Efficacy of turmeric as a preservative on tomatoes

How does the ability of turmeric to act as a preservative for tomatoes (solanum lycopersicum), measured in terms of mass loss, pH and lycopene content depends on the percentage concentration (mass by volume) of the turmeric solution used and the type of tomato used-green tomato and red tomato ?

Use of turmeric as a preservative is a common practice in Indian kitchen. Turmeric has always been a common ingredient in almost all Indian recipes. As claimed by chefs and nutritionists, it is so because of the antibiotic and antiallergic therapeutic effects of turmeric. Being an inquisitive learner, the observation that made my interest about this more innate was the fact that I have seen applying turmeric and salt on fish and meats before they are refrigerated. I was more surprised to know that it is done to preserve the meat and fish for a longer time. I went through some research papers and came to know more about it and the biology behind it which will be explored further in the next section. However, the question that striked me was, will turmeric act as a potential preservative for vegetables as well ? Does the efficacy of a preservative depends on the type of the food substance preserved? In the current scenario of e-commerce, selling of packaged and processed foods contributes largely to the economy. To ensure health and safety, use of safe preservatives and their shelf life has become a major issue in food technology and preservation science genre. This finally brought me to the research question stated above.

Turmeric is a common tropical crop in Asia and Sub-Saharan regions(Shelef et al.). It has various medicinal as well as nutritional benefits. It belongs to the genus- “Curcuma longa” (Yarru et al.). The main ingredient of turmeric is curcumin which can acts as anti-oxidant. Moreover, it also shows abilities to produce vitamins through reductive degradation mechanism and can also acts as a precursor or co-factor of various other enzymes that are involved in the biological reactions happening during ripening of fruits and vegetables(Buch et al.). Curcumin can also degrades to form proteins and polyphenolic compounds which finally converts them to long chain fatty acids and can inhibit various diseases in plants due to deficiency of micro and macro nutrients (Almeida-Doria and Regitano-D’Arce).

Tomatoes of “solanum lycopersicum” is generally claimed to be a perennially grown vegetable albeit botanically it belongs to the class of berry fruit (Singh et al.). It is one of the most popular crop consumed worldwide which started it journey from South America and was then exported to Europe around the 15th century(OECD ). The main constituent of tomato is lycopene which has immense nutritional and medicinal benefits and is a pigment that offers the red colour to the fruit. Apart from this, tomato is also a major source of vitamins like – Vitamin B1 and Vitamin B3(Solanum Lycopersicum, Tomato: Life Cycle, Flower and Fruit Anatomy at GeoChemBio). The major part of tomato is carbohydrate and because of that it is a good source of calorie without unwanted fats for the dieticians (Stevens et al.)

Food preservation is a technique which is used to inhibit or delay the biochemical reactions in a fruit which may lead to the loss of nutritional benefits of the fruit accompanied by loss of texture, colour, aroma and turning rancid which eventually makes the fruit unsuitable for usage either in raw form or for cooking. There are multiple methods approved by the USDA (United States Department of Agriculture) for food preservation(“The Science Behind Food Preservation”). All of these or most of these methods aims to stop the growth of microorganisms and harmful bacteria in the fruits either by interfering with the conditions that are required for their growth or by adding chemicals or foreign substances named as preservatives which may kill the bacteria grown or inhibit their growth. The most popular methods of preservations are(“Science behind Food Preservation Methods”) - adding preservatives (chemical or natural), dehydrating (making the fruit stored in a moisture free condition), atmospheric canning (storing the fruit in a chamber or closed space filled with liquid nitrogen under high pressure), adding salts, adding acids to lower down the pH level that inhibits bacterial growth. In this investigation, use of turmeric a natural and homely method of preserving fruits will be discussed and its efficacy will be investigated.

