The Antimicrobial Potential of Natural...

Jennifer Graham 2014 of 15

The Antimicrobial Potential of Natural Products

1. Abstract

There are an ever growing number of microorganisms that are becoming resistant to current

antimicrobials. Microorganisms can become resistant for a number of reasons such as over

prescription of antibiotics, patients not completing the full course or from random mutation. This

has lead to infections such as MRSA becoming virtually untreatable. There is now a great need from

the health industry for new antimicrobials and the answer may lie within natural products. This

experiment compared four natural products (chives, oyster mushroom, cherry and vanilla) to known

antimicrobials (ethanol, garlic and antibiotics) in a hope that they would produce a significant effect

and show antimicrobial potential. The compounds were tested on two bacteria’s, one gram positive

(S.aureus) and one gram negative (E.coli), this was to show how different bacteria’s behave. All

compounds were suspended in ethanol and a t test was used to show their significance. The

compound that worked best at the lowest concentration (0.1) was cherry on S.aureus, all of the

other compounds produced significant results at the 0.2 concentration. None of the natural

compounds selected produced significant results on E.coli. On neither of the bacteria did any of the

compounds produce a significant result at the highest dilutions (1 and 0.5). In further study,

compounds could be separated into single ingredients to determine which part of the product has

the antimicrobial effect and this could then be used to treat resistant microorganisms.

2. Introduction

Over the past few years the amount of antimicrobial resistant pathogens has sharply increased.

There is now a need to find new compounds that have antimicrobial properties in order to prevent

this from rising further.

Bacteria have developed resistance at a fast rate due to a number of reasons...

1. Doctors have overprescribed antibiotics for minor infections that present no danger. By

overexposing bacteria to antibiotics they are more likely to develop resistance as the more

they come into contact together the less effective the antibiotic becomes.

2. Doctors have prescribed antibiotics for viral infections. As viruses live inside host human

cells they have no metabolism of their own and therefore antibiotics cannot destroy a virus.

Hence it is pointless to prescribe antibiotics to a virus as it will not do anything. However

any bacteria that may be present in the patients system will be exposed to the antibiotic and

so may develop resistance.

3. Patients are not finishing the full course of antibiotics. When patients begin to feel better

they tend to stop taking their prescription but there may be some bacteria left in their

system, these will be the most resistant to the antibiotic. By stopping the course the

bacteria has not been killed and so will reproduce passing on the resistance gained through

the exposure of the antibiotic. This will also lead to the patient returning to the doctors and

getting another prescription which causes more exposure to the bacteria.


Resistance can be caused by a chance mutation in a bacteria’s plasmid. This new gene may be able

to break down a particular antimicrobial before it affects its own metabolism. This increases the

pathogens chance of survival and so when they come to reproduce they pass on this allele to their

offspring. Plasmids are also interesting as they can pass from bacteria to bacteria without

reproduction, this means resistance can spread very quickly throughout a colony and between

different strains of bacteria. This is one of the reasons why antimicrobial resistance has become

such a large issue.

Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the most commonly known

antimicrobial infections. MRSA is a strain of Staphylococcus aureus that is no more infectious than

the normal one but much harder to treat. MRSA is known as a superbug as it is resistant to not only

methicillin but also amoxicillin, penicillin, oxacillin and lots of other antibiotics. MRSA causes mild

skin infections such as sores and boils however can cause serious infections if it enters the

bloodstream or urinary tract. People most vulnerable to catching MRSA are those in hospital, due to

the close contact of other patients on wards, their weakened immune systems and the poor hygiene

from hospital staff. This has led to a large change in the way patients are treated, all patients who

are about to have surgery are screened beforehand for MRSA and if a patient is found to have MRSA

they are isolated on wards with other patients who also have MRSA, this is to reduce the spread.

An antimicrobial agent is something that kills or inhibits the growth of microorganisms whilst causing

little or no harm to the host. An antimicrobial could be an antibiotic, antiviral or antifungal.

Traditional antimicrobials are becoming less and less effective because of the reasons explained

above. Therefore there is an ever demanding need for new antimicrobials to be found to treat these

infections.

