Analysis of The Antibacterial Properties of Oils from The Pits and Peels of Kalma and Cardinal Avocados

Introduction

Escherichia coli can be a harmless inhabitant in the gastrointestinal tract, however, it can also cause significant harm involving diarrhea and extra intestinal diseases. The pathogen has mortality worldwide. Outbreaks are common and fatal, especially to developing countries like the Philippines. They are a major concern for public health as they can be transmitted easily, through food and water intake. That is why the use and source of antibiotics that can cure infections and diseases caused by pathogens are of great importance however, the unrestricted use of traditional antibiotics in the medical field for the treatment of human and animal infections has increased greatly, causing the pathogens to build up a resistance against these compounds, making them less effective. As a result, generations of various drug-resistant pathogens have emerged. Thus, a demand for an alternative method for diminishing pathogens has also emerged.

Persea Americana or most commonly known as avocado is a tree that is classified as a member of the flowering plant family Lauraceae, or the laurel family. The fruit it yields also called an avocado is botanically a large berry containing a single large seed known as a “pit” or “stone.”

Currently, the avocado fruit is being cultivated in various parts of the world, especially in tropical countries like the Philippines. The fruit exists in different shapes, sizes, and colors depending on their varieties. In the Philippines, two specific types of avocados exist. They are the green-fruited and purple-fruited types. In a crop statistic compiled by the Bureau of Agricultural Statistics of the Department of Agriculture showed that in the 1990s the Philippines had a total area of 4, 753 hectares planted with avocado. The average production of the fruit annually is estimated at more than 45, 000 tonnes.

Today only a few varieties of avocados exist in the Philippines. One of them is the cardinal avocado, a fruit that has an average weight of 400 grams. It has a reddish to purple thick skin. The seed is small, weighing 40 grams. Another variety is the calm avocado. It is significantly heavier than the cardinal avocado, with the weight coming up to 600 grams. The skin is also reddish to purple and intermediate in thickness. The seed weighs about 80 grams and is loose in captivity.

The oil that the fruit yields have been reported to have the ability to lower cholesterol level, keep the elasticity of the skin, and reduce coronary heart risk. The 1,2,4-trihydroxyn-heptadeca-16-en found from the avocado fruit and seeds have also been reported to show antibacterial activity. Avocado oil is also globally used in the food industry as well as the cosmetics and medical industry. This is all due to the high content of both unsaturated and saturated fatty acids, proteins, minerals, and fibers avocado oil possesses.

However, studies about the antibacterial activity of Philippine avocado cultivars is not available currently. Therefore, the researchers' main objective is to identify the antibacterial property of two Philippine avocado cultivars, namely the Persea Americana Cardinal and Persea Americana Calma against the pathogen Escherichia coli.

Statement of the Problem

Persea Americana is a highly nutritious fruit that has various compounds that could pose as a benefit to the medical field. However, there is limited to no information about the antibacterial property of Philippine cultivated avocados against the pathogen E. coli. This study aims to investigate the antibacterial property of oils extracted from the seed and peel of Persea Americana Cardinal and Persea Americana Calma against the pathogen E. coli. The oils will be extracted through the Soxhlet Extraction Method; the antibacterial efficacy will be identified using the Kirby Bauer Method. Through this study, the researchers intend to:

1. determine the antibacterial property of Persea Americana Cardinal and Persea Americana Calma against Escherichia coli
2. determine the efficacy of Persea Americana Cardinal and Persea Americana Calma as antibacterial agents
3. determine which extract possesses the greatest antibacterial property against Escherichia coli

Null Hypothesis

The ethanolic extracts of the four samples, namely, both seed and peel of Avocado Persea Americana Calma and Persea Americana cardinal, were of no antibacterial efficacy against Escherichia coli and therefore cannot control the growth of the pathogen

Alternative Hypothesis

The ethanolic extracts of the four samples, namely, both seed and peel of Avocado Persea Americana Calma and Persea Americana Cardinal, have antibacterial efficacy against Escherichia coli and therefore it can control the growth of the pathogen.

