Antimicrobial Activity of Foods with Different Physio-Chemical Characteristics

Abstract

The antimicrobial activities of orange juice, milk, cheese, honey, and yoghurt were determined against some recurrent bacterial pathogens and spoilage agents, including Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Pseudomonas, Bacillus, Micrococcus, and Proteus spp. Orange juice and the combination of yoghurt and orange juice had the broadest spectrum of activity followed by honey and then yoghurt, and then to a lesser extent the combination of yoghurt and honey, and honey and orange juice. In contrast, no inhibition was observed with milk or cheese. Staphylococcus aureus, Salmonella, and Pseudomonas spp. were most resistant to the action of the foods, whereas Micrococcus, Bacillus, and E. coli were remarkably sensitive. Low pH (pH 3.2) was shown to contribute to the inhibitory activity of orange juice. Whereas, water activity (a _w ) was not the major factor in the bacterial growth inhibition caused by honey. In summary, it is good practice to include foods that restrict the growth of microorganisms in prepared foods such as school picnics and foods that may be inadequately stored such as camping meals. Orange juice, yoghurt, and honey were shown to meet these requirements. Their inclusion either individually or in combinations in ‘high risk” products such as salad dressings and sandwiches should be considered.

Introduction

This study was undertaken to assess the antimicrobial activity of a range of foodstuffs that could be included in pre-prepared meals, such as school packed lunches and rations for troops, 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.1 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.2 Changes in our eating habits have also increased the requirements for microbiologically safe foods.3 The use of mass-produced pre-prepared foods, restaurants, and fast food outlets has increased dramatically. Preparation and handling of foods in these situations must be to the highest standards to prevent outbreaks of disease.4

A range of recognized procedures can achieve improved food safety. However, if these practices are not strictly adhered to, then the safety of the food can be compromised. In tropical climates pre-prepared foods can frequently be stored at unsafe temperatures. 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 or heating, 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.

In this study, the feasibility of using foods with antimicrobial physico-chemical properties to improve the safety and shelf life of school meals prepared in local restaurants and canteens was considered. Such meals have a high risk of contamination due to a number of factors including: a large number of meals are prepared at the same time since all of them must be delivered to the schools at break time; limited facilities are available in the preparation kitchens; the educational standards of the employees is low; and transportation and distribution conditions are often inappropriate. Usually, limited attention is given to the food classification whether it is perishable, semi-perishable, or shelf-stable. Since large numbers of students are consuming the same meals, contaminated foods may result in large outbreaks. Some of these meals were analyzed for their microbial quality. The common contaminants isolated were coliforms, Staphylococcus aureus, and to a lesser extent Salmonella typhimurium. It was also noticed that the degree of contamination varied among different types of foods although they were prepared in the same establishment.

All foods derive from living organisms and therefore possess a variety of natural mechanisms for limiting microbial infection.5 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.6 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.

The aim of this study was to identify foods with intrinsic properties that would inhibit the survival and/or growth of contaminants. Preliminary identification of the inhibitory factors was also undertaken. Incorporation of these foods into school meals could help to reduce the incidence of illness by inhibiting the growth of pathogens contaminating the foods.

Materials and Methods

Bacterial Strains and Cultivation Methods

The strains used included Salmonella typhimurium ATCC 14082, Staphylococcus aureus, E. coli, Pseuodomonas, Micrococcus, Proteus, Streptococcus, and Bacillus. The bacteria were cultured on plate count agar (PCA) and in nutrient broth (Oxoid, Basingstoke, UK). Stock cultures were maintained on PCA slopes refrigerated at 2°C to provide standard inocula for each experiment. To standardize the growth phase of the test strains cultures were grown in static 10 mL volumes of nutrient broth for 24 h at 35 ± 2°C, prior to use in sensitivity assays. Growth phase will influence the stress resistance of cultures.7

Foods and Their Physical Characteristics

A range of foods was used in this study including milk, orange juice, honey, processed cheese, and yogurt (bio-yoghurt from which live bacteria were isolated). All food products were produced locally and were purchased from retail outlets. Products available in sealed, labeled packages were selected to ensure high initial quality. The pH values of the foods were determined using a pH meter for the liquid stuffs and yoghurt or pH strips (Fisherbrand, Eu code: FB 3003) for the viscous stuffs. Water activity was determined using a water activity meter (AQUALAB, Decagan devices, USA).