There are various analytical methods to monitor and assess the quality of tomatoes:

• Mass loss
  The dry mass of a vegetable or fruit is an indicator of its nutritional well-being and biological conditions. It is the most accurate measurement of the biomass of the sample. It measures the amount of matter accumulated within the species. Decrease in biomass indicates a decline in the biological content of the sample. Thus, to determine the best possible preservative, it is required to keep all the species in the preservative solutions and measure the dry mass. The case that gives the least decrease in dry mass must be considered as the most effective preservative. The ideal dry mass of a tomato ranges from 110 g to 90 g (H. et al.)
• pH
  pH is a measurement of the acidity or alkalinity of the species. pH is an analytical tool to measure the maturity and ripeness of tomatoes. Ideally, tomatoes are found to have a pH in between 4.2 to 4.6 (Determining Tomato Maturity by Measuring PH). As the fruit ripens or loses its nutritional value, the pH level decreases. Thus, more the decrease in the values of pH from the ideal value, lower the efficacy of the indicator that has been used.
• Lycopene content
  Lycopene is a long chain aromatic compound which can absorb electromagnetic radiations in the visible spectrum and thus imparts red colour to the tomatoes. This is the major nutritional ingredient of the tomatoes. Thus, as the quantity of lycopene decreases, the tomato becomes less suitable for consumption. Using Beer-Lambert law (Chemat-Djenni et al.) the concentration of lycopene can be monitored by measuring it’s absorbance at the wavelength at which it displays maximum absorbance. A UV-Visible spectrum of lycopene shows a peak at 667 nm indicating that lycopene absorbs the maximum amount of radiation in the visible region at 667 nm (Friedman et al.). Absorbance is a measure of the concentration of the sample. Thus, more the value of absorbance at 667 nm as indicated in a colorimeter, more the concentration of lycopene. As lycopene is a nutritional ingredient, decrease in the values of absorbance will indicate a decrease in the quantity of it. So, more the decrease of the value of absorbance of lycopene, lower the efficacy of the preservative the tomatoes were exposed to.

Null hypotheses (Ho): There is no correlation between the percentage concentration of the turmeric solution used and it’s efficacy as a preservative for tomatoes – green and red. Any correlation, if identified is an outcome of a methodological or a systematic error.

 

Alternate hypotheses (H1): There is a positive or negative correlation between the percentage concentration of the turmeric solution used and it’s efficacy as a preservative for tomatoes – green and red. Any correlation, if identified is an outcome of a methodological or a systematic error.

There are two independent variable in the investigation –

• percentage concentration (mass by volume) of the turmeric solution used as a preservative the genus of the tomato – green and red.

The percentage concentration has been chosen to understand if using more amount of turmeric increases the efficacy or not and thus determining the optimum concentration of turmeric solution that can be used. The percentage concentration was varied from 0.00% (control), 5.00%, 10.00%, 15.00%, 20.00% and 25.00%. The values of concentration are expressed as mass by volume. For example, a 5.00% solution of turmeric means 5.00 ± 0.01 g of turmeric was added to 100 cm³ of water. To understand if the effect of turmeric as a preservative depends on the genus or biological variety of the sample or not, two different types of tomatoes-green and red were chosen.

The dependent variable of this investigation is the efficacy of the turmeric solution as an indicator.

 

The efficacy of the indicator will be measured in terms of three quantitative aspects – pH, dry mass loss and lycopene content. Refer to the background information for more details. A pH probe will be used to measure the pH, a digital mass balance will be used to record the mass and a colorimeter will be used to record the absorbance while measuring the amount of lycopene colorimetricaly.

• A laboratory coat and safety gloves were used.
• Social distancing protocols were maintained.
• The solutions were made under the supervision of an expert technician.
• The instruments were handled with care.
• The work station was kept clean and organized.

All unused and waste materials were thrown into the disposal bin and disposed using standard safe procedures.

None of the materials were wasted and minimum possible amount of materials were used. Any toxic or prohibited materials were not used.

The experimental procedure has been self- procured by the experimenter in conjunction with references from laboratory manuals for “measuring pH using pH meters” (What Is a PH Meter? Introduction to Digital PH Meters), “making lycopene extract” (Chiu et al.) and “Using colorimeter in food industry” (Huang et al.)

 

Stage-1 - Preparing the turmeric solution

500.00 ± 0.01 g of turmeric was kept on a watch glass on a top pan digital mass balance and weighed using a spatula. The weighed turmeric was transferred into a clean and dry mixer grinder. The grinder was operated to obtain a fine powder of the turmeric. The powdered turmeric was kept in a 100 cm³ glass beaker.

 

For a 5.00% solution, 5.00 g of turmeric should be added to 100 cm³ of water.

