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. 2020 Mar 14;29(7):1013–1021. doi: 10.1007/s10068-020-00744-4

Effects of phenolic and protein extracts from Melipona beecheii honey on pathogenic strains of Escherichia coli and Staphylococcus aureus

Jesús Ramón-Sierra 1, José Luis Martínez-Guevara 2, Luis Pool-Yam 1, Denis Magaña-Ortiz 1, Alejandro Yam-Puc 1, Elizabeth Ortiz-Vázquez 1,✉
PMCID: PMC7297938  PMID: 32582463

Abstract

Antimicrobial effects of Melipona beecheii honey have been attributed to diverse factors, in this sense, certain components such as proteins and phenolics could explain relevant aspects of its antimicrobial activity. The aim of this study was to evaluate the antibacterial activity of phenolic and protein extracts from M. beecheii honey against two bacterial pathogens: Staphylococcus aureus and Escherichia coli. With respect to phenolic content, HPLC analysis allowed the identification of phenolic acids like chlorogenic acid, caffeic acid, and flavonoids like catechin, myricetin, quercetin and apigenin. On the other hand, seven bands with molecular weight from 7.6 to 95 kDa were detected in protein extract by SDS-PAGE system. It was determined the antibacterial activity of both extracts, with MICs lower than 145 µg/mL and 60 µg/mL for the phenolic and protein extracts respectively. These results indicate that phenolic and protein components of M. beecheii honey contribute significantly to the antibacterial activity.

Electronic supplementary material

The online version of this article (10.1007/s10068-020-00744-4) contains supplementary material, which is available to authorized users.

Keywords: Antibacterial activity, Phenolic compounds, Stingless bee, Melipona beecheii, Staphylococcus aureus

Introduction

The antimicrobial activity of honey has been explored in a scientific viewpoint for many decades and medicinally exploited for centuries. The majority of research has focused on honey produced by the European honeybee Apis mellifera and, until recently, relatively little attention has been directed to honey from stingless bees (Vit et al., 2004). The antibacterial activity of honeys from stingless bees has been studied in different reports (Irish et al., 2008; Miorin et al., 2003; Temaru et al., 2007). In the same sense, antifungal effect has been established; inhibitory activity of honey from the stingless bee Trigona carbonaria has been demonstrated against Candida albicans and Candida glabrata (Boorn et al., 2010). Also, in vitro assays have showed that stingless bee honey of Melipona beecheii showed higher antibacterial activity in comparison with Apis mellifera honey, i.e., minimum inhibitory concentrations (MICs) for E. coli and S. aureus using Melipona honey were significantly lower (from 2.5 to 3.5-fold) than those obtained for Apis honey (Chan-Rodríguez et al., 2012). In contrast, there are no reports about phenolic and protein components of stingless bee honeys with antimicrobial activities.

The components that could be related to antimicrobial activity are hydrogen peroxide, methylglyoxal, flavonoids, phenolic acids and proteins, i.e., isoenzymes that belong to the family of Major Royal Jelly Proteins (MRJP). Several authors have reported the non-peroxide antibacterial activity of stingless bee honey; which could explain the higher activity of these honeys (Nishio et al., 2016; Temaru et al., 2007). Considering the above, the main objective of this study was to evaluate the antimicrobial activity of Melipona beecheii honey of phenolic and protein extracts, in order to determine if these components could confer inhibitory activities against bacteria. Also, it was tested the toxicity of these extracts using hemolysis test, a standard procedure to determine if the extract could damage human blood cells (Asgary et al., 2005; Blasa et al., 2007). In similar experiments, it was tested if bacteria (Staphylococcus aureus and Escherichia coli) maintain hemolytic activity in the presence of extracts because the action of hemolytic enzymes is a relevant aspect of virulence in these pathogenic strains. On the other hand, it was investigated if the extracts could interact with nucleic acids such as DNA and RNA (Kanakis et al., 2005), an aspect that could explain the mechanisms related to antibacterial activity. Finally, it was compared the growth inhibition kinetics in presence of phenolic and protein extracts with the purpose of establishing if the mechanisms involved in antimicrobial action could be similar to the effects caused by available antibiotics. This is the first report of antimicrobial activity of stingless bee honey using phenolic and protein extracts against pathogenic bacteria, two components that are independent of hydrogen peroxide content.

