A NATURAL ANTI-BACTERIAL SOLUTION USING RAINWATER AND PERSIMMON CALYX

Summary

The hygiene products on the shelves; detergents, soaps, disinfectants; are all full of chemicals - and available only to those who can afford them. According to the CDC about 1 million deaths per year are due to a lack of hygiene and poor sanitation. With this in mind I decided to make an affordable - possibly free - anti-bacterial product without the use of any chemicals. 

In my research I learned that the calyx of the persimmon has a high concentration of tannin compared to other fruits - and an effective antimicrobial activity in its pure form. My prediction was that an extraction of tannin from the calyx with water as a solvent and a heat source would give us a solution that is effective against bacterial growth. 

The method I used in this experiment was safe and simple using; persimmon calyx and rainwater heated at 50°C for a short period of time. Because many people lack the access to clean water I used what they could naturally have - rain as a solvent. The resulting tannin extract solution was dropped onto bacteria and within 48 hours the zone of inhibitions were measured to see the effect of the tannin extract on bacterial growth. The findings concluded that the extract does infact inhibit the growth of bacteria.

This discovery can be helpful for the millions of people who lack the resources for a healthy hygiene - they can make their own natural anti-bacterial solution using rainwater and persimmon calyx. 

Question / Proposal

My question is: ''Is it possible for people in least developed and developing countries to find an accessible method to creating an anti-bacterial solution using only rainwater and persimmon calyx to have a more hygienic environment?''

My hypothesis is that if rainwater and dried persimmon calyx are heated at 50°C for a short period of time - a liquid containing tannin can be extracted which then can be used as an effective anti-bacterial solution. Anyone can produce this natural and simple solution with an unused part of the persimmon fruit and have a more hygienic environment. 

Research

Most people throw away the calyx/sepal of a persimmon without thinking about how it can be useful. However, according to the research done on persimmons - the ''seed and calyx extracts showed significantly higher antioxidant activities and phenolic [tannin] contents than peel and flesh extracts.'' (Jang 2011) In other studies the chemical properties of tannin found in the persimmon calyx have been shown to have anti-bacterial activities. (Tomiyama 2016) But, researchers use many different methods to extract the tannin using solvents like acetone, ethanol and methanol to get a high tannin yield. Having done the research I wanted a natural way to extract the tannin using rainwater as a solvent only. The research that has been already done in this field supports the idea that tannin has an anti-bacterial ability - which supports that my solution would be effective and 100% natural simple and easy to access for those in need. With accessibility in mind I used rainwater to support a real world case in which if someone chose to make this natural solution outside of a laboratory; they could. Our world could benefit from this simple but effective idea - and get one step closer to allow anyone from any part of the world to have access to a more hygienic environment. 

Method / Testing and Redesign

1. Making the nutrient agar:

●      First 7 g of nutrient agar was poured into 250 mL of distilled water and boiled at 150°C until the agar dissolved. Then in a clean area in the lab the agar solution was poured into petri dishes. 

2. Growing bacterial colonies:

●      Using a sterile cotton swab bacteria from my hand were collected and gently swabbed onto one nutrient agar plate. The petri dish then was kept in a fume hood for 48 hours for the bacteria to grow.

3.  Making the Tannin extract:

●      Using a mortar and pestle I crushed 2.1 grams of dried persimmon calyx.

●      The crushed persimmon calyx was then added to 15 mL of rainwater.

●      Two 50 ml beakers containing the equal amounts of rain water and the persimmon calyx were placed on a hot plate warmed to 50°C. One beaker was heated for 20 minutes and the other for 30 minutes.

●     The beakers were then cooled down to room temperature and filtered and poured into labeled 15ml centrifuge tubes.

4. Testing the effect of the tannin extract on bacteria:

●      Using a sterile swab one bacterial colony was carefully removed from the bacteria petri dish. The bacterial colony was then gently swabbed onto the center of a new sterile nutrient agar plate. This step was repeated on a second new sterile nutrient plate. 

●      Using a sterile dropper two drops of the 50°C - 20 min heated tannin extract was placed in the center of one petri plate swabbed with bacteria. This was repeated for the second petri plate swabbed with bacteria with two drops of the 50°C - 30 min heated tannin extract. These plates were labeled and left in the fume hood for the next 48 hours.