 

Mass of turmeric powder for 500 cm³ of 5.00% solution = \(\frac{5.00}{100} × 500 = 25.00 \, \,g\)

 

25.00 ± 0.01 g of the turmeric powder was weighed on a top pan digital mass balance and using a watch glass and a spatula. The weighed turmeric was completely transferred to a 500 cm³ glass beaker. The beaker was filled with drinking water till the mark of 500 cm³ . A glass rod was used to stir the solution and dissolve the turmeric. Thus, a 5.00 % solution of turmeric powder was made.

 

The other solutions – 10.00%, 15.00%, 20.00% and 25.00% were made in the same way. For the control – 0.00%, drinking water was used and no turmeric was added.

 

Stage-2: Preserving the tomatoes in the turmeric solution and measuring pH and lycopene absorbance:

•  A 50 cm³ glass beaker was taken and labelled as – 5.00% Trial-1.
•  A clean and dry red tomato was taken and the mass of the tomato was recorded using a digital mass balance.
•  The tomato was kept inside the beaker.
• The beaker was filled with 5.00% turmeric solution till the mark of 50.00 cm³ .
•  After 3 days, the tomato was taken out of the beaker and allowed to dry in open air.
•  The mass of the dried tomato was recorded using a digital mass balance and a watch glass.
•  The tomato was cut into four equal pieces using a knife.
•  The cut pieces of tomato was transferred to a mixer grinder and the grinder was operated for a while until a smooth and uniform paste was obtained.
•  The tomato paste was then transferred to a 100 cm³ glass beaker.
•  The vessel of the grinder used to make the paste was washed with distilled water and the washings were collected in the same beaker to transfer the tomato paste completely.
•  50 cm3 of ethanol was added to the beaker followed by addition of 50cm³ of distilled water. A graduated measuring cylinder was used for this.
•  The beaker was placed on a heating mantle and the temperature was kept constant at 60.0^oC.
•  A thermometer was used to check the temperature of the content of the beaker and heating was continued until a temperature of 60.0^oC is obtained.
•  The content of the beaker was then filtered using a funnel and a filter paper. The residue was discarded and the filtrate was collected in a conical flask. This filtrate is the ethanolic-aqueous extract of tomato.
•  5.00 ± 0.05 cm³ of the filtrate was then transferred to a test tube.
•  The pH probe was taken and calibrated using a buffer solution of pH 10.00
•  The pH probe was inserted into the test tube and the pH of the ethanolic aqueous extract of the tomato was measured.
•  A digital photo colorimeter was taken and the wavelength was set at 667 nm.
•  A cuvette was taken and filled with a mixture of 1.00 ± 0.05 cm³ of ethanol and 1.00 ± 0.05 cm³ of distilled water.
• The absorbance of this mixture of ethanol and water was set at ±0.000 AU to calibrate the photo- colorimeter.
•  A clean and dry cuvette was taken and filled with the ethanolic aqueous extract of the tomato.
•  The absorbance of the solution was measured.
•  Steps 1-4 were repeated for two more times to collect data in triplicates.

The same process was repeated for all other % concentration of turmeric solutions – 0.00%, 5.00%, 10.00%, 15.00%, 20.00% and 25.00%. The same process was repeated for samples of green tomatoes.

• A change in the colour of the skin of the tomato was observed when they were kept immersed in the turmeric solutions. The colour of the red tomatoes started fading with the progress of time from darker shades of red to lighter shades. However, as the percentage concentration of turmeric solution was more, the fading of colour was less significant.
• For green tomatoes, it was observed that the colour of the skin started becoming greenish red from green with the progress of time. As the percentage concentration of turmeric increased, the change of colour was more significant.

Key

• Initial mass of the tomato (measured before putting it in turmeric solution) = m₁
• Final mass of the tomato (measured after taking it out from turmeric solution and drying) = m₂

Sample calculation

 

For Row-1: (0.00% turmeric solution)

 

For Trial-1, Change in mass (m) = Initial mass (m₁) – Final mass (m₂)

 

= 95.30 ± 0.01 g – 91.65 ± 0.01 g = (95.30-91.65) ± (0.01 + 0.01) g = 3.65 ± 0.02 g

 

Average change in mass (m) =\(\frac{(3.65±0.01)+(5.48±0.01)+(4.34±0.01)}{3}= 4.49 ± 0.01 \, \,g\)

 

Standard deviation (SD) = \(\frac{(4.49-3.65)^2+(4.49-5.48)^2+(4.49-4.34)^2}{3} = 0.92\)

