Abstract Malaysian Kelulut honey has been found contain antibacterial activity towards many Gram – positive and Gram – negative bacteria.  This potential is related to its physical and chemical component that presents in Kelulut honey. This study focuses on Kelulut honey capability as a new natural product with therapeutic effect on Mycobacterium tuberculosis clinical samples. Antimycobacterial activity was assessed by broth microdilution and time-kill kinetics assay while the effect of Malaysian Kelulut honey against Mycobacterium smegmatis was investigated using Field Emission Scanning Electron Microscope (FESEM). The minimum inhibitory concentrations (MIC) ranges from 4 % to 8 % (w/v) while the minimum bactericidal concentration (MBC) was recorded at 8 % (w/v) for all honey samples. At 4 % (w/v) Heterotrigona itama (JP) honey demonstrated bactericidal activity within 72 h. Treatment with Kelulut honey resulted in cell deformation and structural irregularity with surface roughening to the M. smegmatis. From this study, it shows that Malaysian Kelulut honey exhibits antimycobacterial activity where it is observed to have bactericidal effect against M. smegmatis at 8 % (w/v). Thus, this study elucidated potential of Malaysian Kelulut honey as a natural therapeutic agent against Mycobacterium species.

Keywords: Kelulut honey, Antimycobacterial, Physicochemical, Phytochemical, Therapeutic effect

Funding: The authors wish to thanks Universiti Tun Hussein Onn Malaysia for supporting this research under Geran Penyelidikan Pasca Siswazah (GPPS Vot No. Q670), UTHM and Multidisciplinary Research Grant UTHM (MDR Vot No. H507).

Citation: Talip, B. A., Kahar, E. E. M., Johar, I., Fauzi, N. A. M., Razali, M. F., Muthalib, K. A., Kodeem, N. N. M. 2025. Investigation of antimycobacterial properties of Malaysian stingless bee (Kelulut) honey using the Mycobacterium smegmatis model. Natural and Life Sciences Communications. 24(2): e2025023.

INTRODUCTION

The bacterium Mycobacterium tuberculosis (Mtb) as a causative agent of the disease tuberculosis (TB) is a phenomenal pathogenic bacterium that can cause life-threatening diseases and latent infections, which can persist for a lifetime in the human host. Patient with positive TB disease means their immune system is not able to stop the proliferation of TB bacteria which makes the person sick. Mycobacterium spp. is a very unique bacteria with a very thick complex cell wall. TB has acquired antibiotic resistance against anti-TB drugs and is worsen by the emergence of multi-drug resistance TB (Smith et al., 2013). The complex cell wall of Mycobacterium spp. consists of a thick layer of mycolic acid and other features that is hydrophobic to solutes and other free lipids. It is almost impermeable for foreign compounds to pass through its cell wall, which act as an effective barrier from the penetration of antimycobacterial compound.

Mycobacterium smegmatis (M. smegmatis) is often used as a model organism in mycobacterial research due to its non-pathogenic nature and rapid growth rate. It shares many genetic and physiological characteristics with pathogenic mycobacteria such as Mycobacterium tuberculosis (M. tuberculosis), making it an ideal surrogate for studying the biology of mycobacteria and drug efficacy (Sparks et al., 2023). M. smegmatis has been used to evaluate the antibacterial properties of honey, including honey from Kelulut bees. This is due to the properties of honey as a broad-spectrum antimicrobial agent. These properties are due to the reducing sugar content, low pH, presence of hydrogen peroxide and other bioactive compounds (Sathammai et al., 2021).

Many studies have been conducted to investigate the efficacy of different types of honey, including Kelulut bee honey, in inhibiting the growth of mycobacteria. These studies benefit the potential of alternative treatments for mycobacterial infections (Mduda, Muruke and Hussein, 2023).

