Antibacterial Activity of Processed and Unprocessed Honey Samples Against the Clinical Bacterial Pathogens from Kanhangad, Kasaragod District, KL, India

Research Article

Austin J Environ Toxico. 2024; 10(1): 1048.

Antibacterial Activity of Processed and Unprocessed Honey Samples Against the Clinical Bacterial Pathogens from Kanhangad, Kasaragod District, KL, India

Usharani K1,2*; Ummul Mainoor1; Fathimath Thahira1; Ayshath Rahida1

¹Department of Environmental Science, School of Energy, Environment and Earth Sciences, Central University of Kerala, India

²Department of Civil and Environmental Engineering, Universidade Estadual Paulista Julio De Mesquita Filho, Bauru, Brazil

*Corresponding author: Usharani K Research Fellow, Department of Civil and Environmental Engineering, Universidade Estadual Paulista Julio De Mesquita Filho, Bauru, Brazil. Email: usharaniks2003@yahoo.com

Received: January 12, 2024 Accepted: February 16, 2024 Published: February 23, 2024

Abstract

The study intended to either kill or inhibit the growth of these pathogens in the milk by adding antibiotic substances from natural sources without denaturing the quality of the food material. The study aimed to determine the broad spectrum of antimicrobial activity of honey from two different sources natural (unprocessed honey) and commercial (processed honey). The inhibitory action of extracts of honey was evaluated against six bacterial pathogenic strains, Escherichia coli, Enterobacter cloacae Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecium, and Staphylococcus aureus by agar well diffusion method. The minimum inhibitory concentration of bactericidal activity was estimated and the presence of bioactive compounds by spectral analysis using a UV-visible spectrophotometer. The results were obtained by measuring the zones around the wells after the diminution of the well size. It was found to be positive results on cultures using honey samples. The growth of the undiluted culture was less inhibited by different honey samples and the diluted was highly inhibited by all honey samples, and the number of bacterial colonies was high in the undiluted culture whereas less in the diluted culture. The extract of honey showed better antibacterial activities contrary to pathogenic bacteria. Hence, it revealed that as the dilution rate increased, the antibiotic sensitivity also increased. The honey samples capable of antibiotic activity against clinical pathogens have the potential to be used as an effective tool for inhibiting the growth of pathogenic microorganisms and potentially used as a promising application in antibacterial agent against pathogens of raw milk.

Keywords: Honey; Agar well diffusion assay; Minimum Bactericidal Concentration

Introduction

According to the World Health Organization (WHO) measurements, up to 80% of the population in some established countries have used natural products in their primary health care [22,38]. Furthermore, 80% of people are influenced by these types of treatment in Asian countries including India [22]. The researchers have found that natural materials are normally more acceptable to users, and if these substitute methods are in effect, this may decrease the support for other synthetic substances [32]. Also, the study of such natural compounds may lead to the finding of a bioactive component that could prevent some ecological threats or effects on a disease process in humans [17]. The growth in the resistance of pathogenic bacteria to antibiotics is too a progressively vital factor behind the growing interest in the use of these natural compounds. The herbs, plant extracts, essential oils, and honey are the supreme collective sources for these bioactive compounds [17], and these products are effective against a range of bacterial infections and inflammatory cases [1,21].

Honey is the natural sweet substance produced by honeybees from the nectar of blossoms or from secretions of living parts of plants or excretion of plant-sucking insects on the active parts of plants, which honeybees gather, convert, and pool with specific substances of their own, store and leave in the honeycomb to mature and ripen. The honey is ranked by color, with the clear, golden amber honey often at a higher trade price than darker varieties. The flavor of honey will vary based on the types of flower from which the nectar was yielded. The presence of its nutritive, therapeutic, and dietetic quality in honey, is used in the food industry, medicine, and many other domains. From the time when prehistoric periods, it was discovered that honey is a medicine and is well known for its antibacterial activity, which was first reported in 1892 [31,20]. And also it has been used for the treatment and prevention of wound infections. With the advent of antibiotics, the medical application of honey was dropped in current Western medicine, though it is still used in traditional ways. For all antibiotic classes, including the major last-resort drugs, resistance is increasing worldwide and even more alarming, very few new antibiotics are being developed. The potential activity of honey against antibiotic-resistant bacteria resulted in renewed interest in its application and has been approved for medicinal application. The inadequate knowledge of the antibacterial compounds and the changeability of antibacterial activity are chief hindrances to the applicability of honey in medicine. In current years, the information on the antibacterial compounds in honey has expanded.