Detection of the Antimicrobial Activity of the Selected Foodstuffs

Under aseptic conditions wells were made in PCA plates using an alcohol sterilized cork borer. A separate well was prepared for each food type. To prepare bacterial lawns on the agar containing the test wells approximately 0.5 mL 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 and a good contact was created between the food and the surrounding agar, but not contaminating the agar surface around the well. Plates were incubated at 35 ± 2°C for 24 h prior to measuring the radii of the interaction (inhibition or enhancement) zones around each well.8

Evaluation of Different Inhibitory Factors

The pH of retail orange juice was modified from pH 3.32 to 4.00, 6.05, and 7.11, using strong KOH (BDH). A concentrated solution of KOH was used so that the dilution factor was negligible. The inhibition zones induced by the modified orange juice were compared using the well procedure. Staphylococcus aureus and E. coli were selected for this experiment. To determine the role of water activity in the antimicrobial action of honey the water activity was modified from the original value of 0.62 to about 0.53 using three different carbohydrates namely; glucose, fructose, and sucrose (BDH).

The Effect of Different Food Combinations

Foods that showed relatively high growth inhibitions were combined in ratios of (1:1), the combined foods were mixed using a stomacher to form a homogenous mix. The combined foods were added to the wells and the resultant growth inhibition zones measured.8 9

Bacterial Growth

Individual 5 g or 5 mL samples of honey, orange juice, and yoghurt and their combinations were artificially contaminated with 0.1 mL of an overnight culture of E. coli, S. aureus, Salmonella, or Micrococcus prepared in 5 mL volumes of nutrient broth (Oxoid, UK). Viable counts of the cultures were determined on PCA incubated aerobically at 35 ± 2°C overnight. The food samples were incubated aerobically at 35 ± 2°C for 24 h. Following incubation of the contaminated food samples viable counts for surviving/growing test contaminating organisms were made.10 11

Results and Discussion

The selected foods had a range of intrinsic physical properties. Both orange juice and yoghurt had low pH values, less than pH 4, whilst the water activity of honey was much lower than the other foods (Table 1). The antimicrobial activity of the different foods was quantified in terms of their ability to restrict the growth of the test strains. In general, areas of complete growth inhibition were detected and the size of these zones measured (Table 2). 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 (Table 2), indeed, enhanced growth of S. typhimurium was seen around the well containing cheese. Milk and cheese were not included further in this investigation as they did not demonstrate useful antimicrobial properties. The Streptococcus spp. and S. aureus were the most resistant to the physico-chemical properties of the food, whereas in contrast, Micrococcus, E. coli, and Bacillus were the most sensitive. The resistance of the Streptococcus spp. and S. aureus was significantly (p < 0.05) greater than that of Micrococcus, E. coli, and Bacillus. Salmonella and E. coli which are closely related were similar in their resistance except for their resistance to orange juice where Salmonella demonstrated a much greater tolerance than E. coli.

Table 1 Physical properties of the foods and beverages

Food/beverage type	pH	a w
Orange juice	3.32	0.98
Bio yoghurt	3.95	0.97
Honey	6.0 ± 0.5	0.62
Cheese	4.0 ± 0.5	0.94
Honey and yoghurt	3.51	0.93
Honey and orange juice	3.71	0.92
Yoghurt and orange juice	3.75	0.99

Table 2 Antimicrobial activities of different foods against a range of bacterial strains

	Mean clear growth inhibition radii (mm)
Strains	Orange juice	Yoghurt	Honey	Cheese	Milk
S. typhimirium	3.7 ± 0.06	6.1 ± 0.13	6.2 ± 0.08	−0.8a	0
S. aureus	4.7 ± 1.4	5.3 ± 0.6	2.7 ± 0.5	1.6 ± 1.1	0
E. coli	9.0 ± 1.4	6.8 ± 0.3	7.7 ± 0.5	0.1 ± 0.1	0
Pseudomonas	7.8 ± 1.3	2.3 ± 2.3	3.5 ± 0.7	2.5 ± 0.5	0
Bacillus	7.0 ± 0.8	3.5 ± 0.5	11.7 ± 3.5	NT	NT
Micrococcus	11.4 ± 1.3	10.4 ± 0.8	5.2 ± 0.9	NT	NT
Proteus	5.3 ± 0.8	5.0 ± 0.7	3.4 ± 1.6	NT	NT
Streptococcus	3.3 ± 0.5	5.1 ± 0.5	3.1 ± 0.8	NT	NT

Values are means ± standard deviations. NT: not tested.

