To assess the antimicrobial activity of natural foods

against food-borne pathogens by agar well technique.

Abstract: The aim of this study was to identify foods with intrinsic properties
that would inhibit the survival and/or growth of contaminants. The antimicrobial
activities of orange juice, milk, cheese, honey, and yoghurt were determined
against some recurrent bacterial pathogens and spoilage agents, including
Salmonella typhimurium and Staphylococcus aureus. Orange juice had the
broadest spectrum of activity followed by honey and then yoghurt. In contrast, no
inhibition was observed with milk or cheese. Staphylococcus aureus was
resistant to the action of most foods. Low pH was shown to contribute to the
inhibitory activity of orange juice. Whereas, water activity (aw) was not the major
factor in the bacterial growth inhibition caused by honey.

Introduction:
This study was undertaken to assess the antimicrobial activity of a range of
foodstuffs, in order to reduce the potential for food spoilage and the incidence of
foodborne diseases. Illnesses due to food contaminated with either chemical or
biological agents are one of the most widespread problems throughout the world.
In the last few years there has been an alarming increase in the number of cases
of food poisoning. The general health status of the host, such as the age, metabolic
disorder, immune competence, pregnancy, or a range of other diseases or
conditions affects the individual’s susceptibility to foodborne diseases. Many
cases of disease are caused because of poor sanitation or improper practices
during food preparation, processing, or storage. For instance, failure to refrigerate
properly, inadequate cooking, use of low quality or contaminated raw ingredients,
employing infected personnel. Extreme care is required for the storage of food,
especially, cooked foods or those that may be eaten with very little or no
subsequent cooking. The common contaminants isolated from foodstuffs were
coliforms, Staphylococcus aureus, and to a lesser extent Salmonella typhimurium.
All foods derive from living organisms and therefore possess a variety of natural
mechanisms for limiting microbial infection. The defense mechanism may be in
the form of a physical barrier, preventing entry to microorganisms or it may be
physico-chemical in nature such as extreme pH levels. In some instances the
inhibitory activity is a consequence of an essential growth factor being limited
e.g., lactoferrin is a protein found in milk that binds iron; also importantly a
proportion of the moisture content of food is bound and microbial growth is
restricted or prevented by low water activity. In addition, many natural food
substances have damaging effects on microorganisms e.g., fruits and spices
contain essential oils that can disrupt enzymes and damage membranes. A range
of enzymes are also naturally found in foods some of which have bactericidal
activity e.g., lysozyme in eggs lyses the cell walls of Gram-positive bacteria.
Materials:
• Stock cultures of Salmonella typhimurium and Staphylococcus aureus, were
maintained on PCA slopes refrigerated at 2 ֯С.
• Two plate count agar (PCA) plates.
• Foodstuffs including: milk, orange juice, honey, cheese, and yogurt.
Method:
▪ Under aseptic conditions wells were made in PCA plates using an alcohol
sterilized cork borer. A separate well was prepared for each food type.
▪ Bacterial lawns were prepared on the agar containing the test wells
approximately 0.5mL of a 24 h culture of test organisms was spread over the
surface of the agar.
▪ One hundred microliters of liquid foods was added to wells, whereas,
viscous foods were added until the well was completely full.
▪ Plates were incubated at 35±2 ֯C for 24 h.
▪ Radii of the inhibition zones were measured around each well.

Observations:
Table 1. Antimicrobial activities of different foods against the bacterial strains.

Values are means±standard deviations.

ᵃA thicker zone of S. typhimurium growth occurred around cheese in all the plates.
Results:
➢ The antimicrobial activity of the different foods was quantified in terms of
their ability to restrict the growth of the test strains. Areas of complete
growth inhibition were detected and the size of these zones measured
(Table1).
➢ Orange juice exhibited greatest growth inhibition followed by honey, and
then yoghurt.
➢ The milk samples used did not demonstrate any antimicrobial activity and
the antimicrobial activity of the cheese was also low, indeed, enhanced
growth of S. typhimurium was seen around the well containing cheese.
➢ S. aureus was resistant to the physico-chemical properties of most food.
➢ Salmonella showed resistance to orange juice.
Discussion:
Orange juice induced the greatest growth inhibition (Table 2) pH of orange juice
is a critical factor in its inhibitory activity. Certain factors in honey damage
bacterial cell walls, and that this probably results in lysis of the cell. The high
sugar content of honey exhibits fairly good antimicrobial activity against both
Gram-negative and Gram-positive bacteria The chemical composition of honey
varies and generally contains high levels of tetracyclines, phenolic compounds,
and hydrogen peroxides. Probably, all these contribute to the antimicrobial action
of honey. Yoghurt was the second most inhibitory food item in this study, the
presence of lactic acid bacteria could be a major cause of the inhibitory action. In
addition to the ability of lactic acid bacteria to lower the pH of foods, they can
release a number of antimicrobial compounds that may inhibit the growth of
pathogenic bacteria. For instance, hydrogen peroxide and free radicals that result
from oxygen metabolism, organic acids, and bacteriocins all exhibit bactericidal
activity. Lactic acid in yoghurt has been shown to have an inhibitory effect against
Salmonella spp., which frequently cause disease. Samples of foods were
bactericidal in all cases, reducing the number of viable bacteria. The sensitivity
of the bacteria varied as did the potency of the different foods. The results of this
experiment indicate that the foodstuffs successfully inhibited the growth of the
test bacteria, in support of the agar well technique.
References:
1.Notermans, S.; Hoogenboom-Verdegaal, A. Existing and emerging foodborne
diseases. International Journal of Food Microbiology 1992, 15, 197–205.
2. Singlenton, P. Bacteria: In Biology, Biotechnology and Medicine, 5th Ed.;
John Wiley & Sons, LDT, 1999.