Physicochemical characterization and antioxidant activity of honey samples of Apis mellifera and different species of Meliponinae subfamily from the Brazilian eastern Amazon region

Abstract

In the present study, we evaluated the physicochemical characteristics (color, humidity, soluble solids, free acidity, pH, ash content, carbohydrate content, and 5-HMF) and antioxidant activity (Folin-Ciocalteu reagent and DPPH free radical scavenging) of honey samples produced by different species of Meliponinae subfamily (n = 16) and A. mellifera (n = 16) from the Eastern Brazilian Amazon region. Considering global averages, the stingless-bee honey had high acidity (93.45 mEq kg^-1), high humidity (27.43%), low content of reducing sugars (55.65%), and darker colors (Pfund scale – 162.17 mm), when compared with A. mellifera honey (62.96 mEq kg^-1, 19.35%, 65.70% and 68.43 mm for the respective parameters). Furthermore, more than 50% of samples of stingless-bee honey had values of 5-HMF above that proposed by Codex Alimentarius (max. 80 mg kg^-1). The two types of honey investigated showed similar results for total phenolic compounds and antioxidant activity. The principal component analysis (PCA) of the physicochemical characteristics of the honey samples, showed that the A. mellifera samples formed a differentiated group, while the multi-species Meliponinae samples were more scattered along the PCA axes. The distinctive characteristics of stingless-bee honey compared to A. mellifera honey produced in the same region, reinforces the need for specific regulations for honey produced by stingless-bees.

Keywords:
Meliponinae; 5-HMF; DPPH radical; principal component analysis, bees

1 Introduction

Although there are more than 20,000 species of bees in the world, only those belonging to the Apidae family produce honey (Silva et al., 2020). Apinae and Meliponinae are the most well-known subfamilies (Silva et al., 2016; Ávila et al., 2018). Apis mellifera (Hymenoptera: Apidae: Apinae) is the species that dominates honey production worldwide. However, Meliponinae has demonstrated growing potential in the bee market. Popularly recognized as stingless-bees (Nordin et al., 2018), this family is divided into more than 500 species which are distributed worldwide in tropical countries, mainly in South and Central America, Africa, southwest Asia, and Australia (Ávila et al., 2018).

Several species of Meliponinae subfamily (usually known as stingless-bees) differ from A. mellifera due to their smaller size, varied body colors, and absence of stinger. Besides, stingless-bees have short flights to collect food, prefer creeping flowers, and do not make large selections of food, that is, collect what is available and easier; as for their hive, they build a nest divided into combs for chicks and a reservoir for honey and pollen in the shape of a pot.

Honey is a natural product produced by bees that contain high content of sugars, water, and several other minor compounds, such as phenolic compounds, proteins, vitamins, free amino acids, minerals, and organic acids, which make it a nutritious and beneficial food to human health (Nordin et al., 2018). In addition, honey is a food appreciated worldwide and the investigation of this physicochemical properties is essential to its classification and characterization (Wen et al., 2017; Zhou et al., 2018; Crăciun et al., 2020). Due to the entomological characteristics, the floral and habitat preferences of each bee, the A. mellifera, and multi-species of Meliponinae subfamily produce honey samples with different characteristics. While multi-species of Meliponinae subfamily honey has a high moisture content (> 20%), acidity (> 50 meqkg^-1), and low sugar content (< 60% w/w) (Ávila et al., 2018), the A. mellifera honey samples have been usually reported with a darker color, higher carbohydrate content (70-80% w/w), and lower moisture content (< 20%) when compared with the honey obtained from different species of Meliponinae subfamily (Rizelio et al., 2020). Consequently, the sensory characteristics of both kinds of honey have also been reported as different: stingless-bee honey has a slightly acidic flavor and a fluid texture. It has been also reported that these characteristics vary when comparing different species of stingless bees (Ávila et al., 2019).