From this experiment it is hoped that some of the products tested will show some antimicrobial

potential. The products chosen were; Chives, Oyster Mushroom, Cherry and Vanilla. Chives were

chosen as they are a member of the allium family and this will be interesting to compare with garlic

which is one of the positive controls. Oyster mushroom was chosen because mushrooms are known

to have lots of health benefits, but also because mushrooms are a fungus like Penicillin. There were

lots of fruits that would have been good to test and many are known to have lots of health benefits

but there wasn’t much about cherries and that is why it was chosen. Vanilla was a more random

choice just to see what happened.

The success of this experiment will be determined from

1. Did the compounds produce any inhibition

2. Was this inhibition down to the compound or the ethanol suspension ( t test)

3. What is the lowest concentration the compounds produce inhibition at, if any

4. Are the natural products selected any better than the positive controls

Bacteria

The bacteria used in this experiment are Escherichia coli (E.coli) and Streptococcus aureus (S.aureus)

which are both a category II hazard. E.coli is a gram negative bacteria and S.aureus is a gram positive

bacteria, this will allow a comparison as some compounds may only work on gram positive bacteria.

The difference between gram positive and gram negative is how the cell wall is structured, gram


negative have a thin peptidoglycan layer and a high lipid content due to the outer membrane,

whereas gram positive have a thick peptidoglycan layer which is what retains the crystal violet dye.

The differences can be seen through a gram stain.

Figures 1 and 2: Gram stain of E.coli and S.aureus

Controls

The negative controls in this experiment are just bacteria and sterile water, this is to check that the

spreads are even and the bugs are growing (no problems with incubation time/temperature-37°C).

The positive controls are ethanol, garlic and antibiotics. Both the ethanol and garlic will be diluted

like the natural compounds so a direct comparison can be made. All compounds will be suspended

in ethanol (including garlic) so this will act as base to measure against in the statistical analysis. The

garlic came in a tablet form, which had an enteric coating, this may affect the results of the

experiment as the garlic may not be able to metabolise properly with it. If this was proved to be a

problem then we would either use fresh garlic cloves or react the tablet with some hydrochloric acid

as it would be in the stomach. The antibiotics used were Ciprofloxacin 1µg (CIP) and Fusidic acid

10µg (FC). CIP is a gram negative antibiotic and FC is a gram positive antibiotic.

CIP is a bactericidal which kills bacteria by inhibiting enzymes responsible for DNA replication,

transcription, recombination and repair. They do not normally cross-resist with other bacteria and

mutations develop slowly which make them ideal for treating bacteria that are resistant to other

antibiotics. CIP can be used to treat both gram-positive and gram-negative bacterias. FC is a

bacteriostatic which inhibits protein synthesis (bacterial replication). FC is only effective for gram

positive bacteria.

Chives- Allium schoenoprasum

Chives are part of the Allium family of vegetable, which contains such foods as onions, garlic and

leeks. Onions and Garlic have known antimicrobial properties and this is one of the reasons why

Garlic acts as one of the positive controls in this experiment. The active ingredient that causes there

antioxidant effects is Allicin, a combination of alliin and the enzyme allinase which is a highly

unstable and a very reactive compound (Kourounakis, 1991). It is believed that alliin and allinase are

kept separate during growth and only when cut/cooked do they mix, giving them their properties

(Focke, 1990). Chives also contain vitamin A, vitamin C, potassium and folic acid which have


numerous benefits such as; lowering blood pressure, aid digestion, stimulate appetite and possess

some antiseptic properties.

Figure 3: Structural formula of Allicin (C6H10OS2)

Oyster Mushroom- Pleurotus osteratus

Oyster mushrooms contain lots of active ingredients including; polypeptides, polysaccharides, alpha-

glucans, pleuran, beta-glucans, lovastatin, natural statins, pelonic compounds and tannins. Due to

the high amount of natural statins, particually lovastatin which makes up about 2.8% of the

mushrooms dry weight, oyster mushrooms have a large affect against reducing LDL cholesterol by

binding to the enzyme, HMG-CoA reductase. Recent in-vivo research has suggested that the extract

may induce apoptosis (programmed cell death) in some cancer lines. Oyster mushrooms are also

known to have anti-inflammatory, antiviral and antibacterial properties and so often used in

preventive medicine.

