Investigating The Effect Of Coffee Solution Biology Essay

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Abstract

This experiment was designed to investigate the effect of coffee solution on the effectiveness of amoxicillin as an antibiotic acting against Staphylococcus aureus. The interaction between amoxicillin and coffee solution is studied using the minimum inhibitory concentrations (MIC) method. Amoxicillin solutions of varying concentrations were incubated in sterile test tubes with Staphylococcus aureus with coffee solution added as the manipulated variable. After 24 hours of incubation, the bacterial growth was measured by measuring the turbidity of the mixture using spectrophotometer. The minimum inhibitory concentration (MIC) was determined. The statistical t-test showed that the MIC for the antibiotic with coffee solution is significantly lower than that without coffee solution. The results support the hypothesis that coffee solution will increase the effectiveness of amoxicillin against Staphylococcus aureus as the lower the MIC, the more effective the antibiotic.

Research and rationale

The aim of this experience is to investigate the relationship between the presence of coffee solution and the effectiveness of amoxicillin against Staphylococcus aureus, a gram-positive bacterium that causes infections and diseases ranging from minor skin infections such as abcesses to critical diseases such as meningitis and toxic shock syndrome as well as a common cause of postsurgical wound infections. [15]

Staphylococcus aureus,infections are often treated with penicillins. [15] Penicillins in particular interfere with the chemicals required by the bacteria to synthesise bacterial cell walls. It works by disrupting the cross-linkages of the polymer that constitutes the bacterial cell wall of Gram-positive and Gram-negative bacteria. Penicillin is bactericidal antibiotics and is mostly broad spectrum antibiotic. [15] The antibiotic used in this investigation is amoxicillin which is semi-synthetic penicillin. It is used particularly in gastrointestinal infections and soft tissue infections caused by Staphylococcus aureus.

625px-Amoxicillin2.svg.png

Figure 1: Structure of amoxicillin [16]

Antibiotics taken orally are commonly consumed with beverages such as coffee and tea. Statistics showed that every year, 400 billion cups of coffee are consumed. [17]

It is believed that coffee has a profound effect on the effectiveness of antibiotics. This is primarily due to the components of coffee. Green coffee does not possess antimicrobial activity while roasted coffee show antimicrobial activity that depends on the degree of roasting.

Component

Arabica

Robusta

Green

Roasted

Green

Roasted

Minerals

3 - 4.2

3.5 - 4.5

4 - 4.5

4.6 - 5

Caffeine

0.9 - 1.2

~1

1.6 - 2.4

~2

Trigonelline

1 - 1.2

0.5 - 1

0.6 - 1.75

0.3 - 0.6

Lipids

12 - 18

14.5 - 20

9 - 13

11 - 16

Total Chlorogenic Acids

5.5 - 8

1.2 - 2.3

7 - 10

3.9 - 4.6

Aliphatic Acids

1.5 - 2

1 - 1.5

1.5 - 2

1 - 1.5

Oligosaccharides

6 - 8

0 - 3.5

5 - 7

0 - 3.5

Total polysaccharides

50 - 55

24 - 39

37 - 47

-

Amino Acids

2

0

2

0

Proteins

11 - 13

13 - 15

11 - 13

13 - 15

Table 1: A summary of compositional data (in %) for green and roasted arabica and robusta coffee beans [18]

Coffee's antimcrobial activity can be attributed to the alfa-dicarbonyl compounds and volatile compounds formed during roasting such as glyoxal, metylglyoxal and diacetyl and caffeine, even in small amount, is able to synergistically enhance dicarbonyl antimicrobial activity as well as that of chlorogenic acids and trigonelline.

Caffeine are methylxanthine drugs and is a powerful stimulant. A cup of coffee contains about 100 and 200mg of caffeine and accounts for almost 50% of coffee's antimicrobial properties. Trigonelline gives coffee the fragrance and bitter taste and has valuable antibacterial properties. [10] These compounds are believed to inhibit phosphodiesterase which potentiates the effect of antibiotics Action of antibiotics allows the compounds to diffuse into the bacterial cells and inhibit synthesis of DNA.

cinact83.jpg

Figure 2: The structures of caffeine, theophylline and theobromine[19]

Chlorogenic acid, an ester of caffeic acid and quinic acid, is a major phenolic compound in coffee; daily intake in coffee drinkers is 0.5-1 g. Chlorogenic acid has been proven in animal studies in vitro to inhibit the hydrolysis of the glucose-6-phosphate enzyme in an irreversible fashion and thus reducing the conversion of new glucose. [12] Besides that, the volatile compounds present in roasted coffee have been found to inhibit the growth of certain gram-positive and gram-negative bacteria. [12]