Significance of the Study

The significance of the study is to contribute factual data and information about the antibacterial properties and efficacy of the said samples, which were found to be of utmost importance and usage to the following:

• To the Students and teachers. This study would provide new information about the antibacterial property of Persea Americana Calma and Persea Americana Cardinal for additional knowledge.
• To the Future Researchers. This study could be utilized as a fine reference to future studies with connection to Persea Americana Cardinal and Persea Americana Calma and their antibacterial property.
• To the Patients. This study would be beneficial for providing an alternative remedy to bacterial infection which is cost-efficient and natural.
• Physicians and Healthcare Practitioners. This study would provide data about avocados which can help them make alternate antibacterial medications to supply the necessities of the patients.

Scope and Delimitation

This study will be conducted at the Angeles University-Main Campus laboratory and the Angeles University Foundation Santa Barbara Campus laboratory. The study will cover only the antibacterial efficacy of the seed and peel of Persea Americana Cardinal and Persea Americana Calma against the pathogen Escherichia coli. The oils will be extracted using the Soxhlet Extraction Method; the antibacterial efficacy will be identified through the Kirby Bauer Method.

In addition, this study will not identify the antibacterial efficacy of the juice or pulp of the two avocado cultivars. It will strictly cover the peel and the seed of the fruit only. Furthermore, the pathogen in which the variables will be tested will be Escherichia coli only and no other.

Definitions of Terms

• Antibacterial Property. Intends to kill or diminish the harmful effects of bacteria on humans and animals.
• Persea Americana. Most commonly known as avocados; a large berry containing a single large seed known as the 'pit'. The main subject of the study.
• Pathogen. A bacterium, virus or any other microorganism that can cause disease.
• Soxhlet Extraction. Process of transferring the partially soluble components of a solid to a liquid using a Soxhlet extractor.
• Kirby Bauer Test. Antibiotic testing that uses disks to determine whether a particular bacteria are susceptible to specific antibiotics.
• Zone of Inhibition. A circular area around the spot of the antibiotic in which the colonies of bacteria do not grow.

Chapter II

Persea Americana

A study by Duarte et al. aimed to introduce a literature review about the attributes, applications, and capability of avocado (Persea Americana) Avocado is viewed as one of the primary tropical fruits, as it contains fat-soluble vitamins which are not that common in other fruits, aside from high levels of protein, potassium, and unsaturated fatty acids. The avocado pulp contains variable oil content and is generally utilized in the pharmaceutical and cosmetics industry, and in the creation of commercial oils akin to olive oil. This fruit has been acknowledged for its medical and health advantages, specifically due to the compounds present in the lipid fraction, for example, omega fatty acids, phytosterols, tocopherols, and squalene. Research done in the past has shown and examined the benefits of avocado related to a well-adjusted diet, especially in lessening cholesterol and preventing cardiovascular diseases. The processed avocado pulp is an alternative to use fruits, which can be utilized in different value-added food products. Liquid extract of the avocado leaves is generally used in pharmaceutical products, mainly due to the diuretic features of the present compounds in plant leaves. With growing research supporting the nutritional characteristics and benefits of avocado, the estimated tendency is to increase the production and utilization of this raw material in Brazil, as likewise observed in other countries.

In addition to the significant vital compounds, avocado possesses generous amounts of bioactive compounds, for example, phytosterols, especially in the lipid fraction, and the major representative, the β-sitosterol.

The utilization of avocado fruit is highly associated to its potential advantages. Although, with the rise of avocado production, limited time of maturation and simple oxidation of avocado fruit are the major problems for manufacturers. The production of oil from the avocado fruit, therefore, is greatly encouraged.

Escherichia coli

A review article by Jang, J., et al aimed to examine the current knowledge on the ecology of E. coli strains in different environments with regard to its role as a fecal indicator bacterium (FIB) and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health are also discussed. E. coli is a rod-shaped, Gram-negative bacterium, and classified as a member of the family Enterobacteriaceae within the Gammaproteobacteria class. it is among one of the well-studied bacteria. It can grow rapidly under optimal growth conditions, replicating in ~20 min. Numerous gene manipulation systems have been developed using E. coli as the host bacterium, producing countless enzymes and other industrial products.

E. coli strain to acquire nutrients, compete with other microorganisms, and survive and grow in the environment is likely to vary by strain and genotype. Thus, differential survival/growth ability among E. coli strains could cause a shift in E. coli populations in wastewater or fecal-impacted sediments.

In addition, Escherichia coli includes not only commensal strains but also pathogenic ones that cause a variety of human diseases—resulting in more than 2 million deaths each year.