Materials and methods

Honey samples and bacterial strains

Melipona beecheii and Apis mellifera honey were collected in the community of Maní in Yucatan, Mexico. The honey samples were collected directly from the hive. Two strains of standard pathogenic bacteria were used in this study, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The strains were grown in LB medium at 37 °C. All chemicals and reagents were either HPLC or analytical grade.

Analysis of the phenolic content of honey

Total phenolic compounds were extracted using Amberlite XAD-2 column (Fluka Chemie; pore size 9 nm, particle size 0.3–1.2 mm) (Ferreres et al., 1994) and stored at − 20 °C. The Folin-Ciocalteu method was used to determine the phenolic content, expressed as Gallic Acid Equivalent (GAE), in honey samples (Giorgi et al., 2011). Analysis of the phenolic extracts was performed on a Liquid Chromatograph-Series 200 Perkin Elmer, equipped with a reversed-phase C18 column (150 × 4.6 mm) and an ultraviolet detector Perkin Elmer 235 Clyndale, using formic acid at 5% v/v and HPLC grade methanol. Detection was performed at 290 nm. The different phenolic compounds were identified by comparing the retention times of HPLC standards.

Analysis of the protein content of honey

A sample of 25 g of honey was mixed with 2.5 mL of acetate buffer (1.6 M, pH 5.3) and 1.5 mL of NaCl (0.5 M), plus distilled water up to 50 mL. Total proteins from the mix were extracted as described previously by Peterson (1977) with modifications. Briefly, sodium deoxycholate (0.015% v/v) were added to the sample, slowly shaken and incubated for 10 min at room temperature. Afterwards, trichloroacetic acid (72% v/v in water) was added and the sample was stored at − 20 °C for 20 min, after this proteins were precipitated by centrifugation at 1900×g for 30 min and washed three times with pure acetone. The sample was maintained at room temperature until the solvents were evaporated. After this, the sample was resuspended in 150 μL of deionized water. The protein content was determined using Coomassie Brilliant Blue G-250 (Bradford, 1976) and the proteins were subjected to SDS-PAGE discontinuous system in polyacrylamide gels (4% and 15%) and run at 100 V for 2 h; after which polyacrylamide gels were stained with silver nitrate.

Antibacterial activity assay

The antimicrobial tests were performed according to the methods described in Schwalbe et al. (2007). Briefly, the two bacterial strains were grown in LB medium for 12 h at 37 °C, after this time the inoculum was adjusted to a 0.5 McFarland scale, equivalent to 1 × 108 CFU/mL. Then, the initial inoculum was diluted to 1 × 106 CFU/mL to perform the test. For antimicrobial activity in disk diffusion 50, 60, 70, 80, 100, 120 µg of total protein extract and 70, 80, 90, 100, 120, 140, 160 and 180 of µg GAE of total phenolic compounds were employed. The plates were incubated for 24 h at 37 °C. After this, the diameters of inhibition were measured and reported in millimeters. The minimum inhibitory concentration (MIC) of antibiotics and samples was determined by the broth microdilution method CLSI procedures (2008). For determination of Minimal Inhibitory Concentration (MIC), the inoculum was prepared as has been described above and similar concentrations of the extracts were tested: 50, 60, 70, 80, 100, 120 µg/mL of protein extract and 70, 80, 90, 100, 120, 140, 160 and 180 of µg GAE/mL of phenolic compounds. The cultures were incubated at 37 °C for 24 h and their absorbances were measured at 600 nm. The concentration of phenolic extract and protein extract that reduced the absorbance to zero in comparison with blank was taken as MIC. All experiments were performed in quintuplicate. Ampicillin (20 µg) and methanol were used as positive control and negative control, respectively. Growth kinetics in presence (up to 15% v/v) and absence of methanol were performed in order to establish if this solvent could have antibacterial activity. Low toxicity of methanol against bacteria has been previously reported (Ganske and Bornscheuer, 2006).