Cherry- Prunus avium

Cherries contain lots of antioxidants which protect against free radicals produced in the body. This is

important as oxidation of the bodies’ molecules can cause arthritis, heart disease and ageing skin

and many other chronic diseases. Cherries contain anthocyanins (350-400mg per 100g) which may

have antiviral properties and could improve vision, however these are not proven. Melatonin

regulates sleep patterns and cherries are a natural source of melatonin, supplements are used to

treat jet lag and in the future reduce chronic cluster headaches and help people who work night

shifts.

Figure 4: Structural fromula of Lovastain (C24H3605)


Vanilla- Vanilla planifolia

Vanilla is a phenolic aldehyde and contains the functional groups; aldehyde, hydroxyl and ether.

Vanilla’s active ingredient is called Vanillin and makes up approximately 2% of a vanilla pods dry

weight. At the moment Vanilla is not needed for any medicinal purposes other than pharmaceutical

flavourings however a synthetically produced version of Vanillin, Ethyl Vanillin is used widely in the

food industry. Although it is more expensive the ethoxy group makes this compound much stronger

than its natural version.

3. Method

Prepare agar plates using aseptic techniques.

Serially dilute bacteria on a streak plate. Take a single colony and make a bacteria emulsion.

Working aseptically, remove 100µl of bacterial emulsion into the centre of a fresh agar plate. Using

a spreader, cover the entire plate with the bacteria. Starting in the middle, take care not to damage

the surface of the agar. Sterilise equipment between different strains of bacteria.

Using good aseptic techniques flame a cork borer (size 4) in ethanol, allow to cool briefly and punch

4 holes into the agar. Gently remove with a sterile picking needle.

Figure 7: Structural formula of Vanillin (C8H8O3)

Figures 5 and 6: Strucural formula of Anthocyanin (C15H11O+) and Melatonin (C13H16N2O2)


Using 0.5g of each compound, grind in a mortar and pestle to a pulp. Pulse spin in a centrifuge,

using the supernatant produce 4 dilutions as shown below.

Concentration (compared to original)

Amount of compound/µl Amount of sterile water/µl

1 200

0.5 110 110

0.2 50 200

0.1 25 225

Pipette 100µl of each compound into the wells. The order of concentrations is relevant for ethanol,

garlic, chives, mushroom, cherry and vanilla. Two plates will not have wells as the antibiotic

compounds come in discs.

Repeat this so results are in triplicate.

Incubate overnight the right way up at 37°C.

Measure the diameter of each inhibition zone in cm using a ruler. Calculate a mean and standard

deviation for each concentration of the compounds. Carry out a t test, comparing all natural

compounds results to ethanol.

4. Results and Discussion

These are the hypotheses for the t test.

H0: no difference between ethanol and natural compounds

H1: some difference between ethanol and natural compounds

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Ster

ile w

ater

Eth

ano

l 1

Eth

ano

l 0.5

Eth

ano

l 0.2

Eth

ano

l 0.1

Gar

lic 1

Gar

lic 0

.5

Gar

lic 0

.2

Gar

lic 0

.1

CIP

1µ

g

FC 1

0µ

g

Ch

ives

1

Ch

ives

0.5

Ch

ives

0.2

Ch

ives

0.1

Mu

shro

om

1

Mu

shro

om

0.5

Mu

shro

om

0.2

Mu

shro

om

0.1

Ch

erry

1

Ch

erry

0.5

Ch

erry

0.2

Ch

erry

0.1

Van

illa

1

Van

illa

0.5

Van

illa

0.2

Van

illa

0.1

Dia

me

ter

of

inh

ibit

ion

zo

ne

/cm

Compound Concentration

A Graph to show how different concentrations of natural products affect the growth of E.coli

***


These are the results for the E.coli bacteria. The largest mean inhibition zone was the CIP antibiotic

with 3.667cm. A t test was carried out to see if the natural compounds had any effect on their own

or if it was down to the ethanol. None of the compounds with concentrations of 1 or 0.5 produced a

significant result and so therefore the null hypothesis can be accepted. Garlic with a concentration

of 0.2 produced a significant result as ethanol 0.2 did not produce any inhibition, this means in this

case the null hypothesis can be rejected (*** = p<0.001).