●      After 48 hours the effect of the tannin solution on the bacterial growth was observed and the zones of inhibition (ZOI) in both the 50°C 20 min/ 30 min tannin extract petri dishes were measured. These results were then compared with each other to determine the time measurements that is best for inhibiting bacterial growth.

Independent Variable: Time (heating the persimmon calyx in rain water for 20 minutes and 30 minutes)

Dependent Variable: Zone of Inhibition (ZOI) on the petri dishes with bacteria.

To ensure that my experiment process was fair, my constant variables were:

●      Type of the bacteria

●      Type of water; rain water; 15 mL for each trial

●      Persimmon calyx from Diospyros kaki L.; 2 grams for each trial

●      Heat: 50°C

●     Equipment: Sterile cotton swabs, sterile droppers, sensitive electronic balance, 15 mL centrifuge tubes, hot plate. 

This experiment took place in the biology laboratory. All safety measures were taken into consideration when working with a hot plate, growing bacteria and using FeCl3 to indicate tannin in a fume hood. I wore goggles, gloves and a lab coat and did the experiment with my biology teacher's supervision.

Results

In this experiment, I made a solution which uses persimmon calyx and rainwater to create a tannin extract solution that can be used as an anti-bacterial - without any chemicals.

For evidence to support the presence of tannin in the rainwater/calyx solution extract I used FeCl3 (Iron III Chloride) to determine the presence of tannin. FeCl3 has an orange-red color - and in the presence of tannin it turned dark green - I had a positive result.

 [Tannin extract before adding FeCl3]   [Tannin extract after adding FeCl3]

In my results I measured the zone of Inhibition (ZOI) where I dropped the tannin extract solution onto bacteria in the petri dishes. The two petri dishes I made were different as my independent variable was two different time periods (heating the calyx for 20 minutes and 30 minutes).

As a result, I observed that the tannin extract that was heated for 20 minutes had a bigger zone of inhibition (ZOI) than the tannin extract that was heated for 30 minutes. While both solutions were effective at inhibiting bacterial growth - heating the calyx for a shorter time might not denature tannin as much. 

TANNIN EXTRACT FROM PERSIMMON CALYX AND RAINWATER HEATED AT 50°C ZONE OF INHIBITION (ZOI) IN CENTIMETERS
20 minutes 2.6 
30 minutes 1.8

Pictures of ZOI:

 CALYX AND RAINWATER AT 50°C 20 MIN

 CALYX AND RAINWATER AT 50°C 30 MIN

Conclusion

In this project my question was: ''Is it possible for people in least developed and developing countries to find an accessible method to creating an anti-bacterial solution using only rainwater and persimmon calyx to have a more hygienic environment?'' The answer is yes - I was able to create a natural and simple way to access a more hygienic solution without using any chemicals; only rainwater and perimmon calyx. The results also supported my hypothesis in that calyx that is not heated above 50°C using rainwater as a solvent can extract tannin - which has anti-bacterial properties.  

I grew hand bacteria to test the effect of the tannin extract solution on common bacterial flora. I dropped the tannin extract into petri dishes that included a bacterial colony that I swabbed from the bacteria petri dish shown above. Where a gap formed in the petri dish meant that the solution was effective. My independent variable was the length of time the persimmon calyx was heated in rainwater. 

  • By changing the time - the zone of inhibition also changed - showing that heating the persimmon calyx for only 20 minutes gave a more effective tannin extract. 

Initially I did other pilot experiments. At first I tried boiling the solution of persimmon calyx at 100°C, 150°C and 200°C. However, the tannin extracts that were obtained did not give any successful results - and did not stop bacterial growth. After further research I learned that while high temperatures can increase tannin extraction - "temperatures above 70°C cause rapid polyphenol [tannin] degradation." (Rajbhar 2014) Therefore boiling the calyx was not the correct way to do this. It was then I realized that I should heat the calyx only at 50°C for different lengths of time. 

This project concluded that anyone in the world can use this accesible method to creating an anti-bacterial solution using only rain water and persimmon calyx to allow them to have a more clean environment. Overall the experiment was conducted successfully and the results are reliable. I observed the gaps in the petri dish where a zone of inhibition meant that the tannin solution has an anti-bacterial property and can stop the growth of bacteria. Last but not least, with Iron (III) Chloride, I proved that the solution contained tannin as it turned a dark green. 