Chlorogenic acid-744161.JPG

Figure 3: Structure of Chlorogenic acid [20]

Therefore, this study will show that coffee can be taken with antibiotics without potentially reducing the effectiveness of antibiotics. In fact, coffee can significantly potentiate its antimicrobial properties. Not only that, Staphylococcus aureus is known to produce β-lactamase which will lead to development in resistance against penicillin antibiotics. [4] Coffee solution will hopefully potentiate the antibiotics's antimicrobial effects and prevent the development of resistance. Further research can be done to investigate if constituents of coffee can be incorporated directly into antibiotic and its other component that is administered to patients.

Experimental hypothesis

There is a significant difference between the effectiveness of amoxicillin with coffee solution and that without coffee solution. The amoxicillin with coffee solution is more effective in its antimicrobial properties to inhibit the growth of Staphylococcus aureus compared to that without.

Null hypothesis

There is no significant difference between the effectiveness of amoxicillin with coffee solution and that without coffee solution.

Variables

Manipulated variable : Presence of coffee solution

Responding variable : Minimum inhibitory concentration of amoxicillin

Fixed variables : Temperature of incubation, amount of nutrient broth, type and amount of bacteria cultured,

Apparatus

250 ml beaker, sterilised spatula, electronic weighing balance, 100 ml measuring cylinder, label stickers, 100µl-1000µl micropipette, micropipette tips, Bunsen burner, sterile test tubes, incubator ( 30°C), spectrophotometer (wavelength 600nm), cuvette, glass rod, graduated syringe, sterile cotton wool

Materials

Ground coffee (coffea canephora) powder, 500 mg amoxicillin trihydrate capsule purchased from Chemical Company of Malaysia (CCM), nutrient broth culture of Staphylococcus aureus, nutrient broth, sterilised distilled water, 70% ethanol, antiseptic solution (Dettol),

PLANNING

Trials

A trial experiment was carried out prior to the actual experiment to determine the most suitable method to carry out the investigation. Four antimicrobial susceptibility testing methods were tested in the trial run - disk diffusion, well diffusion, minimum inhibitory concentration and bacterial colony counting. Well diffusion involves administering a fixed amount of antibiotic solution into a well made using cork borer in the agar while disk diffusion involves applying paper disks soaked in antibiotic solutions on the agar. Bacterial colony counting involves spreading the bacteria over a wide area around the nutrient agar plate and counting the number of colonies that grow. Minimum inhibitory concentration is the highest dilution of antibiotic solution at which it inhibits the growth of bacteria. The growth of bacteria is determined using a spectrophotometer. All the bacterial cultures were incubated at 37°C. It can be seen that determining the MIC has a distinct difference that can be clearly seen.

Methods of determining effect of coffee solution

With coffee solution

Without coffee solution

Disk diffusion

20.0 mm

17.0 mm

Direct mixing of coffee solution with antibiotic solution and bacterial colony counting

No bacterial colony

1 bacterial colony of 2.0mm

Well diffusion

23.0mm

17.0mm

Minimum inhibitory concentration (MIC)

0.5

2.5

Table 2: Results for the different methods of determining the effect of coffee solution

The different concentrations of the coffee solution that will affect the effectiveness of the antibiotics were determined. Two concentrations were used - 5mg/ml and 10mg/ml. The two different concentrations of coffee solution were prepared using 1 g of ground coffee powder in 100 ml of water for 10mg/ml and 0.5 g of ground coffee powder for 5mg/ml. It was shown that 10mg/ml of coffee solution has a marked increase in effectiveness of amoxicillin.

Concentration of coffee solution used

(mg/ml)

Minimum inhibitory concentration

(MIC) (mg/ml)

5

1.5

10

0.5

Table 3: The minimum inhibitory concentration with respect to the different concentrations of coffee solution used.

The minimum inhibitory concentration of different antibiotics against Staphylococcus aureus was also determined. The effectiveness of the antibiotics in the presence and absence of coffee extract may vary. Doxycycline, amoxicillin and ampicillin were used in this trial to determine the antibiotic which will show the most significant difference in its effectiveness in the presence and absence of coffee extract. It was shown that the MIC for amoxicillin showed the most significant difference and has its MIC within the range of concentrations of antibiotics.