These are the results of the S.aureus experiment. The mean inhibition zones for all compounds were

much greater in this experiment than in the E.coli one. The antibiotic FC gave the largest inhibition

zone with 3.2333cm. Like E.coli, none of the concentrations 1 or 0.5 were greater than the ethanol

base, however all were significant at lower concentrations. Cherry was the most significant at 0.2,

closely followed by chives. These were also the compounds that came closest to being significant at

higher concentrations and were only narrowly under the critical values. Cherry was also the only

compound that gave a significant result at the lowest dilution (0.1) from both bacteria’s.

The diameters of inhibition zones for E.coli, decrease as concentration decreases which is what is

expected. However some of the results on S.aureus do not behave this way, these are cherry and

chives between 0.5 and 0.2. This could mean they are anomalous results and would have to be

repeated in order to verify this. These results could be down to the freshness of the ingredients

used as cherry is a fruit it oxidises very quickly. In fact between the two days when we made the

compound dilutions the cherry was a completely different colour, on the first day it was a very bright

red colour and after it had been left for a day and oxidised the colour was a deep brown. This

supernatant was redone with fresher cherry to give more consistency throughout the experiment. A

similar explanation could be seen for chives as they began to dry out.

One of the main reasons for selecting chives was to compare it to the positive control garlic as they

come from the same family. From previous research it has been shown that the Allicin is weaker in

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Ster

ile w

ater

Eth

ano

l 1

Eth

ano

l 0.5

Eth

ano

l 0.2

Eth

ano

l 0.1

Gar

lic 1

Gar

lic 0

.5

Gar

lic 0

.2

Gar

lic 0

.1

CIP

1µ

g

FC 1

0µ

g

Ch

ives

1

Ch

ives

0.5

Ch

ives

0.2

Ch

ives

0.1

Mu

shro

om

1

Mu

shro

om

0.5

Mu

shro

om

0.2

Mu

shro

om

0.1

Ch

erry

1

Ch

erry

0.5

Ch

erry

0.2

Ch

erry

0.1

Van

illa

1

Van

illa

0.5

Van

illa

0.2

Van

illa

0.1

Dia

me

ter

of

inh

ibit

ion

zo

ne

/cm

Compound Concentration

A Graph to show how different concentrations of natural products affect the growth of S.aureus

*** ***

*** *** ***


chives than garlic. The results from the E.coli experiment support this as garlic (0.2) produced a

significant result and had much larger inhibition zones for all concentrations. However on the

S.aureus experiment this is not seen as chives (0.2) produced a significant result and garlic did not.

This may be down to the fact that a garlic tablet was used instead of a fresh clove, this may have

skewed the results leading to this conclusion. It is also interesting that chives (0.2) had a larger

inhibition zone than chives (0.5) which may be anomalous and can only be proved through further

repetitions of the experiment.

The reason the compounds worked better on S.aureus than E.coli may be to do with their structure.

S.aureus is a gram positive bacteria and so doesn’t have a cell wall or extra outer membrane, this

makes it easier to penetrate and affect the metabolism of the cell. It is surprising that the chives had

a larger affect than the garlic in the t test (particularly S.aureus), as Allicin is weaker in chives. This

may be explained by the fact that the garlic used was a tablet and not a natural clove. The enteric

coating on the garlic may have stopped the full reaction as ethanol may not have been able to

dissolve it. It would be better to repeat this part of the experiment with fresh garlic.

Limitations

Although all compounds used had the same starting mass (0.5g) there were very different amounts

of the active ingredient in each sample, due to the water content of each compound, for example

there was very little cherry compared with the amount of chives. Some of the active ingredients

may have reacted with the ethanol and this could have affected the results, to see if this did affect

the results, repeat with suspension in sterile water. In all natural compounds there are a variety of

active ingredients so without further experiment we do not know which were responsible for the

inhibition, but could find this out by separating the compounds. The plates had to be incubated

right side up as otherwise the liquid would run out, this meant that condensation from incubation

may drip onto the agar, possibly causing dilution. Some plates may have had thinner agar than

others, which may affect the results as less compound could fit in the wells and may diffuse quicker.

The spreading method of bacteria was quantitative as 100µl was used each time, however the

amount of bacteria that remains on the spreader will be different and the spread may not be

completely even over the agar. The temperature of the lab at the time of the experiment was very

hot and this may have evaporated some of the supernatant. The garlic tablet and vanilla required

2ml of ethanol to form a liquid, this was twice the amount the other compounds needed and so they

are effectively twice as diluted as the others. The emulsions were made by different technicians.

