Physicochemical composition and antimicrobial potential of stingless honey : a food of differentiated quality

This study aimed to assess the antimicrobial activity of various honeys against strains of gram-negative and -positive bacteria, as well as to determine the physicochemical parameters of these honeys. Seven honeys from various species of stingless bees were evaluated. The Research, Society and Development, v. 9, n. 10, e7099108223, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8223 2 physical-chemical parameters evaluated were pH, moisture, water activity, acidity, ash, electrical conductivity and color. Antimicrobial activity was determined using disc diffusion agar tests and minimum inhibitory concentrations. We found that there was a relationship between the physical-chemical parameters and the antimicrobial activity. The minimum inhibitory concentration of 25% honey was able to inhibit the growth of both gram-positive and -negative bacteria; the greatest efficacy was verified for the species of bees Melipona mondury, M. quadrifasciata, Scaptotrigona bipunctata and Tetragona clavipes. Regarding synergism, Escherichia coli maintained its sensitivity profile in relation to all studied honeys combined with antimicrobials. An important factor to consider is the concentration of honey capable of sensitizing the microorganism, as it has been shown to be dependent on the species of the stingless bee. Nevertheless, all honeys showed antimicrobial activity in various methods of analysis. These data suggest that honey is a promising alternative to sensitize resistant microorganisms, for the health of humans and animals alike.


Introduction
Stingless bees are social insects, belong to the order Hymenoptera and the family Apidae (Meliponini), as well as species that are closely linked to tropical and subtropical regions for honey production (Crane 1990;Michener et al. 2013;Chuttong et al. 2016).
In Brazil, it is very common to use herbal medicines and homemade syrups with the use of honey in popular therapies, mainly by indigenous people and rural areas, because of the belief that this type of honey has healing properties (Posey 1987;Cortopassi-Laurino and Gelli 1991;Madaleno, 2015). Currently, due to the widespread concern about the growing emergence of resistant pathogens, honey has been promoted as an alternative for sensitizing microorganism's resistant to antibiotics and for being a natural product (Boorn et al. 2010;Pimentel et al., 2013;Campeau and Patel, 2014). Nevertheless, data on the antimicrobial activity of Brazilian honeys remains limited (Nishio et al. 2014;Bueno-Costa et al. 2016).
Therefore, the present study aims to evaluate the antimicrobial activity of various stingless honeys against strains of gram-negative and positive bacteria, as well as to determine the physicochemical parameters of these honeys and their relationship with antibacterial action.

Samples and material
The stingless honey samples (SHS) used in this study were collected in various cities in the state of Santa Catarina, totaling 20 honey samples from seven bee species (Table 1).
The samples were collected and stored under refrigeration and were protected from light in sterile plastic bottles with airtight closures. Development, v. 9, n. 10, e7099108223, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8223 (DECAGON, 2003). To determine the ash content, 5 grams of honey were weighed in a previously calcined crucible, and a muffle oven was heated to 550 °C for three hours (IAL, 2008). The pH, acidity and electrical conductivity of the samples were analyzed according to the recommendation Bogdanov et al. (1997). Color determination was performed with the aid of a spectrophotometer (Metrolab 1700 uvvis, JP) by the measurement of absorbance in the visible region at 635 nm in diluted solution of honey and distilled water (50:50) (m / v) being used the glycerin as white (Bogdanov et al., 1997). The determined values were compared to those in the Pfund table, according to the methodology described by Bianchi (1981).

Antibacterial activity
To determine the antimicrobial activity of antibiotic discs, the diffusion method described by CLSI (2015) and Brasil (2003) was used. To verify the synergism between the antibiotic discs and the different types of stingless bees honeys, the agar dilution technique described by CLSI (2003) was modified, using the differents honey in the dilution of the agar in the plate to evaluate the diffusibility and its antimicrobial activity. The technique used with honey for well agar diffusion was performed according to Perez et al. (1990). The minimum inhibitory concentration was determined according to the methodology of CLSI (2012).

Inoculum preparation
To assess the antimicrobial activity of the different techniques employed, three to five bacterial colonies isolated from each of the samples were selected from three to five bacterial colonies isolated from each of the samples, touching the top of each colony with a platinum handle and transferring them to tubes containing Brain Heart Infusion Broth (BHI). To evaluate the minimum inhibitory concentration (MIC), the same procedure was used, however the broth used was Muller-Hinton. The samples were incubated at 36 °C for 18 hours. A turbidity adjustment was performed to obtain an optical turbidity comparable to that of the 0.5 McFarland standard solution resulting in a suspension containing approximately 1 x 10 8 CFU/mL of E. coli (ATCC® 25922). For visual adjustment, a white background card with contrasting black lines was used.