Analysis of the non-peroxide antibacterial activity of Melipona honey

Melipona honey was treated with catalase (3500 U/mg) at ratio of 3500 units per 1 mL of 50% honey solution in sterile water for 2 h at room temperature. The antibacterial activity and the minimum inhibitory concentration of the catalase-treated Melipona honey were determined by disk diffusion and microdilution assay, respectively.

Hemolytic activity assay

The hemolytic activity of the total extracts was determined using the method described previously (Park et al., 2010). The assay was performed with red blood cells obtained from human blood type O positive from a healthy donor. A suspension of red blood cells was adjusted to 0.1% (v/v) with phosphate buffer (0.05 M, pH 7.4) plus 0.9% of NaCl. Phenolic and protein extracts were added in concentrations corresponding to the MIC and S. aureus and E. coli cultures were incorporated to obtain a final concentration of 1 × 106 UFC/mL. Prior to the test, methanol of the total phenolic extract was evaporated at 40 °C and the total phenolic extract was resuspended in sterile water. In the case of protein extract, phosphate buffer and Triton X-100 (1% v/v) were used as negative and positive controls, respectively. In the case of phenolic extract methanol was used as negative control as in the case of phosphate buffer in protein test. Finally, the mixtures obtained were incubated for 1 h at 37 °C and centrifuged for 10 min at 800 g; the absorbance of the upper layer was read at 414 nm. The hemolysis percentage was calculated by the equation:

Hemolysis%=SA-NCA/PCA-NCA×100

where SA = Sample absorbance, PCA = Positive control absorbance, NCA = Negative control absorbance (Park et al., 2010)

Growth inhibition kinetics

The kinetics of the strains exposed to different antimicrobial agents (ampicillin, vancomycin, tetracycline and rifampicin) was performed as has been reported previously (Schneider et al., 2010) with slight modifications. Briefly, bacterial strains were grown overnight in LB medium and diluted in NaCl solution (0.85%) to obtain a final optical density at 600 nm (OD600) of approximately 1. NaCl solution with bacteria was adjusted to obtain a final bacterial OD600 of approximately 0.5 McFarland scale. After this, protein and phenolic extracts, catalase-treated Melipona honey and different antimicrobial agents were added to in concentrations corresponding to 2× of its MIC. The final mixtures were incubated at 37 °C and the OD600 of the samples were read every hour for a total time of 6 h.

Bacterial DNA/RNA-total extracts interaction assay

The interaction assay was performed according to methods established previously (Xu et al., 2007) with slight modifications. Genomic DNA and total RNA of bacterial strains was extracted using standard protocols (Valadez-Moctezuma and Kahl, 2000). In brief, the experiments were performed by mixing 500 ng of genomic DNA or 2 µg of total RNA with increasing amounts of the total extracts and diethyl pyrocarbonate (DEPC)-treated distilled water up to a 7 µL volume. A total extract of 10, 20 and 50 µg of protein extract and 35, 70 and 140 µg of phenolic extract were tested, respectively. The reaction mixtures were incubated at room temperature for 1 h. Subsequently, 3 µL of loading buffer (50% glycerol, 1 mM EDTA and 0.4% bromophenol blue) were added and the reaction mixtures were applied to 1% or 1.2% agarose gel electrophoresis for DNA and RNA, respectively, and run for 40 min at 100 V. Before the test, methanol of the total phenolic extract was evaporated at 40 °C and phenolic extract was recovered in (DEPC)-treated distilled water; the latter to prevent interference caused by methanol.