Honey is a well-known natural product that has been used by humans for many centuries for its nutritional and health purposes. A study by Hannan et al., 2014 has discovered the antimycobacterial activity of honey against Mycobacterium as a candidate for TB drugs (Hannan et al., 2014). For instance, Apitheraphy, a branch of alternative medicine that uses honey for medical treatment (Fratellone et al., 2015) in fact, has been used since ancient Egyptian time (Eteraf-Oskouei & Najafi, 2013). Sharma et al. (2008) reported successful reduction of the adverse effect of anti-TB drugs of Directly Observed Therapy (DOT), for patient newly diagnose with sputum of acid-fast bacilli (AFB) positive pulmonary tuberculosis patients of category I and suggested honey was also an adjuvant for pulmonary TB patients. Malaysian honey including Kelulut bee honey had been acknowledged for their superior antioxidant and antibacterial activity against various microbes (Mohd Kamal et al., 2021). As one of the tropical country, Malaysia is a habitat for many species of honey bee especially the Kelulut bee (Wee et al., 2020). Malaysian honey such as gelam, Kelulut and tualang were proven to have high non-peroxide antibacterial activity, comparable to New Zealand Manuka honey (Zainol et al., 2013). They found that Kelulut honey demonstrated consistent minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) at 20% (v/v) against all strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus. Additionally, the dose-response activity of Kelulut honey showed consistent inhibition activity for all different bacterial strains used in the experiment.

According to Zainol et al., (2013), most of the Malaysian honey was more effective towards Gram-positive bacteria. However, despite all the investigations done on Malaysian Kelulut bee honey, the research had not been carried out extensively, especially investigation on the properties and antibacterial activity. Kelulut honey was reported to have more superior antibacterial activity compared to Apis honey or stinging bee honey (Chan-Rodriguez et al., 2012; Kaewmuangmoon et al., 2012; Suntiparapop et al., 2012; Ewnetu et al., 2013). Furthermore, it has been hypothesized the non-peroxide dependent antimicrobial activity in some honey may be due to their unique phytochemical compounds. The potential of Kelulut honey as anti-TB agents cannot be underestimated. Researchers have reported the antimicrobial activities of honey against many Gram-positive and also Gram-negative bacteria (Demera & Angert, 2004; Snow & Manley-Harris, 2004; Singh et al., 2008; Pimentel et al., 2013).

In general, the antibacterial activities of honey depend on its physical and chemical components (Pimentel et al., 2013). The acidity of honey, and its phytochemical components contribute to the action of oxygen peroxide which involve glucose oxidase enzyme that is produce by the bee hypopharyngeal glands (Pimentel et al., 2013). Factors frequently claimed to be responsible for honey antibacterial value are the physicochemical properties such as high osmolarity, low pH, low water content, low protein and low metabolic by-product - hydrogen peroxide (Molan & Cooper, 2000). Honey has low moisture content which creates a high osmotic pressure that can kill or inhibit pathogenic cells by removing water out from the cell. Other compounds that had shown antioxidant capacity and are also associated with oxidative stress are phenolic acids, flavonoids and the enzymes glucose oxidase and catalase (Pimentel et al., 2013). Hence, studying the physicochemical and phytochemical properties of honey is important in order to assess its potential as natural antimycobacterial agent. This study will help to elucidate their potential as an antibacterial agent and increase their commercial value.

MATERIAL AND METHODS

Kelulut honey sample collection and preparation for antibacterial properties

The Kelulut honey samples were obtained from different parts of peninsular Malaysia (Table 1). All honey samples were collected during dry season and stored in dark sterile glass at 8°C. Kelulut honey was prepared according to method of each experiment while for antibacterial properties, Kelulut honey was prepared at highest concentration which was at 64 % (w/v). The 64 % (w/v) honey solutions were filtered through Whatman filter paper no. 1 then subjected to filter sterilization using 0.45 μm nylon filter (Agilent, USA). Sterile Kelulut honey samples were stored in sterile glass vial at 8°C. Figure 1 showed the Kelulut honey samples in bottle.

Table 1. Nest location of honey samples.