Type of antibiotic used

Minimum inhibitory concentration

(MIC) (mg/ml)

With coffee solution

Without coffee solution

Doxycycline

0.5

0.5

Amoxicillin

0.5

2.0

Ampicillin

0.5

0.5

Table 4: The minimum inhibitory concentration in the presence and absence of coffee solution with respect to the type of antibiotic used.

Actual experimental procedure:

Preparing coffee solution

100 ml of distilled water was measured using a 100 ml measuring cylinder.

1.0 g of coffee powder was measured using an electronic balance.

The 1.0 g of coffee powder was dissolved in 100ml of distilled water in a sterilised 250ml beaker to produce 10 mg/ml of coffee solution. A glass rod was used to stir the mixture.

The mixture was then filtered to remove any residue.

Preparing antibiotic solution

100 ml of distilled water was measured using a 100ml measuring cylinder.

500 mg of amoxicillin trihydrate was measured using an electronic balance.

The 500 mg of amoxicillin powder was dissolved in 100ml of distilled water in a 250ml beaker to produce 5 mg/ml of antibiotic solution. A glass rod was used to stir the mixture.

The mixture was then filtered to remove any residue.

Steps 1 to 4 were repeated by replacing 500mg with 450, 400, 350, 300, 250, 200, 150, 100 and 50mg of amoxicillin trihydrate powder to prepare amoxicillin solutions of concentrations 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0 and 0.5mg/ml. The beakers were labeled with stickers according to concentrations respectively.

Preparations to measure bacterial growth

10 sterile test tubes were labeled to indicate the different concentrations of amoxicillin solution.

4ml of sterile nutrient broth was measured using a graduated syringe and dispensed into a sterile test tube.

600µl of 5mg/ml amoxicillin solution was measured using a micropipette and added to the nutrient broth.

400µl of Staphylococcus aureus inoculums was also measured using a micropipette and added to the mixture. The mixture was vortex.

Repeat steps 2 to 4 by replacing 5mg/ml of amoxicillin solution with concentrations of 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0 and 0.5mg/ml.

Steps 1 to 5 were repeated with addition of coffee solution. 200µl of 10mg/ml of coffee solution is added to each of the 10 sterile test tubes. The 10 sterile test tubes were labeled to indicate the presence of coffee solution.

Another set was prepared in exactly the same way using only nutrient broth and antibiotic solution to act as a control.

A test tube was used to only incubate nutrient broth and Staphylococcus aureus to be used as a standard to determine complete inhibition as the turbidity of bacterial growth may be faint.

Sterile cotton wool was used to stopper the mouth of the test tubes.

The test tubes were kept in the incubator at 30°C for 24 hours.

After 24 hours, test tubes were removed from incubator.

The test tubes were compared with the standard bacterial culture as primary identification of the MIC.

Determination of turbidity to measure bacterial growth using spectrophotometer

The spectrophotometer was preset to a wavelength of 600nm.

2 ml of sterile nutrient broth was dispensed into cuvette using a micropipette. The cuvette was then inserted into the cuvette chamber and the cover is closed.

The machine is calibrated by adjusting the absorbance to read zero (zero absorbance indicates maximum transmission). Cuvette was then removed.

2 ml of the 5mg/ml bacteria-antibiotic mixture without caffeine was dispensed into a new cuvette using a micropipette. It was placed in the cuvette chamber and the cover was closed. The reading was read and recorded.

Steps 2 to 4 were repeated by replacing 5mg/ml bacteria-antibiotic mixture with the other concentrations without caffeine.

Step 5 was repeated by replacing bacteria-antibiotic mixture without caffeine with those containing caffeine. Step 2 was repeated by calibrating with a mixture of nutrient broth and coffee solution with equal proportions as that in the bacteria-antibiotic mixture. (at a ratio of 1/20)

The absorbance readings were noted and recorded.

Graphs of absorbance readings against concentration of amoxicillin solution used in the presence and absence of coffee solution were plotted.

The Minimum Inhibitory Concentration (MIC) of amoxicillin in the presence and absence of coffee solution can be determined accurately from the graph.

Risk Assessment

Aseptic techniques were adopted in this investigation. Hands were washed with antibacterial hand wash and the working area was sprayed thoroughly with bench spray of 70% alcohol to ensure no bacteria were present. A Bunsen burner was lit and placed near the workplace to ensure surrounding bacteria does not affect the experiment. Gloves were worn to prevent direct contact and contamination with bacteria as Staphylococcus aureus may cause skin infections and food poisoning while carrying out the experiment. [14] Sterile apparatus such as capped test tubes, cuvettes and beakers were used to prevent growth of other bacteria that might affect the results. The mouths of the nutrient broth flask and McCartney bottle containing culture of Staphylococcus aureus were flamed to prevent entrance of other microorganisms that might affect the results. The teats of the micropipettes were disposed off in a biohazard disposal container containing disinfectant solution to avoid spreading of bacteria. Surgical masks were also worn while transferring bacteria-antibiotic mixture into cuvette as the mouth of the cuvettes are open to avoid bacterial infections. The open cuvettes were handled carefully to prevent spillage. The cuvettes were then sent for autoclaving before disposal. Bacteria-antibiotic mixtures were immediately disposed to prevent the development of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA).