Results and discussion

Phenolic composition of M. beecheii and A. mellifera honey

According to the results, content of total phenolic compounds of M. beecheii honey was 515 ± 20 μg GAE/g of honey, this is a lower concentration than the phenolic content of Apis mellifera honey (946 ± 15 µg of GAE/g, p < 0.005) collected from the same geographic area (Maní, Yucatán). When comparing the phenolic composition of Melipona and Apis honeys obtained from the same geographical origin; it was found that the phenolic profiles of both honeys were different; the variations may be attributed to differences in source of nectar and bioactive components transferred from plants. Three phenolic acids were identified in M. beecheii honey: chlorogenic acid, caffeic acid, and ellagic acid, and four flavonoids: (+)-catechin, myricetin, quercetin and apigenin (Fig. 1A). Fourteen similar peaks were present in the chromatograms of both honeys from Yucatan. From these peaks, nine phenolic compounds were identified in Apis honey including the aforementioned seven, plus ferulic acid and kaempferol (Fig. 1B). Several of the phenolic compounds identified in both honey types of this study match with those reported by Alvarez-Suarez et al. (2018).

Fig. 1.

Fig. 1

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HPLC chromatograms of phenolic compounds extracted from (A) Melipona beecheii and (B) Apis mellifera honeys. (C) SDS-PAGE profile of proteins extracted from different honeys. Lane M: PageRuler™ Prestained Protein Ladder (Thermo Scientific); Lane 1: Melipona beecheii honey from Yucatan; Lane 2: Apis mellifera from Yucatan

Protein profile of M. beecheii and A. mellifera honey

The protein content was 1.86 ± 0.3 mg/g of honey in M. beecheii honey and in this case the concentration was higher in protein content in comparison with A. mellifera honey (1.38 ± 0.4 mg/g of honey), with a value p < 0.005. Seven protein bands with a molecular weight (MW) of 7.6, 11.6, 24.3, 37.3, 49.7, 55.4 and 95 kDa were found in the total protein extract of M. beecheii honey and seven protein bands with a MW of 5.5, 12, 23.5, 47.5, 55.2, 75.5 and 98.7 kDa were observed in the total protein extract of A. mellifera honey (Fig. 1c). In addition, a comparison of molecular weights of the protein profiles of honey showed that Melipona honeys had no similar bands with A. mellifera, therefore the protein profile could be more associated with the entomological origin than the botanical origin (Ramón-Sierra et al., 2015). Protein content of the honey is related to its production, a process that takes place in the digestive tract of bee, some of the bee enzymes involved in the transformation of honey must remain in this natural product.

Antimicrobial activity and MICs of Melipona honey and extracts

In the disk diffusion assay, Melipona honey showed antimicrobial activity against both E. coli and S. aureus; in the same test, catalase-treated Melipona honey was able to inhibit the growth of both bacteria (Fig. 2A). These results demonstrated that non-peroxide components could be related to antimicrobial activity. Similarly, with respect to the antibacterial activity evaluated by microdilution assay, catalase-treated Melipona honey showed antimicrobial activity against S. aureus and E. coli strains (Table 1).

Fig. 2.

Fig. 2

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(A) Inhibition zone diameters of Melipona honey (MH) and catalase-treated Melipona honey (MHT) against E. coli and S. aureus. (B) Inhibition zone diameters of the total phenolic extract (PHE) and total protein extract (PRE) of Melipona honey against E. coli and S. aureus (*** and **** indicate significant difference between the treatments, p < 0.005). (C) Effect of total phenolic extract (PHE) and total protein extract (PRE) on human red blood cells in the absence or presence of E. coli and S. aureus

Table 1.