In addition, studies have reported differences in the physicochemical and sensorial characteristics of honey samples produced by different bee species; as well as the presence of minority compounds content which may also reflect on the antioxidant activity of the honey (Guerrini et al., 2009; Silva et al., 2013a; Silva et al., 2013b; Crăciun et al., 2020). The antioxidant activity is one of the main biological properties present in some foods, and it is related to the prevention of oxidative stress caused by the free radicals (Zhang, 2005; Vásquez-Espinal et al., 2019; Rizelio et al., 2020; Farias et al., 2021). In addition, this property can vary due to the phenolic compound content present in the food (Nascimento et al., 2020).

It is important to highlight that the composition of honey can also be strongly influenced by edaphoclimatic conditions, flora available for nectar collection, and the maturation period (Dourado et al., 2019; Miłek et al., 2021). Therefore, the investigation of the characteristics of honey obtained from different regions of the world is important to have a broad knowledge of their composition (Sant’ana et al., 2020).

In Brazil, especially in the Amazon region, the diversified flora combined with a hot and humid climate and the presence of high biodiversity of bee species allows the production of different types of honey with unique properties (Bandeira et al., 2018; Dourado et al., 2019). The state of Pará, located in the Eastern Amazon, has an equatorial climate (Am in the Köppen-Geiger classification), with annual thermal averages between 24 and 26 °C and high rainfall. These characteristics might impact the composition of honey and lead to undesirable chemical reactions during its production, maturation, and storage, such as the Maillard reaction which produces the contaminant 5-hydroxymethylfurfural (5-HMF) (Shapla et al., 2018).

Due to the influence of these factors, some studies have compared the honey from different geographical origins and botanical sources (Maione et al., 2019), apiary (Scholz et al., 2020), storage or processing conditions (Silva et al., 2020), aiming to assess the impact of these variables on the quality of honey, especially the physicochemical characteristics. However, most of these studies considered only A.mellifera honey or Meliponinae honey individually (Silva et al., 2020; Biluca et al., 2016).

Thus, analyzes of the characteristics of the honey samples produced among these two groups of bees obtained from the same region have a high relevance due to their economic and nutritional importance. The comparison of sympatric samples of A. mellifera and stingless-bee honey samples allows us to properly discern the species-specific differences related to the production of the honey. In this context, the present study aims to perform a comparative study of the physicochemical properties and antioxidant activity of the honey samples produced by five species of stingless-bees (Meliponinae) and A. mellifera produced in the state of Pará, Eastern Amazon.

2 Materials and methods

2.1 Honey samples

Sixteen samples of honey from different species of stingless-bees (Meliponinae) and sixteen A. mellifera honey samples were obtained directly from producers or local markets in four city cities of Pará, Brazil: Itaituba (04º16’34”. 55º59’01”), Mojuí dos Campos (02º10’17”. 56º44’42”), Belterra (02º32’11”. 54º56’14”) and Santarém (02º26’34’. 54º42’28”) (Table 1 and Figure 1). The collected samples were conditioned and identified in Falcon tubes. The samples were collected and transported at room temperature to the laboratory and stored at 18 ± 2 °C in a darkroom.

Table 1
Identification of species of bees and geographical description of the municipalities of origin of the samples honey.

Code *	Beespecies	City	P/M **
MCM1	Melipona comprensipes manaosenseis	Itaituba	M
MCM2		Itaituba	M
MCM3		Mojuí dos Campos	P
MCM4		Mojuí dos Campos	P
MCM5		Belterra	P
MCM6		Santarém	P
MCM7		Santarém	P
MCM8		Santarém	M
MCM9		Santarém	M
SS1	Scaptotrigona sp.	Mojuí dos Campos	P
SS2		Belterra	P
SS3		Santarém	P
TA1	Tetragonisca angustula	Mojuí dos Campos	P
TA2		Belterra	P
FL	Frisiomelitta longipes	Belterra	P
MS	Melipona sp.	Santarém	M
AM1	Apis mellifera	Itaituba	M
AM2		Itaituba	M
AM3		Itaituba	M
AM4		Mojuí dos Campos	M
AM5		Belterra	M
AM6		Santarém	M
AM7		Santarém	M
AM8		Santarém	M
AM9		Santarém	M
AM10		Santarém	M
AM11		Santarém	M
AM12		Santarém	M
AM13		Santarém	M
AM14		Santarém	M
AM15		Santarém	M
AM16		Santarém	M

• ^*
  Identification code of the samples.
• ^**
  P = honeys collected directly by producers on rural properties; M = honeys collected at the local market.

