Antimicrobial Efficacy and Chemical Profiling Of Tribal And Commercial Honey Varieties: A Comparative Study Of It’s Therapeutic Potential

Honey is a naturally occurring sweet substance made by honeybees. It is known for its high nutritive value and benefits to health. Its usage as a food and medicinal product dates back to 7000BC. This complex mixture exhibits large variations in composition and characteristics. Its key characteristics are closely related to the source of flowers or the nectar that bees gather.
       
The major components of honey are directly dependent on the flower source, season, environmental factors and method of processing. It is composed of a range of nutrients. In honey, the major sugars present include, glucose and fructose as the monosaccharides. There are also 25 forms of oligosaccharides, such as sucrose, maltose, etc. (Bogdanov et al., 2016a). The total amount of fructose (38.5%), glucose (31%), maltose and sucrose (12%) in honey sums up to 81.5% of total carbohydrates (Khan et al., 2007).
       
Proteins and amino acids (proline, glutamic acid, aspartic acid, etc.), enzymes (invertase, oxidase, amylase), minerals (potassium, sodium, iron, copper, etc.), vitamins and organic acids, such as citric acid are also the primary constituents of honey (Hermosin et al., 2003; Jeffry et al., 1996; Alaqrni et al., 2014). The amino acid and protein content is quite less, i.e., about 0.7% in which content of proline, which is the main amino acid, is about 50-85%. The remaining amino acids include glycine, threonine, aspartic acid, histidine, tryptophan, lysine, ornithine, glutamic acid (Engin Gundogdu et al., 2019). Honey is also composed of tiny amounts of vitamins, of which the most composition is that of derivatives of vitamin B, including thiamine (B1), biotin (B8), riboflavin (B2), pantothenic acid (B5), nicotinic acid (B3), along with minor amounts of vitamin C (El-Bialee et al., 2011). Invertase, Amylase and Glucose oxidase are the enzymes which are manufactured in the glands of the worker honeybees called hypopharyngeal glands. Acid phosphatase and Catalase is extracted from plants (Engin Gundogdu et al., 2019).
       
The chemical characteristics affects viscosity, which in turn influences extraction methods, processing, preservation and packaging of honey (Tatjana pavlov et al., 2018). Since honey has a long shelf life, it can be kept for several years without being processed or given preservatives (Szweda, 2017). The honey colour ranges from light yellow or pale yellow to a dark reddish-brown colour (Engin Gundogdu et al., 2019). Suspended materials like pollen, propolis and bee wax is filtered out by straining the freshly harvested honey after it has been harvested for commercialisation. 5-hydroxy methylfurfural (HMF) is a parameter which determines how fresh the honey is. The concentration of this HMF will increase during processing or aging when compared to the fresh extracts of honey (Eshete and Eshete, 2019; Shapla et al., 2018).
       
Honey is also considered for various medicinal purposes. A number of studies conducted showed that being a natural reservoir of antioxidants, honey has the ability to reduce the risk of many diseases, such as heart diseases, cancer, different inflammatory diseases, etc. Components found in small amounts (flavonoids, phenolic acids, carotenoids, etc.) and the resultant substances of Maillard reaction exhibits many biological features, such as action against microbes and the ability to fasten the process of healing of injuries. The antagonistic effect of honey on microbes is associated with its high sugar concentration, low pH, as well as the action of phytochemical antibacterial compounds present in honey (Bucekova et al., 2019; Bogdanov et al., 2008). Since honey has a water activity of about 0.6 and a greater osmotic environment, which inhibits microbial growth, honey is thought to be self-stabilizing for extended periods of time (Raziuddin et al., 2021). The sugar molecules in honey use up the water molecules, hence there will be no water molecules available to assist the growth of microorganisms like bacteria, which die due to dehydration. The water content mainly depending upon many factors such as humidity throughtout the hive, weather (Malika et al., 2004). The natural acidic nature (pH ranges between 3.2-4.5) will inhibit the growth of many pathogens (Varlová et al., 2005). Hydrogen peroxide (H₂O₂) is the product of glucose oxidation by glucose oxidase which is introduced when nectar is being harvested. As H₂O₂ decomposes, it results in the formation of highly reactive free ions which reacts and kills bacteria (Manyi-Loh et al., 2011). The measured turbidity of honey from turbidimeter which is an indicator of honey granulation (Paula A. Conforti et al., 2006; White et al., 1963).        
The geographic region where the honeybees are raised, nectar of the flower, climatic conditions, etc., affect the anti-bacterial strength of the honey (Abd-El Aal et al., 2007). Defensin-1 (Def-1) is one of the main components for anti-bacterial activity generally against gram-positive bacteria (Kwakman et al., 2011; Tseng et al., 2011). Using high performance liquid chromatography (HPLC), several chemical compounds with anti-bacterial action have been found in the ether extract of honey (3,5-dimethoxy-4-hydroxybenzoic acid and methyl-3,5-dimethoxy-4-hydroxyl benzoate) (Selvamohan et al., 2016). Cerumen (made of wax) which is sectreted by the glands present in the abdomen region of worker bee and propolis which is derived from the plant’s resin used in order to seal the extra space between the nest and cavity. This propolis carries antifungal, antibacterial and antiviral properties (depending upon the resin content of the plants) (Temaru et al., 2007).
       