^aA thicker zone of S. typhimurium growth occurred around cheese in all the plates.

When the pH of the orange juice was raised to pH 4 and above no growth inhibition was observed (Table 3). Also, modification of the honey by addition of sugars generally had slightly reduced inhibitory action. When the water activity was further reduced to 0.53 using glucose this did increase the inhibitory effect upon E. coli (Table 4).

Table 3 Growth inhibition of S. aureu s with original and modified pH orange juice

pH	Mean clear growth inhibition radii (mm)
3.32a	5.0
4.00	ND
6.05	ND
7.11	ND

ND: Represents no detectable inhibition zone.

^aOriginal commercial product.

Table 4 Inhibition of the growth of S. typhimurium and E. coli by original honey and honey with reduced water activity

		Growth inhibition radii (mm)
aw	Added sugar	S. typhimirium	E. coli
0.62	Controla	6.0	6.2
0.53	Glucose	1.0	6.5
0.53	Sucrose	1.0	5.0
0.52	Fructose	1.5	5.5

^aOriginal, unmodified honey.

When foods were combined the inhibitory activity was in no way additive, in fact the combinations were usually less effective at inhibiting bacterial growth than the individual components. The only exception was for S. typhimurium when the combination of yoghurt and orange juice produced a somewhat larger inhibition zone than either of these alone (Table 5). Again E. coli was more sensitive to the action of the combinations including orange juice than was S. typhimurium (Table 3). Inhibition by the combination of yoghurt and orange juice was significantly (p < 0.05) greater than the other combinations.

Table 5 Antimicrobial activities of combinations of active foods and beverages

	Mean clear growth inhibition radii (mm)
Strains	Honey and yoghurt	Honey and orange juice	Yoghurt and orange juice
S. typhimirium	0.0 ± 0.0	0.0 ± 0.0	6.5 ± 0.6
S. aureus	0.0 ± 0.0	1.5 ± 0.6	2.4 ± 0.5
E. coli	4.8 ± 0.5	5.6 ± 1.1	6.5 ± 0.6
Micrococcus	4.7 ± 0.5	6.5 ± 0.6	6.3 ± 1.0

None of the test organisms grew when inoculated onto the foods. Counts were all reduced following 24 h incubation (Table 6). The percentage survival was very low in honey, yoghurt, and in combinations of honey and yoghurt and honey and orange juice. Pseudomonas was the least well able to colonize the foods. The least numbers of organisms survived when inoculated onto honey either individually or in combinations. In general, S. aureus was the best able to survive on the different food types.

Table 6 Change in bacterial counts following artificial contamination of different foods

	Proportion of original inoculum surviving (% × 10^4)
Strain	Orange juice	Honey	Yoghurt	Honey and yoghurt	Honey and orange	Yoghurt and orange juice
S. aureus	1.0	1.0	0.4	0.2	0.08	0
E. coli	1.0	0.002	0.02	0.002	0.5	0.1
Micrococcus	1.0	1.0	0.005	0.03	0.007	0
Pseudomonas	0	0.007	0	0.05	1.0	0.01
Salmonella	NA	0.01	1.0	0.01	0	0

Generally, orange juice induced the greatest growth inhibition (Table 2). Canned orange juice was selected, rather than fresh juice, for its ease of use during mass production of meals. No preservatives were present so the observed effects were a consequence of the physico-chemical properties of the orange juice alone. The pH value of the retail sample was low, pH 3.32. When the pH level was raised above pH 4.0 no inhibitory activity was observed (Table 3) clearly demonstrating that the acid pH of orange juice is a critical factor in its inhibitory activity.

Honey also showed good antibacterial activity (Table 2). When the water activity of the honey was further reduced by carbohydrate addition, in all but one case no increased inhibition was detected. This indicates that water activity and consequent osmotic activity is not the main factor contributing to the antimicrobial action of honey. Adding water to the honey to increase the water activity would produce unclear results as all the active ingredients would be diluted. It could be concluded that changing the chemical composition of the honey effects its antimicrobial activity. El-Sukhon and coworkers12 also stated that high osmolarity is not the main cause of the antimicrobial activity of honey. They suggested that certain factors in honey damage bacterial cell walls, and that this probably results in lysis of the cell. In a similar study Wahdan13 investigated the effect of high sugar content on the antimicrobial activity of honey. High sugar content solutions that resembled honey were used together with honey against 21 types of bacteria and two types of fungi. Due to the differences in the antimicrobial activity between honey and the sugar solutions, it was suggested that although the high sugar concentration of honey plays an important role in its antimicrobial activity, a number of other contributory factors are present.