Note: * According to honey supplier, HM honey might be a mixed of honey from more than one farm.

Figure 1. Malaysian Kelulut honey samples. From left to right: A: Heterotrigona itama (JP); B: Humaira honey (HM); C: Geniotrigona thoracica (TH); D: Lepidotrigona terminata (LT).

Determination of moisture content and soluble solid (Brix˚)

Water content of Kelulut honey samples was determined according to standard protocol of International Honey Commission 2009 (Bogdanov, 2009). The moisture content was measured using a refractometer (Atago, PAL-BX/RI, Japan) at 20˚C where the honey was measured as Brix˚ value and refractive index (R.I.) (Yegge et al., 2019). The moisture content was calculated using the formula as introduced by Wedmore (1955) (Equation 1). Samples were homogenized by heating it in water bath at 50˚C until the sugar crystals dissolved, then they were allowed to cool to room temperature. The cooled honey samples were stirred slowly to prevent introducing air bubbles during the process. Few drops of samples were transferred onto the refractometer prism after sample homogenization. Readings were taken after the samples were left to sit for 2 minutes.

Determination of pH and free acidity

The pH and free acidity of samples were determined with reference to standard protocols of International Honey Commission 2009 (Bogdanov, 2009). To determine the pH, ten (10) grams of Kelulut honey samples were dissolved in 75 ml distilled water then the pH was measured using pH meter (Eutech Instrument pH 700, US).

Next, the homogenized solution was titrated with 0.1 M sodium hydroxide (NaOH) solution to pH 8.30 (Ismail et al., 2018). Free acidity, which is the total free acids in honey, is expressed in milliequivalents/kg honey. Free acidity was determined using Equation 2.

Free acidity (milliequivalents acid/kg acid) = volume of 0.1 M NaOH titrated x 10              (Equation 2)

Determination of Kelulut honey colour

The color of Kelulut honey were determined according to the method by Khongkwanmueang et al. (2020). Honey samples at 50% (w/v) were prepared with distilled water. The homogenized mixture was heated to 50°C to dissolve its sugar crystals. The mixture was centrifuged at 3200 rpm for five minutes and then transferred into 1.5 mL cuvette. Spectrophotometer (Thermoscientific, Biomate 3S, US) was used to measure the absorbance of Kelulut honey at 560 nm. The color intensity was determined using the Pfund scale after converting the absorbance values (Iglesias et al., 2012) (Equation 3)

mm Pfund = −38.70 + 371.39 × Absorbance value          (Equation 3)

Determination of carbohydrate content

Quantitative analysis of sugar composition in honey samples were sent for analysis in UKM Unipeq Sdn Bhd using the Harmonised Methods of The International Honey Commission, 1.7.2/ HPLC. All honey samples were sent for quantification of main carbohydrates in honey which were glucose, fructose, sucrose and maltose.

Determination of total phenolic content (TPC)

Total phenolic content in honey samples was determined using Folin-Ciocalteu according to Ooi et al. (2021) method. First, 0.5 mL honey solution at concentration of 0.1 g /mL was mixed with 2.5 mL of 0.2 N Folin-Ciocalteu reagents (diluted 10-fold with distilled water) and incubated for 5 minutes. Then, 2 mL of sodium carbonate solution (75 g/L) was added and the mixture was incubated for 2 hours at 25˚C. The absorbance value was measured at 760 nm using UV-Vis spectrophotometer (Perkin Elmer, Lambda 25, USA). The standard curve was constructed using gallic acid (0 - 1000 mg/L).

Determination of total flavonoid content (TFC)Total flavonoid content in honey samples was referred to Ya'akob et al. (2019) with slight modification. First, 5 mL of honey solution at concentration of 0.1 g/mL was mixed with 5 mL of 2% aluminium chloride (AlCl₃). The mixture was incubated for 10 min at 25˚C. The absorbance value was measured at 415 nm using UV-Vis spectrophotometer (Perkin Elmer, Lambda 25, USA). The standard curve was constructed using rutin (0 - 1000 mg/L). The data was presented in mg of rutin per kg of honey (mg/kg).