The experiment was only repeated three times which is no really enough to draw conclusions from

Figure 8and 9: Inhibition zones of garlic and chives on S.aureus


and so for some of the compounds that came very close to being significant more repeats would be

needed to see if they did have an effect. The compounds needed to be fresh when tested to keep

results as similar as possible, this includes oxidisation of the cherry, as the supernatants of an

oxidised and non-oxidised cherry looked very different and it is not known if this is to impact on its

antimicrobial ability.

5. Conclusion

The results of this experiment showed that none of the selected compounds significantly affected

the growth of E.coli. All four of the compounds had a significant effect on S.aureus at the 0.2

concentration and cherry was the only compound to have an effect at the lowest concentration, 0.1.

Cherry and chives were also very close to being significant at their higher concentrations and only

narrowly missed the critical value. This means that both cherry and chives could have antimicrobial

properties and this could be verified with further research. In future experiment the compounds

could be separated and purified into singular active ingredients and repeating the test as above, with

the singular compounds to see which had the effect on inhibition. Also more compounds could be

tested on a wider variety of not only bacteria but also viruses and fungi. This in turn will provide the

pharmaceutical industry with alternatives to traditional prescriptions and prevent further

antimicrobial resistance developing. Through this research it has been shown that natural

compounds do have the potential to be used as antimicrobials, to treat infections that are currently

untreatable.

6. References

About Pleurotus Osteratus medicinal mushroom

Available at www.mycolivia.com/pleurotus-mushroom (Accessed 8th August 2014)

Anthocyanins and anthocyanidins

Available at www.food-info.net/uk (Accessed 9th August 2014)

Antimicrobials: An Introduction (2011)

Available at www.amrls.cvm.msu.edu (Accessed 14th August 2014)

Ciprofloxacin (13 April 2014)

Available at www.drugs.com/ciprofloxacin.html (Accessed 6th August 2014)

Focke, M., Feld, A., Lichtenthaler, K., February 1990, Allicin, a naturally occurring antibiotic from

garlic, specifically inhibits acetyl-CoA synthetase.

Kourounakis, P.N., Rekka, E.A., November 1991, Effect on active oxygen species of alliin and Allium

sativum (garlic) powder, Res Commum Chem Pathol Pharmacol, 74 (2): 249-252

Sleep disorders health centre: Melatonin-Overview (June 20, 2012)

Available at www.webmd.com/sleep-disorders (Accessed 9th August 2014)