Generally, honey is well known for antimicrobial activity against a number of microorganisms. Nzeako and Hamid14 stated that like any antibiotic, honey might be active against specific microorganisms only. However, Bilal and Al-Falki,15 demonstrated that honey exhibits fairly good antimicrobial activity against both Gram-negative and Gram-positive bacteria which supports the findings of this study. In addition, Obi et al.,16 observed that honey could have inhibitory activity against a range of diarrhoea causing bacterial agents isolated in Lagos, Nigeria. It might be considered that the antimicrobial activity would be insignificant when an oral dose of honey becomes diluted after absorption from the gut. Therefore, the potential for honey as an antimicrobial agent against gastrointestinal infections is best for preventing or reducing the levels of food contamination.

The chemical composition of honey varies and different types of honey exhibit different degrees of antimicrobial activity. In general, honey contains high levels of tetracyclines, phenolic compounds, and hydrogen peroxides. Probably, all these contribute to the antimicrobial action. Bogdanov17 fractionated 10 different types of honey into four basic substance groups including; volatile, acidic, basic, and nonvolatile and nonpolar. The antimicrobial activity of each group was tested against Staphylococcus aureus and Micrococcus luteus. The acidic fraction had the greatest inhibitory activity followed by bases, nonpolar, and nonvolatile group and finally the volatile. It was concluded that the nonperoxide antibacterial activity of honey is correlated with the acid content but not the pH. A number of studies have investigated the role of the different physico-chemical properties of honey in its antimicrobial activity, but the contribution of the different inhibitory factors still remains unclear.

Yoghurt was the second most inhibitory food item in this study. Live bacteria were isolated from the product used; 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.5 Lactic acid in yoghurt has been shown to have an inhibitory effect against Salmonella spp., which frequently cause disease.18 Applying yoghurt as a dressing for salads may reduce health problems associated with salad contamination. Microbial analysis of school meals showed that most of the contaminated meals are sandwiches containing green salad.

Samples of single or combined foods were artificially contaminated with test bacteria. In all cases the foods were bactericidal, reducing the number of viable bacteria. The sensitivity of the bacteria varied as did the potency of the different foods. Combinations of both orange juice and honey and orange juice plus yoghurt reduced the count of Salmonella to below the detection level.

It was essential to confirm the observations on the foods as the complex nature of the food means extrapolation of data from agar plate studies could lead to inaccurate conclusions. Foods like orange juice have considerable ability to diffuse through the agar media potentially giving larger inhibition zones, while viscous foods probably have less diffusion ability. Generally, these results indicate that the foodstuffs successfully inhibited the growth of the test bacteria, in support of the agar well technique.

In general, the activity of the combined foods did not exceed that of either of the foods in isolation. Possibly because in the combination the inhibitory factors are diluted, e.g., honey plus orange juice, the pH of the orange juice is raised above pH 4, whilst the water activity of the honey is raised and all other antibacterial agents in the honey are diluted by a factor of 2. This dilution reduces the effectiveness of the inhibitory agent. This may account for why the combination of yogurt and orange juice is one of the most inhibitory, if pH is the major inhibitory factor in this combination the pH has not been raised and consequently still prevents growth or kills the cells. Therefore, careful deliberation of the intrinsic properties of the foods is required when considering food combinations for inhibitory activity.

Conclusion

In summary, different foodstuffs exhibited varying levels of antimicrobial activity. The sensitivity of different bacterial species also varied. Although the zone method is commonly used it should be combined with more extensive studies using food products incubated under conditions similar to those that occur during food distribution. This study has also highlighted the importance of considering the risk of different food items for disease or spoilage when preparing large quantities of meals for delayed consumption. Foods, such as yoghurt, honey, and orange juice, with physico-chemical properties that inhibit the growth or survival or contaminating pathogenic or spoilage bacteria should be considered for use in pre-prepared meals either directly or as dressing for other foods.