Bacterial culture

The model organism used was M. smegmatis mc2155 70084 ATCC, USA. The bacteria stock and working cultures were maintained on Middlebrook 7H10 (HiMedia, India) agar supplemented with 0.5 % (v/v) glycerol. All M. smegmatis liquid cultures were incubated in shaking incubator at 37˚C and 150 rpm for 72 hours. All Middlebrook 7H9 and Middlebrook 7H10 media were enriched with supplements as summarized in Table 2. Bacterial inoculum was prepared according to method by Wiegand et al. (2008). The purpose of using two (2) types of medias were these media is a selective media that suitable for Mycobacteria experiment.

Bacterial isolates were prepared by streaking a bead from freezer stock onto an agar plate and incubated at 37˚C for 72 hours. Three to five colonies with same morphological appearance were inoculated in 10 mL Middlebrook 7H9 broth (Sigma-Aldrich, USA), then incubated at 37˚C for 20 hours with constant agitation at 150 rpm. The bacterial culture was adjusted to achieve turbidity equivalent to 0.5 Mcfarland standard (Parvekar et al., 2020). Then, a 100-fold dilution was prepared and used immediately. It was assumed that 100-fold dilution of 0.5 Mcfarland standard M. smegmatis culture produced approximately 1 to 2 x 10⁶ cfu/mL.

Table 2. Nutrient supplements of media used.

Determination of minimum inhibitory concentration (MIC)

The assay was conducted to determine the minimum concentration of Kelulut honey required to inhibit ≥99 % growth of M. smegmatis in-vitro. The MIC was determined using broth microdiution method of susceptibility testing according to European Committee on Antimicrobial Susceptability Testing. Method of Khalifa et al. (2013) was employed for the optimization of Resazurin Microtiter Assay (REMA) Plate method. Several two-fold dilutions of Kelulut honey samples (64 % w/v) in supplemented Middlebrook 7H9 were dispensed in 96-well microtiter trays at volume of 50 μL. The final concentration of samples ranged from 0.0625 % to 32 % w/v. Fifty (50) μL of diluted suspensions (inoculum) were inoculated into the samples. Negative controls were prepared with 100 μL of broth media.

For honey samples sterility control, test samples of broth and honey samples were prepared. Positive control was prepared with two-fold dilutions of rifampicin (Sigma, USA). The final concentration of rifampicin ranged from 0.0977 μg/mL to 100 μg/mL (9.77 x 10⁶ % w/v to 0.01 % w/v). The plate was incubated at 37˚C for 72 hours. After 72 hours of incubation, 30 μL of resazurin solution (0.02 % w/v) was added into control well then incubated again at 37˚C until colour change was observed (blue to pink for positive control). Afterwards, 30 μL of resazurin solution was added into each well and further incubated at 37˚C for 12 hours. Colour changes of each honey samples were observed to determine the MIC.

Determination of minimum bactericidal concentration (MBC)

The MBC was performed using streak plate method with inoculum from the REMA Plate method. The negative REMA (wells with no bacterial growth) was tested. Three wells were selected at random for each sample of Kelulut honey. Loopful of bacterial culture was plated on Middlebrook MH10 agar and incubated at 37˚C for 48 to 72 hours. MBC was determined by lowest concentration of samples where no viable cell was observed on the agar plates. Any developed colony were recorded as bacterial growth (+) and no bacterial growth (-) (Hammond & Donkor, 2013). 

Sample preparation for protein profile analysis and morphology plasticity

Inoculum was prepared with previously elaborated method. After the bacteria was incubated for 16 hours or at approximately OD 600, exponentially growing bacterial culture was divided into 50 mL aliquots. Then, Kelulut honey samples and rifampicin were added into the aliquots. Fifty (50) mL of M. smegmatis culture without samples was used as control. The bacterial culture was incubated for 2 hours at 37˚C with constant agitation at 150 rpm.