Key

• Initial mass of the tomato (measured before putting it in turmeric solution) = m₁
• Final mass of the tomato (measured after taking it out from turmeric solution and drying) = m₂

For 0.00% turmeric solution (control)

 

Change in mass (m) = 4.49 ± 0.01 g

 

Percentage error in change in mass = \(\frac{0.01}{4.49} × 100 = 0.22\)

 

pH = 4.13 ± 0.01

 

Percentage error in pH = \(\frac{0.01}{4.13} × 100 = 0.03\)

 

Absorbance of lycopene at 667 nm = 0.663 ± 0.001 AU

 

Percentage error in absorbance of lycopene at 667 nm \(=\frac{0.001}{0.663}× 100 = 0.15\)

 

Total percentage error for efficacy of turmeric as a preservative at 0.00 % (control) = 0.22 + 0.03 + 0.15 = 0.40

 

As the magnitude of percentage error is really less significant, it can be claimed that the results are accurate and precise enough.

The scatter graph above indicates the patterns in the decrease of mass for the red and the green tomato against the various values of percentage concentration of turmeric solution used.

• For both the red and the green tomato, the maximum decrease in mass is found to be maximum for the control , 0.00 % turmeric solution, 5.22 ± 0.02 g for green tomato and 4.49 ± 0.02 g for red tomato. This indicates that the use of turmeric brings down the loss of mass for both the samples of tomato. This confirms that turmeric displays preservative effect.
• For both cases, it has been observed that the values of decrease in mass decreases from 4.49 ± 0.02 g to 0.89 ± 0.02 g for red tomato and 5.22 ± 0.02 g to 1.36 ± 0.02 g for green tomato. Moreover, as indicated in the linear equation of trend -line, the gradient are -0.1426 and -0.1747 for the green and red tomato respectively. These two facts confirms that as the percentage concentration of turmeric increases, the loss of mass decreases. This allows us to claims that more the concentration of turmeric, higher is the efficacy of turmeric as a preservative.
• The patterns of reduction in the magnitude of decrease of mass is same for both the green and red tomato. Albeit, the gradient is higher for green tomato (-0.1747) and thus it can be indicated that turmeric is comparatively found to show more efficient preservative action for green tomato than red tomato.

As shown in Graph-2, the pH values of both red and green tomato ranges from 4.20 to 4.60 which is the ideal range of pH for all values of percentage concentration of turmeric solution. In both cases, the value for the control (0.00 %) is lower than the ideal range, 4.10 for red tomato and 4.13 for green tomato. Thus, it can be claimed that turmeric can act as a preservative and helps tomato to maintain the pH in the ideal range. However, the maximum value of pH is obtained for the percentage concentration of turmeric at 11.00% as indicated in the peak of the curve shown in the graph above. The maximum value of pH is around 4.63 which is slightly more than the ideal value.

The above graph depicts the variation of the lycopene content in red and green tomato against the percentage concentration of turmeric solution.

• For green tomato, the absorbance increases from 0.336 ± 0.001 AU to 0.714 ± 0.001 AU and for red tomato, it increases from 0.663 ± 0.001 AU to 0.995 ± 0.001 AU. This confirms that with the increase in the percentage concentration of turmeric solution, the lycopene content of both the red and green tomato increases resulting in increase in the absorbance of lycopene at 667 nm. Thus, it can be claimed that turmeric or preferably curcumin favours the production of lycopene in tomatoes.
• For both red and green tomatoes, the values at 5.00% turmeric solution and more concentrated solution is higher than the control (0.00%). This again confirms that turmeric helps in the bio-synthesis of lycopene pigment in tomatoes.
• The linear equation of trend line has been displayed in the graph and in both cases the gradient are positive (0.0129 for red tomato and 0.0156 for green tomato). This shows that the turmeric is favourable for the increase in the enzymatic pathway leading to the synthesis of lycopene.

The analysis above clearly shows that with the increase in the percentage concentration of turmeric, the loss in mass of tomatoes decreases, the content of lycopene increases and the pH of the tomatoes lies within the ideal range of 4.2 to 4.6 mostly barring few slight or insignificant variations. This can be justified by two scientific facts:

Turmeric contains curcumin. Curcumin from the turmeric solutions can penetrate into the cells of the tomato through diffusion. Lycopene is produced in a melavonate pathway which involves six stages of enzymatic pathway (Cabral). Curcumin acts as a precursor and maximizes the synthesis of Acetyl-CoA which is involved in this enzymatic pathway of biosynthesis of lycopene (Schwartz et al.). Thus, the addition of curcumin enhances the production of lycopene. This is in accordance with the data displayed in Table-4, as the percentage concentration of turmeric increases, the amount of curcumin penetrating into the cells of tomato is more, more co-factors is released and thus more lycopene is produced which finally increases the absorbance value.