Extraction

According to Gatbonton, G.L high estimation of oil and chlorophyll focus is contained in an avocado or Persea Americana, which is a natural product. with respect to the extraction and improvement of the avocado mash oil yield, numerous investigations had been made. Whereas, the most pragmatic process in separating avocado oil is Soxhlet Extraction, as demonstrated by their examinations. This examination, through Soxhlet extraction with hexane and ethanol as solvents at various extraction times and measures of solvents, explored the oil yield and oleic corrosive substance of the avocado oil. Moreover, properties of the oil, such as the physical and substance properties, were resolved as well. First, the meat of the avocado variety was subjected to stripping, destoning, and freeze-drying. Then, the subject is grounded, of which, 30 grams is used for every exploratory run. Utilizing Hexane and ethanol as solvents at different sums, particularly 250mL to350mL, the steady example mass was extricated. A rotational evaporator was utilized to isolate the extricated oil. Oil yield was figured and after that examined for its oleic corrosive substance utilizing gas chromatography. Best conditions were finished up from the starter tries and were repeated to additionally pressure the impacts of the differing conditions. The most elevated oil content gotten utilizing ethanol was 73.56% while 40.33% (w/w) was acquired utilizing hexane.

A study conducted by Chawla, S. et al evaluated the bioactivities of Citrus Peel Extracts when subjected into different extraction conditions and storage temperatures. Frozen citrus peels were blended into a fine powder and extracted using two methods. The first method was an extraction with 70% ethanol at room temperature for 72 hours with the sample to solvent ratio of 1:10. The second method used the same concentration of the same solvent and same ratio of sample to solvent but utilizes the Soxhlet apparatus at 85 degrees Celsius for three hours. The extracts were subjected into filtration using a Whatman filter paper. The groups were then stored in room temperature level and the other one in 4 degrees Celsius. Both were kept in capped bottles. In the results stated in their study, there was of no effect from the extraction methods implicated and storage period.

Kirby-Bauer Disk Susceptibility Test

The Kirby-Bauer uses disks or wafers containing the antibiotic to determine whether a particular bacteria is susceptible to specific antibiotics. A culture of bacteria is grown overnight on a solid growth media. If the bacteria are susceptible to the particular antibacterial from the disk, a clear area will form surrounding the disk, meaning bacteria were not able to grow around that area, also known as the zone of inhibition. A larger zone of inhibition around the disk indicates that the bacteria are more sensitive to the antibacterial drug in the disk. The test is performed only through standardized conditions. They are typically reported as susceptible, intermediate, and resistant in terms of results. If the observed zone of inhibition falls under the conditions of a standard zone of inhibition, this means the microorganism is susceptible to the antibacterial. Inversely, if the observed zone of inhibition falls short of the standard zone of inhibition, the microorganism is resistant to the antibacterial. This test can be used as for clinicians to choose which antibiotic is appropriate to control particular pathogens and combat infections.

Review of Related Study

In a study conducted by Ogundare, A.O. & Oladejo, B.O., they made use of the leaves and barks of Persea Americana to assess its efficacy against some pathogenic bacteria like S. aureus, E. coli, K. pneumoniae, and P. aeruginosa so as to be a source of antibacterial agent. They collected their samples from the Federal University of Technology, Akure, Nigeria, where they were reportedly growing naturally. They were cleaned using water and then dried for a couple of days. The dried plants were then crushed into fine powder. The pulverized plants were weighed each soaked in methanol for 72 hours and filtered using number one Whatmann filter paper. The filtrates were collected in separate bottles and removed the solvent using a rotary evaporator. The cultures were obtained from the Department of Microbiology, Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Osun State, Nigeria, and Don Bosco Catholic Medical Centre, Araromi Street, Ondo State, Nigeria. All the bacteria used were kept on agar plates and stored in a refrigerator at four degrees Celsius. The antibacterial activity of the extracts was evaluated using he agar well diffusion method or the Kirby Bauer Method. Areas that are clear indicate the zone of inhibition of the bacteria against the extracts and were then measured and noted by millimeters. The results stated that the methanol extracts of the leaf and bark of Persea Americana had the highest inhibitory effect on S. aureus with a zone of inhibition of 6 mm and 12 mm respectively, while the least inhibited by the leaf extract was the K. pneumoniae with a zone of inhibition of 2 mm and P. mirabilis with a zone of inhibition of 3 mm by the bark extract. Furthermore, the results stated that both of the extracts had zero inhibitory effect on S. typhi and E. coli.