Time - kill kinetics assay

For time-kill assay, drop method of Chen et al. (2003) was employed with slight modification. M. smegmatis was cultured in 10 mL supplemented Middlebrook 7H9 broth at 37˚C with constant agitation at 150 rpm with 4 % (w/v) and 8 % (w/v) honey samples concentration respectively. Bacterial culture without honey samples and with rifampicin at concentration of single and double MIC doses were prepared as control. Hundred (100) μL of 1 to 2 x 10⁶ cfu/mL inoculum was transferred into each medium in a 250 mL sterile conical flask. The inoculum was cultured for 72 hours. At time interval of 0, 8, 24, 48 and 72 hours, samples were obtained for plating on Middlebrook 7H10 with serial dilution. The plates were incubated at 37˚C for 72 hours. Colonies formed on agar plates, in the range of 3 to 30 colonies, were counted using cell counter (Rocker Galaxy 230) and recorded.

Morphology plasticity

Procedure of Piroeva et al. (2013) was employed with slight modification. Honey treatment as elaborated in previous section was performed. After 2 hours incubation period, the bacteria cells were harvested by centrifugation at 3500 rpm for 5 minutes. The supernatant was discarded and washed twice with distilled water. Then, 5 to
20 μL distilled water was added and the pellet was homogenized to prepare for fixation. Sterilized 18 x 18 mm cover slips were dipped into agar solution and left horizontally to allow it to dry for 30 min. The cells were fixed onto sterilized cover slips coated with 0.8 % w/v agar and left for 45 min to allow the samples to be embedded in the agar layer. Sample dehydration was done by immersing the fixed samples in ethanol solutions starting from low to high concentration (10, 25, 50, 75, 96 and absolute 99.99 %). For each immersion step, fixed samples were maintained in ethanol for 30 min. the samples were subjected to drying at 37˚C for 1 hour. the samples were coated with gold for 15 seconds to produce thickness of gold at < 10 mm. Morphology of cells were observed under SEM.

Statistical analysis

The physicochemical and phytochemical analysis were performed in triplicates and the results were expressed as mean values with standard deviations (SD) except for carbohydrates composition analysis. The significant differences were analysed with one-way analysis of variance (ANOVA) followed by Tukeys honesty significant difference (HSD) post hoc test (P < 0.05). Correlations analysis between properties of samples were conducted using Pearsons correlation coefficient (r) in bivariate linear correlations (P <0.01). All statistical analysis was conducted using IBM SPSS 19.0.

RESULTS

Moisture content, colour, soluble solid, pH and free acidity

The physicochemical properties of each honey sample were summarized in Table 3. The colour, pH and free acidity of honey samples differed significantly (P <0.05) while there was no significant difference with the moisture content (P >0.05). Among the four honey samples, JP exhibited darkest colour, highest moisture content, lowest pH and highest acidity. Positive correlation was found between colour and acidity of honey samples (P<0.01) with negative correlation observed with colour and pH (P<0.01).

Table 3. Physicochemical characteristics of honey samples.

Note: * a-d superscript letters in the same column denote significant differences (ANOVA p<0.05)

Carbohydrate content

Carbohydrate contents of honey samples are summarized in Table 4. The overall carbohydrate composition of Kelulut honey samples were characterized by lower contents of monosaccharides and elevated level of sucrose. High sucrose content in honey has been associated with the activity of honey enzymes such as diastase (amylase), invertase and glucose oxidase (Missio et al., 2016). The role of invertase is to break down sucrose into fructose and glucose along with glucose oxidase which later will produce hydrogen peroxide and gluconic acid from glucose (Bogdanov
et al., 2008).

Table 4. Carbohydrate composition of Kelulut honey samples.