Agar disc diffusion
Using a sterile swab moistened in the standardized bacterial suspension, the sample was wiped gently in all directions on petri dishes containing Muller-Hinton agar. The placement of antimicrobial discs in the petri dishes was carried out using forceps that had been flame-sterilized and then cooled. The plates were incubated in a bacteriological oven at 37 °C for 24 hours. Antimicrobial activity was measured by the diameter of the halos in millimeters, from the zone of inhibition formed around the discs according to CLSI (2015).

Agar dilution and synergism
The agar dilution technique described by CLSI (2003) was modified, each honey was diluted with agar and poured in a petri plate to assess diffusibility and synergism for the effect of antimicrobial activity. Muller-Hinton agar was autoclaved and cooled in a 45-50 ºC water bath. The volume of 5% honey of each species of bee was added in relation to the total volume, obtaining a concentration of 124.54 mg/ml of honey. The mixtures were placed in Petri dishes until a 3-4 mm thick layer was obtained. Importantly, the plates were produced on the day of the test. The procedure for spreading the inoculum, placing the antimicrobial discs, and for reading and classifying the bacterial isolates was carried out in a manner similar to that described for disc diffusion (CLSI, 2003).

Agar diffusion using the well technique
The culture medium used was Muller-Hinton agar; microorganisms were inculcated on the surface using a swab. Then, the wells were drilled using a sterile mold with a diameter of 6 mm. The honeys were tested in separate plates, dispensing the volume of 100 μL/well in the concentrations of 417.3 mg/mL (25%), 550.84 mg/mL (37.5%), 924, 61 mg/mL (50%), 1312.84 mg/ml (75%) (w/v) and 1525.09 mg/ml (100%). As a control (without adding honey), one well per plate was filled with 100 μL of autoclaved distilled water. The plates were incubated at 37 ºC for 24 hours, for later reading of the halos (Perez et al., 1990).

Minimal inhibitory concentration
The susceptibility to various stingless bee honeys was determined using the microdilution method in MH broth with a 96-well sterile plates, according to the CLSI Research, Society and Development, v. 9, n. 10, e7099108223, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8223 8 standards (2012). The honeys were tested at concentrations of 25%, 37.5%, 50%, 75% (w/v) and pure honey in total volumes per well of 200 μL (MH broth and honey). The added volume of each standardized inoculum was 10 μL for each well. The plates were incubated at 37 °C for 24 hours. Subsequently, 20 μL of 2,3,5 -1% triphenyltetrazolium chloride was added per well. If the color red appeared after 3 hours of incubation, it was considered to indicate microbial growth. The minimum inhibitory concentration was defined as the lowest concentration of honey with no visible growth after incubation (CLSI, 201;Mercês et al., 2013).

Statistical analysis
The triplicate averages of the data of the physical-chemical analyzes were subjected to the Kolmogorov-Smirnov standard testing, indicating a normal distribution. The studied variables were grouped by bee species and compared using analysis of variance; if there was a significant difference (P <0.05), the Tukey test at 5% significance was used. For the analyses of honey diffusibility x antimicrobial activity, the averages of the inhibition halos were grouped by species and presented with their respective standard deviations. All statistical analyses were performed using the OriginPro 8 software (Northampton, Massachusetts, USA).

Physico-chemical characteristics of honey
The pH of the honey samples ranged from 3.37 to 3.93, with no significant difference between the species Tetragonisca angustula and Tetragona clavipes (Table 2). There was a difference (P<0.05) of these two species in relation to Melipona spp. and Scaptotrigona bipunctata. Melipona marginata did not differ between the species evaluated.

Agar dilution and synergism
E. coli showed no change in the susceptibility profile, remaining sensitive against antimicrobials and when evaluating the synergism of stingless honey versus antimicrobials (Table 3). S. aureus was sensitive to all antimicrobials tested in isolation; however, there was a change in their susceptibility profile compared to the stingless honey versus antimicrobial test. When evaluated the honey of M. quadrifasciata, S. bipunctata and M. mondory versus ciprofloxacin, for S. aureus it changed its profile to intermediate.  When tetracycline was tested, there was no change in the profile that remained resistant even when associated with honeys, and the halos increased their diameter.

Agar diffusion using the well technique
Using this technique, we observed that the strains of gram-positive and -negative bacteria showed different behaviors in relation to honey samples, ranging from highly sensitive to totally resistant (    Research, Society and Development, v. 9, n. 10, e7099108223, 2020 (CC BY 4.