http://www.mycolivia.com/pleurotus-mushroom

http://www.food-info.net/uk

http://www.amrls.cvm.msu.edu/

http://www.drugs.com/ciprofloxacin.html

http://www.webmd.com/sleep-disorders


7. Appendix

Product

E.coli

Expt 1/cm

Expt 2/cm

Expt 3/cm mean SD SEM UB LB significance

lawn 0 0 0 0 0 0 0 0

Sterile water 0 0 0 0 0 0 0 0

Ethanol 1 1.55 1.3 1.6 1.483333 0.160728 0.093 1.884 1.083

Ethanol 0.5 N/A 0 1.1 0.55 0.777817 0.55 2.917 -1.817

Ethanol 0.2 N/A 0 0 0 0 0 0 0

Ethanol 0.1 N/A 0 0 0 0 0 0 0

Garlic 1 2.4 1.3 2 1.9 0.556776 0.321 3.281 0.519 1.245

Garlic 0.5 1.3 0 1.4 0.9 0.781025 0.451 2.841 -1.041 -0.492

Garlic 0.2 1.3 0 1 0.766667 0.680686 0.393 2.458 -0.924

Garlic 0.1 0 0 0 0 0 0 0 0

CIP 1µg 3.3 3.7 4 3.666667 0.351188 0.203 4.543 2.796

FC 10µg 0 0 0 0 0 0 0 0

Chives 1 0 1.7 1.3 1 0.888819 0.513 3.207 -1.207 0.926

Chives 0.5 0 0 1.1 0.366667 0.635085 0.367 1.946 -1.212 0.277

Chives 0.2 0 0 0 0 0 0 0 0

Chives 0.1 0 0 0 0 0 0 0 0

Mushroom 1 1.5 1.1 1.8 1.466667 0.351188 0.203 2.341 0.593 0.074

Mushroom 0.5 0 1 0 0.333333 0.57735 0.333 1.766 -1.1 0.337

Mushroom 0.2 0 0 0 0 0 0 0 0

Mushroom 0.1 0 0 0 0 0 0 0 0

Cherry 1 1.9 1.4 1 1.433333 0.450925 0.26 2.552 0.314 0.181

Cherry 0.5 0 0 0.9 0.3 0.519615 0.3 1.591 -0.991 0.399

Cherry 0.2 0 0 0 0 0 0 0 0

Cherry 0.1 0 0 0 0 0 0 0 0

Vanilla 1 2.5 1.6 2 2.033333 0.450925 0.26 3.152 0.914 -1.99

Vanilla 0.5 0 1.2 0 0.4 0.69282 0.4 2.121 -1.321 0.221

Vanilla 0.2 0 0 0 0 0 0 0 0

Vanilla 0.1 0 0 0 0 0 0 0 0

Product

S.aureus

Expt 1/cm

Expt 2/cm

Expt 3/cm mean SD SEM UB LB significance

lawn 0 0 0 0 0 0 0 0

Sterile water 0 0 0 0 0 0 0 0

Ethanol 1 2.25 2.5 2.8 2.516667 0.275379 0.159 3.201 1.833

Ethanol 0.5 N/A 1.4 1.1 1.25 0.212132 0.15 1.895 0.605

Ethanol 0.2 N/A 0 0 0 0 0 0 0


Ethanol 0.1 N/A 0 0 0 0 0 0 0

Garlic 1 3.1 2.1 3.4 2.866667 0.680686 0.393 4.558 1.176 -0.826

Garlic 0.5 1.6 0 2.1 1.233333 1.096966 0.633 3.957 -1.491 0.025

Garlic 0.2 0 0 0 0 0 0 0 0

Garlic 0.1 0 0 0 0 0 0 0 0

CIP 1µg 1.9 3.1 3.2 2.733333 0.723418 0.418 4.532 0.934

FC 10µg 2.1 3.6 4 3.233333 1.001665 0.578 5.72 0.746

Chives 1 1.4 1.4 1.1 1.3 0.173205 0.1 1.73 0.87 3.919

Chives 0.5 0.8 0 0 0.266667 0.46188 0.267 1.416 -0.882 3.214

Chives 0.2 1.3 0 0 0.433333 0.750555 0.433 2.296 -1.43

Chives 0.1 0 0 0 0 0 0 0 0

Mushroom 1 1.6 1.5 2.2 1.766667 0.378594 0.219 2.709 0.825 2.775

Mushroom 0.5 1.8 0 1.1 0.966667 0.907377 0.524 3.222 -1.288 0.52

Mushroom 0.2 1 0 0 0.333333 0.57735 0.333 1.766 -1.1

Mushroom 0.1 0 0 0 0 0 0 0 0

Cherry 1 1.5 1.6 2.1 1.733333 0.321455 0.186 2.533 0.933 3.206

Cherry 0.5 0 0 1 0.333333 0.57735 0.333 1.766 -1.1 2.508

Cherry 0.2 1.4 0 0 0.466667 0.80829 0.467 2.477 -1.543

Cherry 0.1 1.1 0 0 0.366667 0.635085 0.367 2.377 -1.643

Vanilla 1 2.9 1.9 2 2.266667 0.550757 0.318 3.635 0.899 0.703

Vanilla 0.5 2.1 1.4 1.3 1.6 0.43589 0.252 2.684 0.516 -1.195

Vanilla 0.2 1.2 0 0 0.4 0.69282 0.4 2.121 -1.321

Vanilla 0.1 0 0 0 0 0 0 0 0

Orange cells (ethanol) are the comparison in the t test.

Purple cells are compounds with overlapping bounderies to ethanol, so will be tested for their

significance.

Green rows are significant to p<0.001 degree of accuracy.

Critical value- 4.303

Ethanol- E.coli


Ethanol- S.aureus

Garlic- E.coli

Garlic- S.aureus

Antibiotic- E.coli


Antibiotic- S.aureus

Chives- E.coli

Chives- S.aureus

Oyster Mushroom- E.coli


Oyster Mushroom- S.aureus

Cherry- E.coli

Cherry- S.aureus

Vanilla- E.coli


Vanilla- S.aureus

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