Results

Set

Minimum Inhibitory Concentration (MIC) (mg/ml)

With coffee solution

Without coffee solution

A

1.0

2.5

B

1.0

2.0

C

1.5

2.5

D

1.5

2.5

E

1.0

1.5

Mean

1.2

2.3

Table 5: Minimum inhibitory concentrations in the presence and absence of coffee solution

Example of Spectrophotometer Reading (Set A)

Concentration of amoxicillin solution (mg/ml)

Spectrophotometer reading (Absorbance)

With coffee solution

Without coffee solution

5.0

0.040

0.060

4.5

0.056

0.065

4.0

0.059

0.073

3.5

0.049

0.062

3.0

0.057

0.074

2.5

0.043

0.076

2.0

0.064

0.158

1.5

0.063

0.170

1.0

0.070

0.175

0.5

0.171

0.188

Table 6: Spectrophotometer reading indicating the absorbance in the presence and absence of coffee solution with respect to the different concentrations of amoxicillin.

Figure 4: Bar chart of minimum inhibitory concentration of amoxicillin with respect to presence of coffee solution

Figure 5: Line graph of absorbance against concentration of amoxicillin concentration in the presence and absence of coffee solution to determine its respective minimum inhibitory concentrations (MIC)

Statistical Analysis

The minimum inhibitory concentration of amoxicillin in the presence of coffee solution is significantly lower than that without coffee solution. The calculated t-value (4.59) shows that is it significant whereby it exceeds the tabulated t-value (critical value) which is 2.306. (Level of significance, p <0.05, degree of freedom = 8). The calculated value is above the highest value from the table which shows that it is more than 99% significant. Therefore, the experimental hypothesis is accepted and the null hypothesis is rejected.

Formulae

With coffee solution

Without coffee solution

6.0

11.5

n

5

5

xÌ„ = / n

1.2

2.3

Σd2=Σ(x- xÌ„)2

0.30

0.85

s2 =

0.075

0.2125

t =

t =

= 4.59

Where = mean of sample 1 (with coffee solution)

= mean of sample 2 (without coffee solution)

n1 = number of subjects in sample 1

n2 = number of subjects in sample 2

S12 = variance of sample 1

S22 = variance of sample 2

Data Analysis

The minimum inhibitory concentration of amoxicillin in the presence of coffee solution is 0.5mg/ml lower than that without coffee solution. This shows that coffee solution shows a profound improvement in the effectiveness of amoxicillin against Staphylococcus aureus as there is about 21.7% difference between both sets of data. The error bars for that with coffee solution does not overlap with the errors bars for that without coffee solution. Therefore, it can be inferred that the two mean MIC values have significant difference.

The minimum inhibitory concentration (MIC) method is the basic laboratory measurement that involves measuring the lowest dilution of antibiotic that will inhibit the visible growth of bacteria after incubation. This is judged by the turbidity of the solution incubated. In this experiment, Staphylococcus aureus was incubated with amoxicillin solution of varying concentrations in the presence and absence of coffee solution. The different components (bacterial culture, respective concentrations of antibiotics and coffee solution) are able to interact with each other in the nutrient broth whereby the synergistic effect of respective antibiotic solutions and coffee solution in inhibiting bacterial growth could be investigated. The MIC was determined first by comparing the turbidity of the mixtures with the standard bacterial culture using the naked eye. It was then later substantiated by measuring the turbidity using spectrophotometer as indication of the bacterial growth.

The MIC is the concentration at which there is a big jump in absorbance in the graph of absorbance against concentration of amoxicillin concentration. As can be seen from the graph, there are slight fluctuations at higher concentrations and at the concentration after the MIC, there was an increase in absorbance by almost 144% for that with coffee solution and by almost 108% for that without coffee solution. The big jump is due to the increase in turbidity which indicates bacterial growth. A higher turbidity is shown by a higher absorbance which indicates bacterial growth. The MIC is the concentration before the big jump which is the maximum dilution of antibiotic solution that will inhibit bacterial growth. Thus, the MIC can be determined accurately. A control was also used and showed no bacterial growth.