Note: *N.D Not detected. *JP contained the highest level of fructose and glucose but lower level of sucrose compared to the other samples. Overall, all Malaysian Kelulut honey samples mainly contained sucrose compare d to fructose and glucose which is not common for honey to have high content of sucrose. However, nectar sources that are high with sucrose may contribute to Malaysian Kelulut honey samples carbohydrates composition

Total phenolic content (TPC) and total flavonoid content (TFC)

The phytochemical analysis showed that TPC of all honey samples differed significantly (P <0.05) while TFC of HM and TH showed no significant difference (P >0.05) (Table 5). Even though both TPC and TFC were representative of phytochemical content of a material, there was no correlation observed between the two in our study (P >0.01);

Table 5. Phytochemical content of honey samples.

Note: * a-d superscript letters in the same column denote significant differences (ANOVA P >0.05)

The samples JP, HM and TH showed bacteriostatic effect against M. smegmatis at concentration of 4 % (w/v) while sample LT recorded doubled their MIC at 8 % (w/v) (Table 6). On the other hand, bactericidal effect of all honey samples was recorded at 8 % (w/v). The bactericidal effect of Kelulut honey against M. smegmatis was lowered by 3.2 times when compared to the tested antibiotic, rifampicin.

Table 6. Susceptibility of M. smegmatis mc2155 to Malaysia Kelulut honey and rifampicin.

From time-kill kinetics assay of Kelulut honey samples against M. smegmatis, it was observed that JP sample (4% w/v) exhibited bactericidal activity after 72 hours even though its MBC was determined to be at 8 % (w/v). On the other hand, HM and TH samples resulted to substantial cells reduction after 6 to 24 hours of incubation period. At concentration of 12.5 μg/mL, rifampicin inhibited growth of M. smegmatis gradually and the inhibition rate became constant after 48 hours incubation period while 25 μg/mL rifampicin (MBC) killed ≥ 99 % of the cells after 48 hours.

When JP, HM and TH were applied at 8 % (w/v), cell decrement was observed at 6 hours and the bacteria were completely killed at 24 hours incubation period in contrast with bactericidal effect of 25.0 μg/mL rifampicin after 48 hours. At 3.2 times dosage of rifampicin, JP, HM and TH honey samples could achieve bactericidal effect at twice the speed. All Kelulut honey samples and rifampicin showed clear concentration-dependent killing activity and substantial time-dependent antimycobacterial activity; except for LT sample which showed moderate killing activity that was time-dependent.

The morphologies of M. smegmatis after being treated with JP sample, HM sample, TH sample and LT sample at 8 % (w/v) and rifampicin at 0.025 % (w/v) were shown in Figure 2. The untreated cells were in the shape of thin straight rods with slight curve, with typical cell length (3.0 - 5.0 μm) and intact surfaces (Figure 2A). In contrast, the morphology of cells treated with Kelulut honey (Figure 2B-2E) and rifampicin (Figure 2F) appeared deformed, shorter and wider, and suffered structural irregularity as compared to untreated cells. It was observed that the average length of treated cell was less than 2.0 μm, which was approximately 50 % the length of untreated cells. Surface roughening was observed with cells treated with JP, HM and LT samples. Besides, formation of septum with cell division as indicted with arrow in Figure 2B, D and E was observed with M. smegmatis treated with JP, TH and LT samples.

Figure 2. SEM micrograph at 8000 x magnification of exponential phase M. smegmatis - untreated and treated with Kelulut honey samples and rifampicin at MIC. The bacterial cells were incubated for 2 h at 37˚C. A: untreated M. smegmatis; B: M. smegmatis treated with JP at 8 % (w/v); C: M. smegmatis treated with HM at 8% (w/v); D: M. smegmatis treated with TH at 8% (w/v); E: M. smegmatis treated with LT at 8 % (w/v); F: M. smegmatis treated with rifampicin at 25 μg/mL. Indicated with arrow: formation of septum with cell division.

Correlation study between characteristics of honey samples

Colour and acidity of honey samples were found to be positively correlated with TFC of honey samples (P <0.01). This study demonstrated that with increment of colour of honey samples, there was an increment with its acidity concurrent with the increment of TFC of the samples. Among all physicochemical and phytochemical characteristics as determined, only TPC showed positive correlation with MIC (P <0.05). Results for correlation study is shown in Table 7.