Figure 1
Map of eastern Amazon region of Brazil indicating the sampling sites of honey of Apis mellifera and Meliponinae bees (this figure is in color in the electronic version).

2.2 Reagents

All reagents were analytical grade and deionized water was purified in the Milli-Q system (Millipore, Bedford, MA, USA). 5-Hydroxymethylfurfural (5-HMF), caffeine, sodium tetraborate (STB), sodium dodecyl sulfate (SDS), sorbic acid, and cetyltrimethylammonium bromide (CTAB) were obtained from Sigma-Aldrich (Santa Ana. CA, USA). D-(+)-glucose monohydrate, D-fructose, and sucrose were obtained from Merck (Rio de Janeiro, RJ, Brazil). 2,2-diphenyl-1-picrylhydrazyl (DPPH), monobasic potassium phthalate, α-tocopherol, and Folin-Ciocalteu were obtained from Sigma Aldrich (Santa Ana, CA, USA). Sodium hydroxide, gallic acid, sodium carbonate, and reagent ethanol reagents from Vetec (Rio de Janeiro, RJ, Brazil).

2.3 Physicochemical parameters

Moisture and soluble solids

Moisture (% m/m) and soluble solids (ºBrix) were determined by refractometry using Abbe Tropen model I refractometer (Carl Zeiss Jena, Germany) according to protocols 969.38 and 932.12 of Association of Official Analytical Chemists (2005), respectively.

Free acidity and pH

To evaluate the pH of the honey, 10 g of each sample were diluted in 75 mL of deionized water and then the pH was measured in pHmeter model mPA-210 (MS Tecnopon, Piracicaba, Brazil). Then, the solutions were titrated with 0.101 mol L^-1 NaOH solution (standardized with monobasic potassium biftalate) until pH 8.5 (Association of Official Analytical Chemists, 2005).

Ash content

The total ash content of the honey was measured by gravimetry, according to the method described by AOAC (Association of Official Analytical Chemists, 2005).

2.4 Analyzes in capillary electrophoresis (EC): 5-hydroxymethylfurfural (5-HMF) and carbohydrates

The contents of 5-HMFhydroxymethylfurfural and carbohydrates were determined by capillary electrophoresis (EC) (EC-Model 7100, Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array detector and a temperature control device maintained at 25 °C. Data acquisition and processing software were provided by the manufacturer (HP ChemStation rev. B 0.4.03).

The capillary micellar electrokinetic chromatography technique was used for the quantification of 5-HMF. The running electrolyte (BGE) was composed of 5 mmol L^-1 STB and 120 mmol L^-1 SD at pH 9.3 and stored at room temperature. The analyzes of the samples were conducted in uncoated fused silica (Polymicro Technologies, Phoenix, AZ, USA) capillary of 32.0 cm total length (8.5 cm effective length × 50 μm ID × 375 μm OD). The separation voltage of 30 kV with positive polarity was applied at the injection end and the detection apparatus at 284 nm (Rizelio et al., 2012; Biluca et al., 2014).

The contents of fructose, glucose, and sucrose were quantified using the capillary zone electrophoresis technique, via indirect detection. For these analyzes, the running electrolyte (BGE) was composed of 20 µmolL^-1 sorbic acid, 0.2 µmolL^-1 CTAB, and 40 mmolL^-1NaOH at pH = 12.2. For the analysis of the samples, a capillary with 60 cm of total length (8.5 cm of effective length × 50 μm ID × 375 μm of OD) was applied with injection done by hydrodynamic mode using a voltage of 25 kV and indirect detection at 254 nm (peak inversion at 360 nm) (Biluca et al., 2014; Rizelio et al., 2012).

2.5 Color evaluation

For color evaluation, the honey samples were diluted in deionized water to obtain a concentration of 50% (v/v). Then, the solutions were centrifuged at 300 rpm for 10 min in a centrifuge model NT815 (Nova Técnica, Piracicaba, Brazil). These solutions were read in a UV/Vis spectrophotometer (Nova 3300, Nova Instruments, Piracicaba, Brazil) at 635 nm. Finally, the absorbances were transformed to the Pfund scale (Gomes et al., 2017).