 

Curcumin also shows anti-carcinogenic activities by killing pathogens and microbes. As indicated in a research based on antibacterial action of curcumin against S.aureus, “ Curcumin may bind into FtsZ proteins, thereby inhibiting the assembly of FtsZ protofilaments. This, in turn, suppresses the formation of Z-ring leading to inhibition of cytokinesis and bacterial proliferation” (Teow et al.). This shows how curcumin can delay the pathways through which the biological content of tomato can decompose and thus make it loss it’s biomass. This fact supports the data collected in Table-1 and Table-2, where it is clearly evident that the values of decrease in mass is reducing as the percentage concentration of turmeric increases.

From Graph-1, the value of regression coefficient is 0.95 for red tomato and 0.99 for green tomato which indicates that there is a strong negative correlation (negative as the value of gradient is negative) between the change in mass of tomato and the percentage concentration of turmeric solution used as a preservative. From Graph-3, the values of regression coefficient is 0.97 for both the red and green tomato confirming a strong positive correlation (positive as the magnitude of gradient is positive) between the absorbance of lycopene at 667 nm (an indicator of the lycopene content) and the percentage concentration of the turmeric solution as a preservative. Both of these factors allow us to confirm that there is a correlation between the percentage concentration of turmeric solution and it’s efficacy as a preservative for tomato. However, in the case of pH any such correlation is not significant. Considering that a correlation is observed in two cases – absorbance and mass loss and not in one-pH, the null hypotheses is rejected and the alternate hypotheses has been accepted.

This section aims to explore if there is any significant difference between the efficacy of turmeric as a preservative for red tomato and green tomato. As here the data has been collected for two different categories – red and green and the categories are mutually independent of each other, a T-test of independence will be done.

 

Though three different variables- mass loss, pH and absorbance of lycopene has been chosen to measure the efficacy of turmeric as a preservative, the T-test (“T Test (Student’s T-Test)”) will be conducted on data for change of mass and absorbance of lycopene at 667 nm as in case of pH any significant correlation between the percentage concentration of turmeric and the pH was not observed (refer to Graph-2).

Null hypotheses(H_o) The change in mass against % concentration of turmeric in red and green tomato are independent of each other.

 

Alternate hypotheses(H₁) The change in mass against % concentration of turmeric in red and green tomato are not independent of each other.

 

Significance level (∝) = 0.05

 

Degrees of freedom (N) = 6 - 1 =5

 

Calculated t value \(\)\(\frac{\frac{\sum(X-Y)}{N}}{\sqrt{\frac{\sum(X-Y)^2-\frac{(\sum(X-Y))^2}{N}}{(N-1)N}}} = −1.09\)

 

According to the T-Table, the value of t at degrees of freedom = 5 is 2.015 (“T Test (Student’s T-Test)”) As the calculated value (- 1.09, negative sign being ignored) is less than the calculated value, the null hypotheses is accepted and the alternate hypotheses is rejected. This means that the efficacy of turmeric as a preservative for tomatoes when measured in terms of mass loss does not depend on the genetic variety of the tomato used.

Null hypotheses(H_o) The absorbance of lycopene at 667 nm against % concentration of turmeric in red and green tomato are independent of each other.

 

Alternate hypotheses(H₁): The absorbance of lycopene at 667 nm agaisnt % concentration of turmeric in red and green tomato are not independent of each other.

 

Significance level (∝) = 0.05

 

Degrees of freedom (N) = 6 - 1 =5

 

Calculated t value = \(\frac{\frac{\sum(X-Y)}{N}}{\sqrt{\frac{\sum(X-Y)^2-\frac{(\sum(X-Y))^2}{N}}{(N-1)N}}} = 2.763\)

 

According to the T-Table, the value of t at degrees of freedom = 5 is 2.015 (“T Test (Student’s T-Test)”) As the calculated value (2.763, negative sign being ignored) is less than the calculated value, the null hypotheses is rejected and the alternate hypotheses is accepted. This means that the efficacy of turmeric as a preservative for tomatoes when measured in terms of absorbance of lycopene or lycopene content depends on the genetic variety of the tomato used.