The study of Raymond Chia, T.W&Dykes, G.A. made us of the microtiter broth microdilution assay by their reference to establish the antibacterial activity of the crude epicarp and seed extracts from mature avocados. On a microtiter plate, tryptic yeast soy glucose broth was put. Then a diluted solution of each avocado extract were were joined in a different well of the plate. A multichannel pipette was used to make serial two-fold dilutions from column one until certain concentrations of the antimicrobial agents were obtained. The seventh column and eighth were removed of antimicrobials while some content of column six was junked. On the other hand, the extracts’ antifungal activity against two molds was established by the Hole plate method using potato dextrose agar or PDA as the growth medium. Disinfected PDA vaccinated with every one of the organisms was vortexed and aseptically filled sterile Petri plates and permitted to coagulate. Five openings of aseptically punched into the Petri plates with a treated steel borer of uniform edge and size. The gaps were loaded up with epicarp or seed separate from the three distinct cultivars of avocado at centralization. There were two negative controls for each concentrate, one containing ethanol and the other containing sterile refined water. Petri plates were hatched at 30°C for as long as 5 days. Clear zones of restraint around the gaps were recorded as having hostile to the parasitic movement. Every antifungal movement was performed in triplicate as above. No inhibition by either of the extracts was observed against the pathogens.

A study conducted by Pereira, J. A. et al compared the antibacterial properties of different cultivars of walnut leaves extract (Cv. Lara, Franquette, Mayette, Marbot, Mellanaise, and Parisienne) grown in Portugal. In the results stated in their study, all of their extracts presented high antibacterial activity, Lara being the most effective one.

Chapter III

Research Design

The researchers will use the experimental type of research in this study. Experimental research design is the method that will allow the researchers to test their hypothesis to have valid conclusions regarding independent and dependent variables. This will also manipulate and control the tests to understand causal processes.

Research Materials and Instruments

The materials and instruments that will be needed for the study are: the seed of Persea Americana Cardinal and Calma, the peel of Persea Americana Cardinal and Calma, Escherichia coli, 70% ethanol, filter paper, Petri dish, Mueller-Hilton agar, cotton swabs, puncher, forceps, micropipette, masking tape, incubator, and Personal Protective Equipment (PPE).

Collection of Avocado

The avocados were acquired from a local farm in Metro Manila. The variety of avocados used for this study is the cardinal and Calma because of its peel and seed characteristics and it being found in the Philippine Islands. They were then ripened and cleansed with water. The avocados were kept at room temperature till further use.

After being ripened, the avocados were then carefully sliced in the middle. The seed and peelings were separated from the flesh. The collected seeds and peelings were then cleaned and powdered through the use of a blender for the extraction.

Collection of Avocado

The process of transferring the partially soluble components of a solid to the liquid phase is through using a Soxhlet extractor. Researchers set up and performed the extraction to increase direct understanding of extraction strategies. The powdered peel and seed of the Persea Americana Cardinal and Persea American Calma were set in a filter paper thimble, which was set inside the Soxhlet extractor. The solvent was warmed at 85 degrees Celsius utilizing the isomantle and started to dissipate, and traveled through the contraption to the condenser. The condensate at that point dripped into the reservoir containing the thimble. When the level of the solvent (ethanol) reached the siphon it emptied once more into the cup and the cycle started once more. The procedure kept running for an aggregate of 3 hours per sample. When the procedure was completed, the ethanol was evaporated utilizing a rotary evaporator, leaving a little yield of extracted plant material (around 2 to 3 ml) in the glass base jar.

Agar Preparation

The agar used in the experiment was Mueller-Hilton agar. The measurement of agar powder needed is 9.5-g which was heated with 250 mL of distilled water in a laboratory hot plate and waited until it turned into a yellowish viscous mixture. After heating, the agar was placed inside the autoclave for 15 minutes with a temperature of 121 degrees Celsius. Then, the agar was put aside until it cooled down. After the preparation of Petri dishes, the agar was put inside the container and let it cool down.

SWABBING OF BACTERIA

Cotton swabs were utilized in the experiment for the process of swabbing the Escherichia coli in the agar plate. It was done by following different directions for the precise of covering all the area of the agar plates with the bacteria. The study used 12 Petri dishes which were labeled into four parts: Cardinal Peel, Cardinal Seed, Calma Peel, and Calma seed. The Petri dishes were reversed facing downwards to prevent the bacteria from jumping down outside the Petri dishes.