Nowadays, a lot of people group honey for its antibacterial and anti-inflammatory properties. The complete practitioners think through its unique nature's greatest versatile remedies [27]. The high sugar concentration, hydrogen peroxide, and the low pH are renowned antibacterial influences in honey, and further lately, methylglyoxal and the antimicrobial peptide bee defensin-1 and also phytochemical compounds, such as aromatic acids and phenols [2,6,27] Hegazi et al. 2017) were recognized as vital antibacterial compounds in honey. The antibacterial activity of honey is extremely composite due to the association of manifold complexes and due to the great difference in the concentrations of these composites among honey [28,29]. The information that honey has antibacterial properties has been documented for more than a century since it therapies for infections [34]. The honey’s resistance has not ever been stated nor has any toxicity effects, low cost of maintenance and local availability confer valuable advantages to using honey as a substitute antimicrobial therapy [40]. There is much information on the antimicrobial activity of honey against a wide range of bacterial and fungal species [7,14]. The antibacterial activity of honey performance has been stated first by van Ketel (1892), followed by Dustmann (1919). The following statement was by Sacket (1919), he also described that the antibacterial potency was augmented by limited dilution of honey, a comment that was hard to describe. The additional serious study did not begin until the work of Dold et al (1937). They make known the term 'inhibin' for the antibacterial activity of honey, a term which has been extensively used subsequently in the literature on honey. Later then there have been several accounts, some have been of simple testing that has revealed honey to have antibacterial activity, and these have often been done without credit of the prior finding of this by others. Furthermost, the study involved the activity spectrum of honey (i.e. defining which species of microorganism are sensitive to the action of honey) or comparison of different types of honey for the effectiveness of their action against one or additional species of bacteria. The practice of milk is quite common in our day-to-day activities. While using raw milk or even after boiling some of the pathogens remain in it and lead to some common infection along with other immune weakness either directly or indirectly. Therefore, the study intended to either kill or inhibit the growth of these pathogens in the milk by adding antibiotic substances from natural sources without denouncing the quality of the food material. The study aimed to determine the broad spectrum of antimicrobial activity of honey from two different sources natural (unprocessed honey) and commercial (processed honey). The screening and selection of pathogens are based on the literature survey and report from the Milma dairy industry (Kanhangad, KL, India). Hence, honey was selected as a natural antibiotic compound with the following properties; its medicinal properties, immunomodulatory properties, presence of glucose oxidase (Peroxide effect), and its availability.

Materials and Methods

Sample collection

The honey samples of three unprocessed and three processed were obtained from different sources and coded as follows respectively, Natural (N1), Aralam (N2), Jamun (N3), Lion (A1) Begood (A2), Dabur (A3), from Kasaragod District. All samples were kept in sterile screw cap tubes and stored at dark at room temperature.

Pathogen Selection and Collection

The pathogenic bacteria used in this study were Escherichia coli (a), Enterobacter cloacae (b), obtained from Kanhangad Diagnostic Centre (KDC) Lab (Kanhangad). These were subcultured on Nutrient agar and incubated aerobically at 37°C. Organisms were maintained in the laboratory on nutrient broth at 37°C for 24 h and then kept at 4°C before further experiments.

Serial Dilution and Estimation of Microbial Concentration

The serial dilution and pour plate method was used for the study using samples from pure cultures of bacteria. A liquid bacterial culture was inoculated and spread onto the surface of the agar plate. To ensure uniformity of sample distribution on the plate spreading in this instance, it should be done with a glass rod and not by the heavy streak method. The plate was incubated to allow bacterial growth and colonies were counted. Since every cell in the population will proliferate to a visible colony, the colonies on the plate characterize the number of cells that exist in the sample taken from the population. A 100μl sample was taken from the flask and placed in 900μl of water in tube A; the contents of tube A would represent a 10-fold dilution of the original sample. That is, the cell number per μl would be 1/10th of the original concentration. If 100μl was taken from tube A and placed into 900μl of water in tube B, that is another 10-fold dilution and represents a total concentration decrease of 1/100 from the original. If 100μl from tube B is placed in 900μl water in tube C, that is another 10- fold dilution now represents a 1/1000 concentration decrease of the original. Such dilution can be prepared up to 10 times dilution. Spread 100 μl samples from each tube into a culture plate and incubate for 24 hours to count the number of colonies.

Agar Plate Assay

In agar plate assay, for determining the concentration of bacteria used for the antibiotic activity of honey samples, therefore different dilutions were performed and serial dilution was done. For that, 100μl of broth was added into 900μl of deionized water and serially diluted. The 100μl of samples were transferred into sterile Petri dishes. The 20ml of nutrient agar medium was added. Also, the concentrations of pathogens at different dilutions were examined.

Broth Assay

Similarly for broth assay, to determine the bacterial concentration, 100μl of honey was added to 900μl of broth and serially diluted. The 100μl of each dilution was poured into a sterile Petri plate. Add 20ml of nutrient agar medium was added and left into laminar airflow for 24 hours. Then the concentration of pathogens at different dilutions was calculated.