2.6 Reducing capacity activity and free radical scavenging activity

To evaluate the reducing activity and DPPH free radical scavenging activity, the honey samples were accurately weighed (2.5 g), and dissolved in deionized water using the proportion 1 : 2 (w : v).

The total reducing activity of the samples was determined by the Folin-Ciocalteu method (Singleton & Rossi, 1965), and aliquots of 0.1 mL of an aqueous solution of honey (0.5 g mL⁻¹) were homogenized with 2 mL of deionized water, 0.5 mL of Folin-Ciocalteu, and 1.5 mL of sodium carbonate solution (20% w/v), then adjusted to 10 mL volumetric flask. After 2 h the absorbance was measured at 765 nm in spectrophotometer SB 1810-S (Spectro Visium, Brazil). Deionized water was used as a blank. The results expressed as mg gallic acid equivalent (GAE) 100 g⁻¹.

The DPPH free radical scavenging activity of the samples was determined according to a previously published protocol (Gomes et al., 2017). An aliquot of 0.4 mL of honey solution (1: 5, v/v) was diluted with1.6 mL of ethanol and 0.2 mL of 1.2 mmol L^-1 DPPH solution. The mixtures were allowed to stand for 30 min under light and at room temperature. The absorbance was measured in UV/Vis spectrophotometer (NOVA 3300, Brazil) at 555 nm. The sequestration capacity of DPPH was expressed as a percentage. For calibration, a standard curve of α-tocopherol (vitamin E) was performed in a 0.2 mmol L^-1 ethanolic solution, with concentrations ranging from 0 to 148 2.50 mg L^-1.

2.7 Statistical analysis

All analyzes were performed in triplicate with values expressed as mean ±standard deviation and submitted to analysis of variance (ANOVA). The Tukey test at the 5% probability level was applied to evaluate the difference between the analyzed samples. The principal component analysis (PCA) was used to explain the interdependence of the analyzed physicochemical properties. All statistical analyses were performed using Minitab 14 software (Minitab, Pennsylvania, USA).

3 Results

The physicochemical properties obtained for the investigated honey samples are shown in Table 2 (moisture, free acidity, 5-HMF, pH, ash, and color) and Table 3 (soluble solids, glucose, fructose, and sucrose).

Table 2
Phisicochemical characteristics of Meliponinae and A. mellifera honey samples.