In terms of improving the method and my approach to doing this experiment - the next time I would have more petri dish trials in testing the length of time the calyx is heated. 

Because the experiment was succesfull my results inspired me to wonder; can we make the tannin extract solution even stronger/more effective? So perhaps now I will try other solvents in the tannin extraction; for example - ethanol - to compare it to the results I got from rainwater. 

 

 

 

 

 

About me

My name is Nida Memnune Totu. I am a 17 year old IB student at Nun High School in Istanbul, Turkey. I enjoy doing taekwondo, writing Islamic calligraphy and playing the violin. I have always been interested in the science aspect of STEM - but more specifically I have always wanted to study gynecology to help and provide mothers with care for them to be healthy to create healthy generations. I have always believed that the best medicine is with natural nutrients that are found in our surroundings. 

An individual person cannot live the experience of others. But we can benefit and learn from the experiences of significant figures from the past and the present. Marie Curie and Elon Musk are two people who continually influence me. Marie Curie, who inspired so many women after her time, was someone who dedicated her life for science. I also really respect Elon Musk's passion; his cool ideas and his drive to do what others only dream of. 

With the Google Science Fair, I wanted to see whether I could prove how nature can provide a simple way to live healthily; something to meet the needs of humanity. Participating in the Google Science Fair is the first of many steps I will take to get closer to my dreams. The award will enable me to access the world to be discovered from different aspects; and give me the opportunity spread the news that natural remedies are better for us. 

Health & Safety

I worked in the biology laboratory of Nun High School, where the rules of the laboratory were carefully followed and my biology teacher always monitored my experiments.

During the experiment I grew bacteria - for this I made sterile petri dishes using nutrient agar - and I sourced the bacteria from my hands with a sterile swab. When making the nutrient agar solution I carefully used a hot plate. I made sure to wear goggles, lab gloves, and a lab coat at all times during the experiment. The petri dishes that were used to grow bacteria were always stored in a fume hood throughout the experiment. Last but not least as evidence to indicate that the persimmon calyx extract contains tannin I carefully performed the ferric chloride test in a fume hood while Ms. Akcay monitored.

  • The Safety Guidelines attachment:
  • https://drive.google.com/file/d/1MtEw-AzQ7sT6ywzRyaOEQApW51GB7FYG/view?usp=sharing

My mentor biology teacher / lab supervisor: Hatice Akcay

Location: Nun High School biology laboratory

Contact Email: hatice.akcay@nunokullari.com

 

Bibliography, references, and acknowledgements

References:

  • Teach.Genetics. https://teach.genetics.utah.edu/content/microbiology/plates/
  • NCBI. https://www.ncbi.nlm.nih.gov/pubmed/9759559
  • NCBI. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817420/
  • SRCC. https://www.tsijournals.com/articles/polyphenols-methods-of-extraction.pdf
  • Encyclopedia Britannica. https://www.britannica.com/science/tannin
  • Encyclopedia Britannica. https://www.britannica.com/plant/persimmon
  • Tomiyama, Kiyoshi et al. “Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms” BioMed research international vol. 2016 (2016): 5730748.
  • Jang, In-Cheol et al. ‘’Antioxidant and antigenotoxic activities of different parts of persimmon (Diospyros kaki cv. Fuyu) fruit.’’ Journal of Medicinal Plants Research, Vol. 4(2), 18 January, 2010, pp. 155-160. http://www.academicjournals.org/JMPR.
  • Hayashi, Hideo et al. ''Callus Growth Inhibitors from Persimmon Calyx.'' 30 September 1977. https://www.tandfonline.com/doi/pdf/10.1080/00021369.1977.10862893 
  • Jo, Kyung-Jin et al. ''Anticancer activity of persimmon (Diospyros kaki L.) calyx extracts on human cancer cells.'' Journal of Medicinal Plants Research, Vol. 5(12), 18 June 2011, pp. 2546-2550. https://academicjournals.org/journal/JMPR/article-full-text-pdf/EBE355323544
  • Salih, Aidin. Gerçek Tıp: Yitik Şifanın İzinde. June, 2007.

Acknowledgements:

Throughout the experiment, my biology teacher, Hatice Akcay helped me a lot. I would like to thank her. In each stage of experiment, she shed light on what I was learning. She gave me access to use her laboratory and the equipments available in the lab.