Table 7. Correlation study of physicochemical and phytochemical characteristics and antibacterial properties of honey samples.

Note: * Correlation is significant at 0.01 level (2-tailed), c: cannot be computed because at least one of the variables is constant

DISCUSSION

Colour is an important parameter that reflects floral sources of honey besides its phytochemical profile due to pigmentation from chlorophylls, carotenoids, flavonoids and derivatives of tannins and polyphenols (A-Rahaman et al., 2013; Pontis et al., 2014). Previous study had demonstrated positive correlation between color and phenolic and flavonoid content and between phenolic vs flavonoid content besides proving that darker honeys exhibited higher antimicrobial activity (Alvarez-Suarez et al., 2010) and high amount of polyphenolics compound (Jenkins et al., 2015). In the present study, positive correlation was found between acidity (r=0.967) and colour (r=0.914) with TFC of Kelulut honey samples. Flavonoids from plant source had been proven to exhibit antimycobacterial activity through inhibiting cell wall and biofilm formation besides inhibiting bacterial DNA synthesis and efflux mediatied pumping systems (Mickymaray et al., 2020). Even though there is lack of evidence for the correlation between TFC and antimycobacterial activity, it was observed that JP sample (108 ± 0.0 mgrutin/kg ; highest TFC compared to other samples) was the sole honey samples that could achieve bactericidal effect against M. smegmatis within 72 hours at its MIC dosage (4 % w/v).

It is generally known that the antibacterial activity of diluted natural honeys are derived from the activity of H₂O₂ from enzymatic oxidation of glucose molecules (Kwakman et al., 2011). Antibacterial activity from non-peroxide factors could be attributed by Def-1 (Defensin 1), the bee antibacterial peptide, lysozyme, polyphenolic compounds and flavonoids (Alvarez-Suarez et al., 2010; Kwakman et al., 2011). Bucekova et al. (2018) postulated that polyphenolic compounds in honeydew honey enhanced their antibacterial activity by acting as pro-oxidants to accelerate radical formation and oxidative stress to the targeted microorganisms. Contradictory to the postulation, our study provided evidence that TPC was positively correlated with its MIC against M. smegmatis, by which LT samples required highest dosage (8 % w/v) against M. smegmatis despite exhibiting highest TPC content (317 ± 4.3 mggallic acid/kg) among other Kelulut honey samples. Zainol et al. (2013) reported similar findings where the increment of equivalent phenol concentration resulted in increment of MIC value. The authors speculated that the presence of other organic antibacterial factors by Kelulut bee might have contributed to different antibacterial properties of Kelulut honey. From this study, it could be deduced that polyphenolic content in Kelulut honey was not related to its antimycobacterial properties, however, further works are required to identify the phytochemical compound acting as major antimycobacterial factor in Kelulut honey.

Moisture is another physicochemical properties associated with antibacterial activity of honey. The osmotic stress exerted due to low moisture content and high sugar concentration could preserve honey from biological deterioration. The Kelulut honey samples studied were characterized by lower content of monosaccharides and elevated level of sucrose as compared to previous studies (Bogdanov et al., 2008; Saba, Suzana and Yasmin, 2013). The high sucrose content might be associated with enzyme activities which produce H₂O₂ for antibacterial activity. However, dilution up to 30 to 40 % negates the antibacterial activity of sugar concentration (Kwakman and Zaat, 2012). It was inferred that the high sucrose content of Kelulut honey samples might have exerted stress to the microorganisms but it was not the sole reason for its antimycobacterial activity. On the other hand, the probiotics properties of honey had been investigated and associated with its antibacterial properties (Lee et al., 2008; Rosli et al., 2020). Even though correlation between bacterial diversity in eight Kelulut bee honey and their antibacterial activity could not be drawn, Rosli et al. (2020) had mapped out that Lactobacillus bacteria, which is the microorganisms responsible for producing bacteriocins, were present in all investigated Kelulut bee species. In the present study, although the honey samples had been filter-sterilized prior to the antibacterial tests, antimycobacterial activity still could possibly can derived from the probiotics remained after filtration with presence of traces bacteriocins.