Minimal inhibitory concentrations (MIC) of Melipona honey, catalase-treated Melipona honey a non-peroxide extracts of Melipona honey

Minimal inhibitory concentration
S. aureus E. coli
Melipona honey (MH) 190 ± 10 mg/mL 150 ± 10 mg/mL
Catalase-treated Melipona honey (MHT) 200 ± 10 mg/mL 210 ± 10 mg/mL
Total phenolic extract (PHE) 145 ± 3 µg GAE/mLa 100 µg ± 2 GAE/mLa
Total protein extract (PRE) 54 ± 2 µg/mLa 60 ± 2 µg/mLa
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aData are reported as Mean ± SEM with N = 5

Notably, the growth of E. coli and S. aureus was inhibited by both the phenolic and protein total extracts (Fig. 2B). A major susceptibility to protein extract was observed in S. aureus strain, this could be related to certain components of Gram-positive bacteria (Fig. 2B). The antibacterial effect of phenolic extract of honey has not been previously studied, however de Queiroz Pimentel et al. (2013) reported antibacterial activity of Melipona compressipes manaosensis honey against a wide range of Gram positive and Gram negative bacteria, that honey contains several phenolic compounds (catechin, apigenin, kaempferol, myricetin and quercetin) which were found in the M beecheii honey of this study. Although methanol did exhibit temporal antibacterial activity for the first 24 h during bacterial growth, the effect of the total phenolic compounds dissolved in methanol was maintained meanwhile the antibacterial activity of methanol disappeared after this time. Furthermore, for these concentrations of the total phenolics, the bacteria were able to grow up to 15% of methanol used as control in liquid media (Fig. S1). Hence, this confirms that the compounds of the total phenolic extract have antibacterial activity and the effect of methanol is negligible.

With respect to the antimicrobial potential of the protein extract of Melipona honey, this inhibited the growth of both evaluated pathogens. Several authors have suggested that the antimicrobial activity of honey proteins are due to peptides and proteins with low molecular weights, as is the case of the extract of Melipona honey (Kwakman et al., 2011b; Temaru et al., 2007).

The minimum inhibitory concentrations of the total phenolic extract were 100 μg of GAE/mL for E. coli and 145 μg of GAE/mL for S. aureus (Table 1). Furthermore, the minimum concentration of the total protein extract to inhibit the growth of E. coli was 54 µg/mL while 60 µg/mL were required for S. aureus (Table 1). Catalase-treated Melipona honey MICs against E. coli and S. aureus were 210 mg/mL and 200 mg/mL, respectively (Table 1).

The results obtained using catalase-treated Melipona honey suggested that the influence of other components such as the phenolics and proteins have a relevant role in antimicrobial activity of this natural food (Ortiz-Vázquez et al., 2013; Zainol et al., 2013). Dissimilar effects observed in disk diffusion method in comparison with MIC determination could be explained by the fact that diffusion of honey components is not the same in agar media compared with liquid media and this diffusion causes changes on antimicrobial activity against bacteria (Table 1 and Fig. 2A).

Hemolysis assays

The phenolic extract exhibited a hemolytic activity of 0.36% meanwhile the protein extract did not show any hemolytic activity (Fig. 2C). On the other hand, cultures of E. coli and S. aureus strains without treatment showed hemolytic activity of 0.63% and 0.83%, respectively. The hemolytic activity of these bacteria may be explained by their ability to produce metabolites with different bioactivities in order to colonize a host. S. aureus and E. coli increased their hemolytic activity when combined with total phenolic extract.

However, the hemolytic activity of both bacteria had a significant reduction when mixed with the total protein extract; this reduction of hemolysis in E. coli and the total elimination of this activity in S. aureus could be explained by the effect of proteins against toxins that are produced by these bacteria. In this sense, ability of certain peptides to abolish the hemolytic activity based on electrostatic interactions been reported previously for hemolysin E of E. coli (Yadav et al., 2008).

Growth inhibition kinetics

The kinetic measurements of S. aureus exposed to Melipona honey revealed that these cultures showed a kinetic behavior very similar to the cultures grown in the presence of phenolic extract (Fig. 3A). An increased in absorbance compared with the control was observed at the beginning of growth, maybe caused by the presence of sugars in honey or methanol in phenolic extract, however; a dramatic reduction of growth was detected after 200 min and 300 min of incubation in presence of honey or phenolic extract, respectively. This could indicate that the diffusion of components of honey or compounds present in phenolic extract is a critical point for antimicrobial activity and certain time is required to interrupt bacterial process in presence of honey or phenolics. In the same way, cultures exposed to protein extract exhibited similar kinetics to cell-wall interfering agents such as ampicillin (Fig. 3B). In contrast, when S. aureus was exposed to catalase-treated Melipona honey, a kinetic profile similar to tetracycline was observed. Due to this, is tempting to speculate that honey components could interfere with the bacterial translation process (Fig. 3C).