The current study is done to gauge the bactericidal properties of honey against various bacterial strains, i.e., Enterobacter aerogenes (E. aero.), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Klebsiella pneumoniae (K. pneumoniae), Bacillus cereus (B. cereus), Streptococcus, Pseudomonas aeruginosa (P. ae), Shigella and Proteus. The above-mentioned strains are typed under microbial type culture collection, research institute in Chandigarh (MTCC). Along with the test for confirmation of anti-bacterial activity, the test for some anti-bacterial compounds and health promoting nutrients was also carried out. 

Collection of honey
 
To perform this experiment three samples of processed honey from renowned honey brands, namely Dabur(P1), Saffola(P2), Lion(P3) along with one unprocessed honey, our primary test sample were extracted and the origin, source and location of the test sample was recorded. The sample was taken from different botanical sources in northern Karnataka. A tribal group from Haryana who procure honey directly from the bee hives provided this sample, which is also used to feed bears at Bannerghatta bear rescue centre (BBRC) as a refreshment. The experiment was conducted at the microbiology laboratory of Jain (deemed-to-be) University during December 2021. The honey sample was stored in Eppendorf vials at room temperature in the dark.
 
Tests for biochemical characterization and bactericidal activity
 
The biochemical tests were conducted with known techniques and the results were recorded. The antimicrobial activity was tested using the agar-well diffusion technique.
 
Agar-well diffusion method-Following the solidification of the media, 10 ml of pure bacterial culture from test tubes were inoculated onto the solidified nutrient agar media using the spread plate technique (Fig 1, Fig 2, Fig 3, Fig 4, Fig 5). The plates were then incubated for 24 hours. Following a 24-hour incubation period, honey samples were added to the wells that had been made using sterile gel puncture. The plates were then incubated again in the incubator for 24 hours at 37°C. The zone of inhibition/clearance developed around each well following incubation was observed, photographed and the zone’s diameter was measured. (Table 4).
 

 

 

 

 

Composition of honey
 
Anti-bacterial effect of honey
 
Moisture content
 
The FSSAI prescribed limit for moisture content of the honey is 20% (20 g/100 g). The analysis of the test samples presented that all the honey samples lie within the range of permissible limits except for sample P1. The sample’s content was found to range between 11.2% and 25.7% (Table 1), with sample P1 having the highest of 25.7, slightly exceeding the limits.
 

 
 
Protein content
 
The protein content of the tribal sample (96mg/10g) was comparable to two of the processed samples, Saffola (100 mg/10 g) and Lion (125 mg/10 g). Whereas Dabur (12 mg/10 g) sample showed significantly low amount of protein. The proximate analysis of the four different samples of the processed and unprocessed honey showed that the specific gravity ranged between 1.36-1.38 mg/10 g. The protein content ranged between 12-125 mg/10 g.
 
Total fat
 
The fat content in all the samples was found to be nil which is comparable to the standard range 0 g/100 g of the honey. Honey is mainly just sugar; it has almost no or very minute amount of fat, with little amount of fibre and protein. There were no significant differences (P>0.05) in the value obtained for specific gravity of the processed and unprocessed honey samples.
 