Preparation of Filter Paper Disk

Filter papers were punched into disks for the application of the extract. Discs were equally distributed in each section forming two quadrants in one Petri dish.

• APPLICATION OF EXTRACTS

The extract was put in the discs by the use of a micropipette.

• INCUBATION OF PLATES

The agar plates were put inside the incubator for 24 hours at a temperature of 36 degrees Celsius.

• EXAMINATION OF PLATES

After the process of the incubation of plates, the variables of the study were ready for the analysis of data and results.

• MEASUREMENT OF ZONE OF INHIBITION

Zone of inhibition Testing is a fast, qualitative means to measure the ability of an antimicrobial agent to inhibit the growth of microorganisms. The inhibition zone executed a rounded shape which was examined by the naked eye. The diameters were measured using a caliper ruler.

Chapter IV

Results and Discussion

The data that was collected from the previous chapters' experiment against Escherichia coli was discussed and analyzed in this chapter.

Presentation of Data

The tables displayed the antibacterial properties of the Persea Americana Cardinal and Calma seed and peel based on the inhibition zones. Each Petri dish contains two samples that were compared with the positive control ( Cefuroxime )

Data Analysis

Data gathered from this study indicates that the extracts from the seed and peel of Persea Americana Calma and Cardinal are not effective in inhibiting the growth of E. coli. Due to the absence of the zone of inhibition of the samples, there was no need for the researchers to do statistical analysis with regards to the millimeter of growth of inhibition against E. coli. In addition, efficient inhibition of the E. coli is shown by the positive control, cefuroxime, with a zone of inhibition of 18mm.

Interpretation of Data

The statistical analysis of Data was not implied after reading the inhibition zone because the data in the tables showed that none of the samples have antibacterial properties against E. coli. According to antibacterial sensitivity testing of CLSI, the standard performance should have an 18 mm zone of inhibition for the pathogen to be considered susceptible to the drug and a zone of inhibition of 15-17 mm to be considered intermediate, unfortunately, 0mm inhibition was observed. Therefore, the data gathered of having no inhibition zone implies that the oils extracted from the seed and peel of Persea Americana Calma and Persea Americana Cardinal have no antibacterial properties and efficacy against Escherichia coli.

Chapter V

In this chapter, the summarization of the findings of the study, conclusions, and recommendations based on the information gathered is presented. During the experimentations, some limitations were discovered.

Summary

The avocado peel and seed were separated from the flesh and powdered. To extract the oils from the samples, the said samples were placed inside a filter paper thimble which was placed inside the soxhlet extractor respectively. The acquired extracts were then rotary evaporated to remove the ethanol and only the pure extract will remain. Agar powder was then heated with water and left to slightly cool. The agar was then poured into 12 Petri dishes, each with respective amounts. The culture of bacteria was then swabbed at the top of the agar plates. Paper disks made from filter papers were then soaked with the extracts respectively and lightly placed on top of the agar plates. The plates were then incubated for 24 hours and studied and measured using a caliper ruler. The data gathered stated that the extract showed no zone of inhibition and is therefore not efficient as an antibiotic.

Conclusion

The study was conducted by following the procedures and the researchers concluded that the seed and peel extracts of Persea Americana Calma and Cardinal have no antibacterial properties against Escherichia coli. According to the data, the inhibition zone of the extract showed zero (0) inhibition.

In order for the variable to be viable as an antibacterial agent, it must show an inhibition zone. The degree of sensitivity of bacteria to a drug is indicated in its size of inhibition. This means that the more sensitive the bacteria is to the drug, the bigger area of inhibition surrounding the antibiotic disc should be.

Based on the data gathered, the positive control which is a proven and tested drug showed a zone of inhibition of 18mm, which indicates the pathogen to be susceptible to the antibiotic, whilst the extract has no inhibition zone, which indicates the pathogen to be resistant to the extract.

Recommendation

The antibacterial property of the seed and peel of Persea Americana Calma and Persea Americana Cardinal is not effective to eradicate Escherichia coli. Therefore, the researchers would like to recommend future researchers to try other variables that have antibacterial properties such as tangerine, lemon, lime, and grape concentrates. The researchers also recommend trying other extraction solvents such as distilled water, ethanol, acetone, chloroform, and hexane.