According to Ortiz-Vázquez et al., (2013), the antibacterial activity of honey towards bacteria depends on its floral, nectar sources and entomological origin. From our study, it was deduced that high antimycobacterial activity of Kelulut honey might be correlated with the amount of non-peroxide compound signifying Malaysia Kelulut honey may also contained high concentration of non-peroxide compounds (Zainol et al., 2013). Other factors that may contribute to the antimycobacterial activity is low pH value of samples which indicate high concentration of gluconic acid (product of glucose oxidase). Hence, if the glucose oxidase concentration is high, it will increase the concentration of hydrogen peroxide in honey.

Form the SEM images, surface roughening was observed with M. smegmatis treated with Kelulut honeys. This revealed that M. smegmatis exhibited morphological plasticity under mild starvation condition where long log-phase rod of starved M. smegmatis cells developed into small resting cell (SMRC) morphotype with threefold to four-fold shorter compared to log-phase bacilli. Besides, it was also reported that large resting cells (LARCs) were unaffected with no substantial morphological plasticity (Wu et al., 2016). The cellular targets and mechanisms of action of honey as antibacterial agents are still ambiguous. Brudzynski and Sjaarda (2014) reported morphological plasticity of E. coli after treated with various concentration of honey and ampicillin, where filamentous phenotypes and spheroplasts were observed. Cell wall disruption and increase permeability of lipopolysaccharides of E. coli were reported referring to microscopic analysis. In addition, Nishio et al. (2016) reported division inhibition and cell wall disruption with methicillin-resistant S. aureus (MRSA) N315 after being treated with Scaptotrigona postica and bipunctata honey. It was inferred that with increment of incubation period, division inhibition shall be observed with M. smegmatis after being treated with Malaysian Kelulut honey samples.

CONCLUSION

The color analysis of samples revealed variety of color, ranging from extra light amber (HM) to dark amber (JP). The carbohydrate composition of samples were characterized by the lower contents of monosaccharides (glucose and fructose), and elevated level of sucrose. JP which had the highest free acidity, moisture content and lowest pH showed the highest antimycobacterial activity compared to the other samples.  Malaysian Kelulut honey exhibits antimycobacterial activity where it is observed to have bactericidal effect against M. smegmatis at 8 % (w/v). JP Kelulut honey showed the highest antimycobacterial activity compared to TH, HM and LT. The SEM images indicated that Malaysian Kelulut honey had significant bactericidal agents and the in vitro antimycobacterial activity performed greater than rifampicin at MIC and MBC which is use in the TB treatment. Susceptibility of M. smegmatis to Malaysian Kelulut honey may indicate the potential of these natural products to be a promising candidate for anti-TB drugs.

ACKNOWLEDGEMENTS

This research was supported by Universiti Tun Hussein Onn Malaysia through Geran Penyelidikan Pasca Siswazah (GPPS), (vot Q670) and Multidisciplinary Research Grant UTHM (MDR Vot H507). The author also express gratitude to the university for providing working space and enough laboratory equipment during completing the experiment.

AUTHOR CONTRIBUTIONS

Balkis A. Talip: Conceptualization, methodology, supervision, project administration, funding acquisition, writing – review and editing, resources. Ernna Erynna Mohamad Kahar: Validation, data curation, writing – original draft, visualization. Izzat Johar: writing – original draft, formal analysis, investigation. Noor Akhmazillah Mohd Fauzi: Supervision, writing – review and editing. Muhammad Faiz Razali: Resources, data curation. Khairunnisa Abdhul Muthalib: Visualization. Nadia Nabila Mohd Kodeem: Formal analysis.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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