Antibiotic Activity by well Diffusion Assay

The nutrient agar medium was prepared and poured into a sterile petri dish and left for solidification. Wells of 1cm diameter were made smoothly using a sterile test tube with 1cm diameter. Add 100μl of pathogenic bacterial culture as inoculum to nutrient agar medium into each petri plate and mix by using the spread plate method. After that, 100 μl of collected honey samples were poured into the wells individually and kept at 37°C for 24h. The results were expressed by measuring the zones around the wells after the diminution of the well size. The experiment was done in duplicate and the mean with standard deviation was calculated. Conferring to CLSI guidelines, these methods are often active in microbiological laboratories. For example, the agar diffusion assay procedure comprises smearing a petite volume of honey or a honey solution to the center of a well bored into a nutrient agar plate that has previously been inoculated with a bacterial culture. The honey disperses out into the agar from its application position while the plate is incubating. The zone of inhibition (ZOI), a clear zone adjacent to the honey application location, is a measure of the honey's efficacy [22].

Evaluation of Antibacterial Activity for the Presence of Hydrogen Peroxide or Proteinaceous Composites

The MIC values of the honey types treated with bovine catalase or proteinase K were evaluated and related to those of unprocessed honey. Concisely, 50% v/v honey in Muller- Hinton broth containing 100 μg/ml proteinase K or 600 U/ml bovine catalase was incubated for 16 h at 37°C, and then it was two-fold diluted and tested. The raised MIC values of the processed honey compared to the unprocessed honey showed the presence of hydrogen peroxide or proteinaceous compounds which supported the antibacterial activity of the verified honey types [24].

Influence of Individual Components on the Antibacterial Activity of Honey by Spectral Analysis

The diluted honey samples were analyzed for bioactive compounds by spectral analysis using a UV-visible spectrophotometer (UV-2600, SHIMADZU). The diluted samples were centrifuged at 5000 rpm for 4 hours and the catalyzed samples were subjected to spectral analysis.

Results and Discussion

The antimicrobial activity and Inhibition Zone Diameter of (IZD) of two different sources of Natural (unprocessed honey) and Commercial (processed honey) were determined for Escherichia coli and Enterobacter cloacae. The pathogens used in this study included Escherichia coli, Enterobacter cloacae Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecium, and Staphylococcus aureus. Both natural and commercial were effective against Escherichia coli and Enterobacter cloacae. The effect on Escherichia coli is somewhat more than Enterobacter cloacae. In this study, six honey samples were tested for their antimicrobial activity on Escherichia coli and Enterobacter cloacae. The present study showed varying degrees of growth inhibition activity of Natural and Commercial honey samples against the tested organisms. These might be due to the osmotic effect and the sensitivity of these organisms to hydrogen peroxide which are unsuitable for bacterial growth and represented as an inhibition factor in honey [19,30].

Honey has several well-known characteristics that are generally accepted as contributing to total antimicrobial activity. These include low pH, an osmotic effect, hydrogen peroxide production, and phytochemical factors [9]. The antimicrobial activity of many honeys can be attributed predominantly to hydrogen peroxide activity [13], evidenced by a decline in antimicrobial activity after treatment with the enzyme catalase. The medicinal effects of honey date back to the days of Aristotle (384-322 BC) for the treatment of sore eyes and wound infections [16,36]. The antimicrobial characteristics of honey have been established for a long time especially for wound healing [8,36]. Its activity may be due to its complex composition and its ability to generate hydrogen peroxide by the bee-derived enzyme glucose oxidase [4,12,15,33,36]. Regarding the number of strains sensitive to the action of honey, a relationship, and interactions between the origin of honey and its antibacterial activity was observed. The natural (N1, N2, N3) samples present a high antibacterial activity against all bacterial strains. The commercial (A1, A2, A3) also have high antibacterial activities. These findings confirm the ability of various types of honey to inhibit strains of pathogenic bacteria whether susceptible or resistant to standard antibiotics. The antibacterial activity of honey was different according to the area of origin. To maximize the therapeutic effects of honey, a careful selection of those with high levels of antibacterial activity must be made. This is possible by identifying the factors that increase the antibacterial effect. In the meantime, the isolation of honey’s active constituents may underlie the synthesis of new drugs with antibacterial effects.

Serial Dilution Method

The concentrations of the bacteria present in different dilutions were determined using serial dilution. Table .1, shows the concentration of pathogens Escherichia coli, Enterobacter cloacae Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecium, and Staphylococcus aureus present in different dilutions. The table reveals that the concentration of pathogens decreases when going for dilution, which means the number of bacterial colonies was more at dilution 10-1 with 356x10 (CFU/μl) in Escherichia coli and 252x10(CFU/μl) in Enterobacter cloacae. Similarly, less number of bacterial colonies are present at 10-5 dilution with 40x105(CFU/μl) in E.coli and 32x105(CFU/μl) in Enterobacter cloacae.