Discussion
Stingless honey has the intrinsic characteristic of being more hygroscopic, even in environments with lower humidity (Nascimento et al. 2015). The moisture content of the samples was above 20%, a parameter considered ideal by Brazilian legislation (Brasil, 2000).
Nevertheless, this difference did not negatively influence the growth inhibitory capacity of microorganisms. M. quadrifasciata honey had the highest moisture content found, corroborating results of the study by Alves et al. (2005), who found that this species, even in a dry climate, produces honey with high levels of moisture. Bogdavov and Blumer (2001) explain that, because water is essential for the oxidation process, hydrogen peroxide is produced significantly even in immature honeys that have high water content. In cases were moisture content is low in the mature honey, the oxidation process is limited by glucose oxidase being practically inactive. Honeys with low moisture content will have a small amount of hydrogen peroxide, which is known to prevent microbial growth (Laallam et al., 2015). Peralta (2010) found pH values for honey melon nectar between 3.2 and 3.9; Nascimento et al. (2015), obtained pH values between 2.93 to 4.08, values very close to those presented in this work. Campos et al. (2010) commented that pH is a parameter that assists in the assessment of total acidity and its variation may be related to the nutritional composition of the bee's diet, depending on the pH of the nectar and the mandibular substances. Lage et al.  Bogdanov (1997) states that the antimicrobial activity of honey correlates significantly with acidity, recognizing that the acid fraction positively influences biological activity, because pH acts as an antimicrobial factor.
Research, Society and Development, v. 9, n. 10, e7099108223, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8223 Based on our conductivity data, we classify the honey of the studied species as being derived from floral sources. Conductivity is closely related to the concentration of minerals, organic acids and proteins, with great variability depending on the floral honey source (Alves 2005;Suntiparapop et al. 2012;Nascimento et al. 2015). Nascimento et al. (2015) found values between 586.20 μS.cm -1 and 539.60 μS.cm -1 for various species of meliponids in Brazil. Chuttong et al. (2016), found a variation between 0.325 μS.cm -1 to 2.8 μS cm -1 for various species of stingless bees from Thailand. Bogodanov et al. (1999) stated that conductivity should be used as a criterion for the botanical determination of honey, replacing the analysis of ash content, because the former would be proportional to the ash content. The data from the present study suggest that electrical conductivity positive correlates with acidity, corroborating the statements of Bogdanov et al. (1999) and Alves et al. (2005); however, the same was not found with respect to ash content. Peralta (2010) found different values of acidity for stingless bees, ranging from 17.8 to 116.7 meq Kg -1 , similar to the data we obtained. The same author showed that there was a net negative correlation between acidity content and minimum inhibitory concentration for E.
coli, assuming that there was slight action of acidity on the variation of biological activity.
The present study also verified this relationship; the honey of T. clavipes and S. bipunctacta had the highest acidity levels and the lowest minimum inhibitory concentrations, by contrast with the honey of M. bicolor that presented the highest value of minimum inhibitory concentration and the lowest acidity value.
The ash content indicates the amount of minerals found in honey (Alves et at., 2005); in the present work, we observed that the ash values varied between 0.38% to 4.88%.
National legislation recommends up to 0.6% of ash in honey samples from Apis mellifera (Brasil, 2000). A factor that is correlated with the color of honey is the mineral content; according to the constitution of honey, a variety of various spectra and colors can be found. We observed that the various honeys of stingless bees had an inhibitory effect on the growth of gram-positive and -negative strains. The effect was related to the concentration and type of honey. This is different from findings reported by Basualdo et al. (2007), who found that the bactericidal effect of honey depended on the concentration of honey used and the nature of the bacteria.
With the diffusion test using the well technique, Fikselová et al. (2014)   In another study carried out by Jenkins and Chapagain (2014), the clinical isolate was more susceptible to manuka honey compared to the standard strain.
Our study was conducted only in vitro; however, we bacteria that are highly pathogenic to humans and animals. The results were positive; however, the mechanisms involved need to be clarified. Further studies using honey in vivo are needed to understand how the components of honey act against various microorganisms; the goal is to increase the efficacy of antibiotics toward previously resistant strains.

Final Considerations
All our tested honeys have antimicrobial activity, regardless of the stingless bee species. The effect of this activity is related to the concentration of honey used capable of sensitizing gram-positive and -negative bacteria, in addition to the technique used in the antimicrobial test. In relation to synergism, there are indications that confirm the influence of the type of stingless honey and tested antibiotic, changing the susceptibility profile of the bacteria. Other factors that influence antimicrobial activity are physical-chemical parameters, because the interaction between the constituents that make up honey contributes to the inhibitory capacity and changes the susceptibility profile of bacteria of both clinical origin and reference strains.
This food of animal origin is interesting and has antimicrobial potential. The development of new research must seek to know which biochemical mechanisms are related to the capacity of antimicrobial and make it a product with wide use, as for example in nutraceutical food in human and animal health.

Funding
There is not any funding sources or support for this study