The results obtained from my investigation was compared with the investigation published in the June 2008 Tropical Journal of Pharmaceutical Research conducted by the University of Benin, Nigeria. The data obtained are very similar to that obtained from my investigation. In this investigation, 5mg/ml and 10mg/ml of caffeine was used to investigate the difference in effect due to the different concentrations. It is evident that 10mg/ml of caffeine which is the higher concentration has a more profound positive effect.

Antibiotic alone

Antibiotic + 5 mg/ml

caffeine

Antibiotic + 10 mg/ml

caffeine

Amoxicillin

2.0178

0.0910

0.0792

Ampicillin

0.0355

0.0060

0.0019

Benzyl penicillin

1.9002

112.61

75.514

Table 7: MIC (mg/ml ± SEM) of the antibiotics against Staphylococcus aureus [4]

Amoxicillin is semi-synthetic penicillin which is a broad-spectrum antibiotic which acts directly on the cell-wall synthesis of bacteria. [1, 16] It works on a variety of bacteria. Amoxicillin might cause the lyses of the cell wall as cell wall formation is disrupted. This will enable coffee extract such as caffeine and chlorogenic acid to diffuse into the bacterial cell thus acting directly on cell function. Components of coffee are able to hinder DNA synthesis and disrupt the bases in the DNA causing protein synthesis and thus cell function to be interrupted. [6] This is mechanism of how coffee can boost the effectiveness of antibiotics acting against Staphylococcus aureus which is a Gram-positive bacterium.

Evaluation

The antibiotic-bacteria mixture was incubated at 37°C and for only 24 hours as rate of growth of Staphylococcus aureus is very rapid. The test tubes were prepared in the shortest time possible and all the apparatus and materials used were kept possibly sterile to prevent contamination by microorganisms in the surroundings. There will be slight or minute amounts of airborne microorganisms and bacteria present in the apparatus and workplace. There might be small amounts present in the hands of those conducting the experiment. This cannot be avoided as no disinfectant can kill all the microorganisms present in the surroundings.This may affect the results. The experiment was also repeated 4 times to have 5 samples to detect any anomalies and decrease the possibility of inconsistent data and inaccuracies due to human error.

There could be limitations due to the coffee and antibiotic not dissolving fully. The coffee and antibiotic solutions were stirred thoroughly to ensure that most of it dissolves. It was then filtered to remove the impurities or other insoluble compounds present in it as it will affect the reading of the spectrophotometer.

The determination of the MIC might not be very distinct as turbidity can be faint. Therefore, it is first compared with a standard to determine complete inhibition and that of MIC. After that, a spectrophotometer was used to verify it by measuring the absorbance which indicates turbidity and bacterial growth. It was calibrated with nutrient broth as standard. However, for the set with coffee solution, it was calibrated with nutrient broth and coffee solution as the colour of the coffee solution may increase the absorbance. This is done to prevent any systematic errors due to the coffee solution.

The results of the experiment can be improved further by extracting the components of coffee such as caffeine, theophylline and chlorogenic acid and testing it directly on the effectiveness of amoxicillin to demonstrate optimum effect. Coffee solution may be impure and the action of compounds present may not be as expected. Pure extracts of the compounds can act directly chemically on the semi-synthetic amoxicillin which is quite structurally similar to ampicillin.

Conclusion

The amoxicillin with coffee solution is more effective in its antimicrobial properties to inhibit the growth of Staphylococcus aureus compared to that without. This was shown by the lower MIC with coffee solution compared to that without coffee solution.

Source Evaluation

Source 1, 2 and 3 are reliable books which have been published and have been subjected to review by credible experts.

Source 4, 5, 6 and 7 are journals which have been published in reliable publications. They are mostly written by well-known university professors and experts at their respective fields with high level of scientific knowledge. All these journals must have undergone reliable intensive review.

Sources 9 is obtained from a manual on microbiology techniques used in the laboratory published by an expert who is a university professor so it should be substantiated by concrete scientific background.

Source 11 and 17 is the national association dedicated to coffee and features coffee consumption research. There must have been extensive research done on this field of study.

Source 16(Wikipedia) is a web-based encyclopaedia which has a wide range of collections of information constantly updated. It is known for its reliability in providing up-to-date information and can be regarded as a credible source.

Source 10, 13, 14, 15, 18, 19, 20 are information sites which features collections of articles on scientific and medical issues. They must have been peer-reviewed by the science community and have scientifically-backed knowledge.

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