Are Grapefruit Seed Extract real antimicrobial components

Are Grapefruit Seed Extract real antimicrobial components ?

(Comparison of the Gentamicin and  Grapefruit Seeds Extract antimicrobial effects on Escherichia coli.)

 

The all story started from a compost. The rumors told that Jacob Harich, (1919-1996) a german researcher in physics but also a passionate gardener, would have observed one day a grapefruit without any kind of microbial rot around it, in his compost. From this observation started the idea that Grapefruit Seeds could be antimicrobial, preventing it from decomposition. This is the beginning of the great and controversial adventure of the Grapefruit Seed Extract. Let's call it GSE.

Today, GSE had been commercialized all over the world and can be bought in internet to treat microbial and fungal infections.

GSE is composed of Grapefruit (Citrus paradisius) Seeds grinded, dissolved with ethanol or water and added to glycerin.

But since more than twenty years, the scientific community are in disagreement about the possible antimicrobial capacity of GSE. Why ?  

In 1999, a German scientific team observed an antimicrobial activity in all the commercially available  GSE they tested but not in their several self-made GSE. Moreover, they found preservatives known to be bactericidal such as benzethonium chloride, triclosan and methyl paraben in the commercialized GSE, letting them to suppose that the antimicrobial capacity of GSE would be merely due to these synthetic agents.  (Woedtke T, Schlüter B, Pflegel P, Lindequist U, Jülich WD (June 1999). "Aspects of the antimicrobial efficacy of grapefruit seed extract and its relation to preservative substances contained". Pharmazie. 54 (6): 452–6. PMID 10399191).) From this moment until today, some studies support this conclusion while other still demonstrate the real antimicrobial capacity of GSE.

    “If you can’t agree each other, I will attempt to resolve this mystery on my own ! (modestly)”

    What was my experimentation ? 

I made my own GSE extract :

I decided to study the impact of GSE on  the growth of Escherichia coli, a bacteria very used in microbiology. To determine the growth rate, which is the number of colony forming units per ml according to the time, I used a colorimetric method, using spectrophotometer measures at 620 nm.  Indeed, while bacteria are growing and cloning, the opacity of a solution is increasing. To detect Absorbance of a solution according to time can let us determine the growth rate.  The main idea was to compare the E coli growth rate in different condition : E coli with bacteria / E coli with gentamicin ( a well known antibiotic ) / E coli with GSE. 

Each conditions has a different growth rate which is the slope of the curves.  The 6th measurement in all the differents conditions and replicates seems to be  a technical error of the spectrophotometer so we will not taking it in account.

The test with only E coli is a positive control, which allow us to compare this growth rate with the growth rate of the two other conditions. We observe that the Absorbance according to the time in the solution (5) composed of Gentamicin+E coli and LB is approximately constant. There is an insignificant growth rate, meaning that the bacteria were killed or couldn’t divide since the first contact of the gentamicin.

We did the average of the 6 replicates GSE+E coli +LB (6)  coli growth rate  and it was equal to  3.81×10⁻4 while the growth rate average of the 6 replicates E coli+ LB was equal to 1.97*10⁻4.

This two different growth rate are statistically different, by using a bilateral average comparator.

What is striking : E coli alone grows slower than E coli in contact with GSE !

It means the growth of E coli isn’t inhibited by the GSE, and is even faster ! What could be the different reason leading to this result ?

1. The “home made” Grapefruit Seeds Extract without any kind of preservatives has no  antimicrobial activities.
2. The GSE concentration in this experimentation was to low. I couldn't determine the concentration I used. Maybe I could redo the experimentation using a way for my GSE to be at the  Minimum Inhibitory Concentration determined by this paper : (“Antimicrobial activity of grapefruit seed and pulp ethanolic extract”, ZDENKA CVETNI, SANDA VLADIMIR-KNE@EVI, Acta Pharm. 54 (2004) 243–250, 2004”. )
3. The “home-made” GSE get more opaque over time ?

The next step of my experimentation would be to redo the spectrophotometric measures and determination of the growth rate but with a commercial GSE in order to compare it with the “natural” GSE+ E coli growth rate. If this growth rate would be lower than the control one,  this study would be really in favour of all the researcher thinking that the natural  Grapefruit  Seeds extract is not antimicrobial, and that this capacity is merely due to synthetic preservatives agents.

Acknowledgement : Petnica Center, Centre de Recherche Interdisciplinaire, Fondation Bettencourt Schueller, licence Frontière du Vivant, my wonderful tutor Gaspard, the assistant team, Bore promotion…

If you want to know more about this subject :

Reference :

-1(Woedtke T, Schlüter B, Pflegel P, Lindequist U, Jülich WD (June 1999). "Aspects of the antimicrobial efficacy of grapefruit seed extract and its relation to preservative substances contained". Pharmazie. 54 (6): 452–6. PMID 10399191).)

-  2 “Antimicrobial activity of grapefruit seed and pulp ethanolic extract”, ZDENKA CVETNI, SANDA VLADIMIR-KNE@EVI, Acta Pharm. 54 (2004) 243–250, 2004”

- “EFFECTS OF OREGANO ESSENTIAL OIL, GRAPEFRUIT SEED

EXTRACT AND THEIR COMBINATION ON THE GROWTH

AND SURVIVAL OF Salmonella Typhimurium AND Listeria

monocytogenes IN POULTRY FILLETS UNDER MODIFIED ATMOSPHERE PACKAGING

Tolga Kahraman 1 , Ghassan Issa 4 , Sema Sandikci Altunatmaz 2 , Beren Basaran Kahraman 3 , Filiz Aksu 2 , Ali Aydin 1 *, Harun Aksu ,

-Armando C, Maythe S, Beatriz NP (December 1997). "Antioxidant activity of grapefruit seed extract on vegetable oils". Journal of the Science of Food and Agriculture. 77 (4): 463–7.

-Takeoka G, Dao L, Wong RY, Lundin R, Mahoney N (July 2001). "Identification of benzethonium chloride in commercial grapefruit seed extracts". J. Agric. Food Chem. 49 (7): 3316–20. doi:10.1021/jf010222w. PMID 11453769.

-Cvetnić Z, Vladimir-Knezević S (September 2004). "Antimicrobial activity of grapefruit seed and pulp ethanolic extract". Acta Pharm. 54 (3): 243–50. PMID 15610620

-Wikipédia : https://en.wikipedia.org/wiki/Grapefruit_seed_extract

- Takeoka GR, Dao LT, Wong RY, Harden LA (September 2005). "Identification of benzalkonium chloride in commercial grapefruit seed extracts". J. Agric. Food Chem. 53 (19): 7630–6.

-Food and Bioprocess Technology,pp 1–13, Grapefruit Seed Extract and Lemon Essential Oil as Active Agents in Corn Starch–Chitosan Blend Films, M. J. Bof, A. Jiménez, D. E. Locaso, M. A. García, A. Chiralt, 13 August 2016, DOI: 10.1007/s11947-016-1789-8

- Why I Don’t Use Grapefruit SeedExtract :

http://www.mommypotamus.com/the-ugly-truth-about-grapefruit-seed-extract/