Fig. 3.

Fig. 3

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Growth kinetic measurements of S. aureus (A) and E. coli (B) exposed to Melipona honey (MH), total phenolic extract (PHE), total protein extract (PRE), catalase-treated Melipona honey (MHT) and antibiotics with known cellular targets, such as Ampicillin (AMP), Rifampicin (RIF), Vancomycin (VAN) and Tetracycline (TET)

Growth inhibition kinetics of E. coli demonstrated that kinetic behaviors of cultures exposed to protein extract were comparable to cultures grown in the presence of vancomycin (cell wall interfering agent) (Fig. 3D). Furthermore, a comparison of the growth inhibition kinetic of E. coli exposed to catalase-treated Melipona honey showed a similar behavior to the kinetic of cultures exposed to rifampicin, an antibiotic which primary target is the transcription process (Fig. 3E). Enhanced antimicrobial activity of catalase-treated Melipona honey was observed in kinetics of growth (Fig. 3C, E), this could be caused by the production of reactive oxygen species after the hydrogen peroxide has been removed, an effect that has been observed in other reports when bacteria is exposed to catalase activity (Bogdanov, 1997; Kono, 1995; Kwakman et al., 2011a). Differences in the kinetics of growth of S. aureus in comparison with E. coli employing catalase-treated Melipona honey could be explained by the composition of cell wall and metabolism of nucleic acids in each species. However, more research is needed in this sense to determine the specific mechanisms of antibacterial activity. Finally, growth inhibition kinetic of E. coli exposed to phenolic extract exhibit similarities with ampicillin (Fig. 3F).

Based on the results of growth inhibition kinetics, it is highly probable that phenolic and protein extracts could affect cell wall synthesis as well as transcription and translational process in bacteria (Brudzynski and Sjaarda, 2014). Difference in the profiles of growth inhibition kinetics could be explained by different susceptibility of bacteria to honey extracts, phenolics have higher activity against Gram-negative bacteria such as E. coli and lower effect on growth of Gram-positive bacteria like S. aureus. Puupponen-Pimiä et al. (2001) reported that small phenolic compounds inhibited the gram negative E. coli and Salmonella; inhibitory effects involved the disruptive action of these compounds on the outer membrane. At this respect, comparable results have been reported in Apis mellifera honey produced in Canada where growth inhibition kinetics showed a remarkable effect on bacterial growth during log-phase, resembling the antimicrobial activity of ampicillin, and morphological changes in cell wall (Brudzynski and Sjaarda, 2014). Also, the experiments performed using nucleic acids could explain a probable mechanism about the antibacterial effect of Melipona honey extracts.

Bacterial DNA/RNA-total extracts interaction assay

The DNA binding assay demonstrated the ability of the total phenolic extract to interact with genomic DNA of E. coli and S. aureus. As shown in Fig. 4, genomic DNA bands of these bacteria showed a specific interaction that caused extremely low mobility when 500 ng of DNA plus 140 µg GAE of total phenolic extract was tested. Accumulation of DNA was observed in the initial point of migration, nucleic acids were retained in wells due to interaction with extract. Similarly, total protein extract exhibited interaction with the genomic DNA of E. coli and S. aureus, thus, bands of genomic DNA of these bacteria was retained in the superior zone of electrophoresis gel in presence of 50 µg of total protein.

Fig. 4.