How does the ability of turmeric to act as a preservative for tomatoes (solanum lycopersicum), measured in terms of mass loss, pH and lycopene content depends on the percentage concentration (mass by volume) of the turmeric solution used and the type of tomato used-green tomato and red tomato ?

• As indicated in Graph-1,magnitude of change in mass reduces from 4.49 ± 0.02 g to 0.89 ± 0.02 g for red tomato and 5.22 ± 0.02 g to 1.36 ± 0.02 g for green tomato as the percentage concentration of turmeric solution increases from 0.00% (control) to 25.00%. As indicated in Graph-2, the values of pH lies within the ideal range 4.20 to 4.60 for all percentage concentrations of turmeric solution except the value being slightly less than 4.20 (around 4.10, slightly more acidic than expected) in control in absence of turmeric and reaches the maxima at 11.00 % turmeric solution. Graph-3 shows that for green tomato, the absorbance increases from 0.336 ± 0.001 AU to 0.714 ± 0.001 AU and for red tomato, it increases from 0.663 ± 0.001 AU to 0.995 ± 0.001 AU. This confirms that with the increase in the percentage concentration of turmeric solution, the lycopene content of both the red and green tomato increases resulting in increase in the absorbance of lycopene at 667 nm. Thus, it can be easily concluded that turmeric displays significant efficacy as a preservative for both green and red tomato. The efficacy increases as the percentage concentration of turmeric solution increases.
• Statistical analysis shows that whether the efficacy of turmeric as a preservative has any dependence on the genetic variety of the tomato or depends on how we measure the efficacy. Considering mass loss of tomatoes as an indicator of efficacy, it is found that the efficacy of turmeric to preserve red and green tomatoes are same while considering lycopene content (measured in terms of absorbance of lycopene at 667 nm), the efficacy of turmeric as a preservative for green and red tomatoes are not the same. Understanding that lycopene is a major ingredient of tomatoes and constitutes a major part of its nutritional value and bioassay, it is concluded that the efficacy of turmeric as a preservative do depends on the genetic variety of the tomatoes.
• Interpreting the values of regression coefficient (R² ) in Graph-1 and Graph-3, the null hypotheses has been rejected and the alternate hypotheses has been accepted.
• The qualitative observations are in agreement with the conclusion made. For example, the darkening of red colour in the red tomatoes were observed with the increase in the percentage concentration of turmeric solution used. This indicates that more amount of lycopene is synthesized in the biochemical pathway imparting more red colour to the tomatoes. This supports the fact that the values of absorbance of lycopene at 667 nm increases with the increase in percentage concentration of turmeric solution as shown in Table-4.
• Turmeric is the chief source of tomatoes. It helps in maintaining the nutritional health of tomatoes in two ways- by acting as a precursor for the biosynthesis of lycopene in the melavonate pathway and by inhibiting the cytokinesis and bacterial proliferation of pathogens and microbes in tomatoes that may make the tomatoes lose their biomass and nutritional values.

• The investigation uses three different analytical methods – mass loss, pH and lycopene content to measure the efficacy of turmeric as a preservative. This makes the result more acceptable and generalized as it takes into account the changes in the bio-content of the sample as the biochemical assay of it in terms of its major constituent lycopene.
• A wide range of independent variable – from 5.00 % to 25.00 % to provide a more wide understanding of the effect of the turmeric as a preservative and a control has been used for a better comparison.
• The value of percentage error is really low indicating high value of precision and accuracy in the data set collected.
• All factors like temperature, moisture which may interfere with the reliability of the result has been controlled.

Like turmeric various other substances like neem leaves, tulsi (Indian herb) leaves also has antibacterial effect. I would like to compare the antibacterial effect of these substances with the chemical anti-bacterial alternatives like laevocetrizine, allegra and Doxycycline. To do this, an extract of the natural substances and a solution of the chemical antibacterial drugs can be prepared. These can be applied on a petri dish where E.coli is grown in a agar agar medium and the zone of inhibition can be measured. A colony count method can also be used. Larger the zone of inhibition, better the antibacterial effect of the substance used. This investigation would allow us to compare and contrast the antibacterial effect of various natural and chemical substances that are claimed to have antibacterial effect.

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