacteria colonies and not parts of the residue because they didn't move when I tilted the petri dishes.

Banana test 1                                 Banana test 2                             Banana test 3

Mango test 1                                       Mango test 2                            Mango test 3

Pomegranate test 1                        Pomegranate test 2                      Pomegranate test 3

Control

Ferric chloride test results:

Ferric chloride turns black in the presence of tannins, it did this for all of the antibacterial solutions. When I only put cut up fruit peel pieces in the ferric chloride, only the pomegranate peel pieces made the ferric chloride change colour, confirming that the extraction process was effective and that pomegranates have the most tannins in them compared to the other fruit peels.

Analysis Summary:

• Compared to the control sample, all three fruit peel antibacterial solutions were very effective in inhibiting the growth of bacteria.
   
• The first test for the mango peel antibacterial solution might have been an outlier.
   
• After the heating and tannin extraction process on the electric burner, all three of the fruit peel antibacterial solutions made the ferric chloride turn black, confirming that the extraction process was effective and that they contain tannins. 
   
• When the fruit peel pieces without the whole heating and tannin extraction process were added to the ferric chloride, only the pomegranate peel pieces made the ferric chloride turn black. Also, the petri dishes with the pomegranate peel added didn’t grow bacteria. Therefore, this makes me infer that the pomegranate peel had more tannins than the other fruit peels, and this greater amount of tannins made the most bacteria growth be inhibited.

Full analysis:

In the petri dishes with the banana antibacterial solution added, the number of bacteria colonies were about the same in each test, and the average number of grid paper squares with bacteria colonies touching them was 4.3. 

In the petri dishes with the mango antibacterial solution, the average number of grid paper squares with bacteria colonies was 4.6. 

However, the first test for the mango antibacterial solution might have been an outlier because 0 colonies grew in it, while the other two tests had 6 and 8 bacteria and fungi colonies. This might have happened because the nutrient agar poured into the petri dish for the first test didn’t have as much nutrients in it as the nutrient agar poured into the other petri dishes. 

The mangos might have also been contaminated with fungi before I did the testing because test 2 and test 3 for the mango antibacterial solution were the only petri dishes that grew fungi.

If you only count the second and third test for the mango antibacterial solution, the average number of grid paper squares with bacteria colonies touching them becomes 7.

The number of bacteria colonies in the petri dishes with the pomegranate antibacterial solution was consistent in all the tests, with 0 colonies growing in all three tests.

Compared to the control sample, all three fruit peel antibacterial solutions were very effective in inhibiting the growth of bacteria. You can confirm this because the control sample had 247 squares with bacteria colonies touching them, and the petri dishes with the fruit peel antibacterial solutions had 0-8 colonies of bacteria or fungi.

After the heating and tannin extraction process on the electric burner, all three of the fruit peel antibacterial solutions made the ferric chloride turn black, confirming that the extraction process was effective and that they contain tannins. 

When the fruit peel pieces without the whole heating and tannin extraction process were added to the ferric chloride, only the pomegranate peel pieces made the ferric chloride turn black. Also, the petri dishes with the pomegranate peel added didn’t grow bacteria. Therefore, this makes me infer that the pomegranate peel had more tannins than the other fruit peels, and this greater amount of tannins made the most bacteria growth be inhibited. 

My hypothesis was correct because no bacteria colonies grew in the petri dishes with the pomegranate peel antibacterial solution added. Also, even before the tannin extraction process, the pomegranate peel pieces changed the colour of the ferric chloride to black, while the other fruit peel pieces didn’t. Therefore, I can infer that the pomegranate peel has more tannins than the other fruit peels, and the pomegranate peel having a higher tannin content was also in my hypothesis.

Some commercial cleaning products can release chemicals while they’re being used, and these chemicals can cause chronic respiratory problems, allergic reactions, chemical burns, and headaches. Some common store bought cleaning products have also been found to have carcinogenic substances in them. 

For these reasons, if people use natural cleaning products with no dangerous chemicals, like the fruit peel antibacterial solutions in my project, it would be safer and also effective at inhibiting the growth of bacteria. Also, making the fruit peel antibacterial solutions aren’t difficult, and they don’t require many uncommon materials, so it’s accessible to almost anyone. Even if they don’t have pomegranates, which were the best in inhibiting bacterial growth, they could also use more common fruits like bananas or mangoes because they also were very effective.

Tannins are slower in destroying Gram-negative bacteria compared to Gram-positive bacteria because of the outer membrane in Gram-negative bacteria. However, they’re still capable of destroying them, which could be functional because Gram-negative bacteria are becoming increasingly resistant to multiple drugs and antibiotics. Because of this, using the tannins in the fruit peel antibacterial solutions, which were confirmed to contain tannins, can be very useful for doctors, researchers, pharmacies, or other people that want to get rid of bacteria that is resistant to a lot of antibiotics.

One of the sources of error I could have had was bacteria from outside sources contaminating the nutrient agar, so that some of the petri dishes contained more bacteria before I put the antibacterial solutions in them than the others. 

Some of the antibacterial solutions could have also been contaminated with bacteria and not the others, causing some tests to grow more bacteria than other ones.

The light switches I collected bacteria from to put on the petri dishes could have also had varying amounts of bacteria each time I swabbed them.

The size of the fruit peel pieces could have also varied slightly, which could have caused more surface area of some fruit peel pieces to be exposed to the heated water, causing more tannins to be extracted.

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I am incredibly grateful to my parents for helping me with this science fair project, and buying all the materials. Thank you!
I am also very grateful to Ms. Davis and other teachers at my school for providing feedback on my project.