Calcium and potassium contents
 
A similar observation as protein was made in the calcium and potassium contents of the samples. The levels of calcium and potassium were significantly different in P1 sample as compared to other samples. The calcium content of the P1 sample was lower than the others. The value lied in the range of 12-23 mg/10 g. Whereas, the potassium levels of P1 sample was much higher than the others. The range was observed to be 10-65 mg/10 g. (tribal sample having the lowest of 10 mg).
       
From the results obtained by this study, the levels of potassium found in tribal honey samples was less compared to the other processed honey samples. As nutritionists mainly recommend the addition of honey to the low-potassium diet of a patient, the main reason behind this is because the potassium content of good-quality honey is low. This diet is mainly recommended for patients who are on haemodialysis. From this study, it was observed that potassium levels for other processed samples of honey were higher, even reaching up to 65. But the value for the tribal honey samples remained consistent at 10. This shows that the use of tribal honey in a low-potassium diet is much more effective rather than using the other commercial samples of honey (Brancati FL et al., 1996; Choi et al., 2013).
 
Sodium and phosphate content
 
Sodium content lies in the range of 3-40 mg/10 g, P3 having the least of 3 mg. The phosphate levels lied in the range of 0.02-0.18 mg/10 g (Table 1).
 
Colour analysis
 
The colour of the honey depends on various parameters which includes geographical origin, floral source, method of extraction, etc. Generally, the colour of the honey can range from being yellowish to reddish or greenish. The colour of the test sample was observed to be yellowish brown (tribal sample) to darker shades of brown (processed samples). The colour might also depend on the temperature, time of storage and the presence of antioxidant pigments like flavonoids and carotenoids (Kaur et al., 2016).
 
Vitamin C and total phenols
 
Table 2 represents the tests for phenols, terpenoids, flavonoids, tannins, alkaloids and vitamin C. The results showed the presence of vitamin C and all the polyphenols in the test samples. The antioxidant content in honey is made up of the existing polyphenols and flavonoids. The antioxidant content confers many health benefits to the honey. It keeps in check the ROS reactive oxygen species in the cells, whose accumulation may cause damage. The accumulation of this may also lead to premature aging, type 2 diabetes and heart diseases (Poljsak et al., 2013) (Table 2).
 

 
Antibacterial activity of the honey samples
 
The primary screening with the agar-well diffusion assay disclosed the anti-bacterial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Klebsiella pneumoniae (K. pneumoniae), Shigella and Streptococcus (Table 3). The antagonistic effect against other strains was not prominent since it did not show distinct zones. Considerable anti-bacterial activity of the tribal honey sample was seen against Bacillus subtilis(1.5cm) and Klebsiella pneumoniae (1.5 cm) which was marked by eminent zones of inhibition in the agar-well diffusion assay. Whereas, sample P1 was found to be more prominent against other strains of the test bacteria (Table 4).
 

 

       
From the study made on the antibacterial properties of honey, it was found that the tribal samples were the only honey samples that showed effective anti-bacterial activity against two bacterial species, i.e., Bacillus subtilis and Klebsiella pneumoniae. The other honey samples showed anti-bacterial activity against either one of the bacterial species or none. Bacillus subtilis is mainly found on the skin and in the intestinal tracts of human beings. Along with certain enzymes, it produces subtilisin, which is a toxin that causes irritation in the lungs. This toxin also causes skin allergies and continuous exposure can also lead to bronchitis (You et al., 1996). Hence, the use of the tribal honey sample can effectively produce an anti-bacterial activity against Bacillus subtilis and prevent the production of subtilisin. Klebsiella pneumoniae is one among the most significant pathogenic bacteria that causes urinary tract infections on   entering urinary tract. This bacterium is mainly found in hospital settings and can spread efficiently from the site of wound infection to other parts of the body, even causing septicaemia. K. pneumoniae  which is foodborne has shown resistance to certain antibiotics, making them more dangerous (Wyres et al., 2020). This study has shown that tribal honey samples are capable of producing anti-bacterial activity against K. pneumoniae indicating that the consumption of this honey is suitable for fighting off and preventing infections by K. pneumoniae.

This research has been made possible through the support and funding provided by Wildlife SOS and the Bannerghatta Bear Rescue Center in Bannerghatta, Bangalore. Gratitude is extended to Dr. A Sha Arun for his invaluable support and guidance throughout the course of this work. Additionally, we express our appreciation to Jain (deemed-to-be) University for generously allowing us access to their laboratory facilities for conducting our research.

All authors declared that there is no conflict of interest.