Fig. 4

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DNA and RNA binding assay. (1) DNA (500 ng) or RNA (2 µg) in the absence of extracts, (2) 35 µg GAE of total phenolic extract (PHE) or 10 µg of total protein extract (PRE) were added to nucleic acids, (3) 70 µg GAE of PHE or 20 µg of PRE were added to nucleic acids, (4) 140 µg GAE of PHE or 50 µg of PRE were added to nucleic acids

Furthermore, total phenolic and protein extracts also showed a strong interaction with RNA of E. coli and S. aureus, they caused a decrease in the ability of migration of these bands of bacterial RNA in the presence of 70 µg GAE of phenolic extract or 20 µg of protein extract.

Notably, similar results have been obtained when antifungal and antibacterial compounds are exposed to nucleic acids, this could explain the antimicrobial activity of the referred compounds, interaction may avoid the correct processing of nucleic acids (Hsu et al., 2005; Xu et al., 2007).

The results suggest that honey flavonoids and proteins could be associated with cell wall inhibition and interactions with nucleic acids. It can be hypothesized that the main reason for the antibacterial activity of phenolic compounds is their reaction with DNA and their potential mutagenic effect; which is higher in gram negative bacteria (Puupponen-Pimiä et al., 2001). The germicidal effect of phenolic compound is dependent of the type of phenol and the conditions of honey, especially the pH, which could stimulate the activity. Several reports document that phenolic compounds could either inhibit the protein synthesis or oxidize membrane proteins in gram positive and gram negative bacteria (Bouarab-Chibane et al., 2019; Puupponen-Pimiä et al., 2001).

The difference of susceptibility to polyphenols between Gram-positive and Gram-negative bacteria is still a controversial issue. Recently Bouarab-Chibane et al. (2019) reported strain-dependent effect since the same polyphenol could be effective on one type of Gram-positive (or Gram-negative) strain and ineffective on the other ones. The wide variety of phenolic compounds in Melipona honey means that it can have different mechanisms of antibacterial action.

These results reveal for the first time that the antibacterial activity of the Melipona honey is strongly influenced by its phenolic and protein content. In conclusion, Melipona honey and its components are able to exhibit multiple antimicrobial mechanisms, which could hamper the development of resistant bacterial strains.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10068_2020_744_MOESM1_ESM.tiff (190.3KB, tiff)

Figure S1. Effect of methanol (15% v/v) in the growth of cultures of E. coli (a) and S. aureus, the kinetics of bacterial growth showed a minimal effect of inhibition in presence of methanol (TIFF 190 kb)

10068_2020_744_MOESM2_ESM.tiff (3.7MB, tiff)

Figure S2 Colony-formed Unit vs protein concentration (a) and phenolic concentration (b) (TIFF 3757 kb)

Acknowledgements

The authors would like to thank Dr. Luis Cuevas for providing the stingless bee honey used in this study, National Council for Science and Technology (CONACYT, México) Grant of Basic Science CB-221624 and National Technological Institute of Mexico (TecNM) Grant No. 6748.18-P for financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Not required.

Footnotes

Publisher's Note

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Contributor Information

Jesús Ramón-Sierra, Email: jesus.ramon.sierra@gmail.com.

José Luis Martínez-Guevara, Email: josmtz@ibt.unam.mx.

Luis Pool-Yam, Email: felipe-19201@outlook.com.

Denis Magaña-Ortiz, Email: dmagana@yahoo.com.mx.

Alejandro Yam-Puc, Email: alejandro_yam@yahoo.com.

Elizabeth Ortiz-Vázquez, Email: eortiz@itmerida.mx.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10068_2020_744_MOESM1_ESM.tiff (190.3KB, tiff)

Figure S1. Effect of methanol (15% v/v) in the growth of cultures of E. coli (a) and S. aureus, the kinetics of bacterial growth showed a minimal effect of inhibition in presence of methanol (TIFF 190 kb)

10068_2020_744_MOESM2_ESM.tiff (3.7MB, tiff)

Figure S2 Colony-formed Unit vs protein concentration (a) and phenolic concentration (b) (TIFF 3757 kb)

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