Samples	Moisture (% w/w)	Free acidity (mEq kg-1)	5-HMF * (mg kg-1)	pH	Ash (% w/w)	Pfund scale (mm)	Color
MCM1	31.93 ± 0.20ab	15.88 ± 1.00r	157.50 ± 4.00c	3.25 ± 0.01op	0.01 ± 0.01o	73.95 ± 3.00m	Light amber
MCM2	30.30 ± 0.10ab	17.21 ± 0.02.00qr	167.00 ± 4.00b	3.08 ± 0.01q	0.02 ± 0.01mno	173.61 ± 3.00f	Dark amber
MCM3	31.27 ± 0.02ab	36.68 ± 1.00o	< LOQ	3.94 ± 0.1gh	0.03 ± 0.02fgh	39.91 ± 0.90n	Extra-light amber
MCM4	29.18 ± 0.04bc	37.25 ± 9.00no	< LOQ	3.81 ± 0.07hi	0.01 ± 0.01o	39.42 ± 7.00l	Extra-light amber
MCM5	32.13 ± 0.01ab	132.36 ± 2.00d	57.12 ± 0.40l	3.16 ± 0.06pq	0.06 ± 0.03k-o	130.65 ± 4.00r	Dark amber
MCM6	25.05 ± 0.01ab	104.00 ± 3.00j	83.87 ± 0.20f	3.32 ± 0.02lmn	0.08 ± 0.02k-o	154.18 ± 1.00p	Dark amber
MCM7	25.91 ± 0.01def	187.2 ± 10.00f	94.22 ± 1.00g	3.23 ± 0.01nop	0.11 ± 0.04j-m	14.53 ± 0.2.00p	Extra white
MCM8	30.78 ± 0.01de	62.69 ± 2.00b	92.14 ± 1.00f	3.48 ± 0.02opq	0.06 ± 0.01i-l	82.50 ± 3.00b	Light amber
MCM9	22.21 ± 0.06fgh	34.79 ± 1.00o	4.90 ± 0.60q	4.16 ± 0.04ef	0.31 ± 0.01bc	29.64 ± 0.7.00a	White
SS1	25.87 ± 0.01de	159.90 ± 2.00c	147.20 ± 3.00d	3.78 ± 0.03hi	0.08 ± 0.01j-m	362.52 ± 4.00no	Dark amber
SS2	29.39 ± 0.01bc	122.30 ± 1.00e	< LOQ	3.52 ± 0.02j-m	0.25 ± 0.01def	151.82 ± 6.00g	Dark amber
SS3	32.64 ± 0.01a	328.6 ± 6.00a	20.42 ± 5.00o	3.39 ± 0.06l-o	0.26 ± 0.02cde	414.27 ± 13.00g	Dark amber
TA1	27.11 ± 0.04cd	18.57 ± 2.00qr	80.49 ± 0.60gh	3.98 ± 0.04g	0.01 ± 0.01o	31.00 ± 5.00h	White
TA2	26.90 ± 0.01cd	122.00 ± 2.00e	12.65 ± 3.00p	3.51 ± 0.01klm	0.21 ± 0.01efg	189.83 ± 0.90p	Dark amber
FL	20.21 ± 0.01hij	72.54 ± 2.00hi	92.68 ± 6.00f	3.55 ± 0.07jkl	0.22 ± 0.02efg	456.24 ± 2.00c	Dark amber
MS	18.01 ± 0.05j	43.03 ± 4.00mn	195.40 ± 6.00a	4.35 ± 0.02cd	0.49 ± 0.01a	250.61 ± 6.00d	Dark amber
Maximum	32.64	328.56	195.43	4.16	0.49	456.24	Dark amber
Minimum	18.01	15.88	< LOQ	3.08	0.01	14.53	Extra white
Mean	27.43	93.45	75.35	3.60	0.14	162.17	Dark amber
AM1	19.42 ± 0.01hij	25.01 ± 3.00p	< LOQ	4.75 ± 0.02a	0.36 ± 0.1b	105.28 ± 3.00j	Amber
AM2	18.15 ± 0.10k	38.84 ± 3.00no	< LOQ	4.52 ± 0.02ab	0.12 ± 0.03ijk	75.69 ± 0.01m	Light amber
AM3	19.36 ± 0.06hij	38.17 ± 3.00no	< LOQ	4.55 ± 0.07b	0.13 ± 0.01hij	93.89 ± 2.00k	Amber
AM4	21.30 ± 0.08ghi	54.73 ± 0.70k	< LOQ	4.30 ± 0.07de	0.19 ± 0.05fgh	73.34 ± 1.00m	Light amber
AM5	19.34 ± 0.01hij	48.27 ± 2.00lm	68.58 ± 0.60jk	4.58 ± 0.01ab	0.08 ± 0.01j-m	72.96 ± 0.60m	Light amber
AM6	23.51 ± 0.02efg	23.31 ± 2.00pq	113.26 ± 1.00e	3.90 ± 0.07gh	0.02 ± 0.01no	42.76 ± 6.00n	Extra-light amber
AM7	19.31 ± 0.01hij	46.96 ± 3.00m	47.55 ± 1.00m	3.45 ± 0.09lmn	0.06 ± 0.03k-no	30.38 ± 0.01p	White
AM8	18.84 ± 0.08ij	69.44 ± 0.50i	36.86 ± 1.00n	3.32 ± 0.06nop	0.05 ± 0.02mno	33.47 ± 0.20op	White
AM9	19.32 ± 0.02hij	53.92 ± 0.20kl	70.82 ± 0.10ijk	3.37 ± 0.09mno	0.02 ± 0.01mno	23.20 ± 0.40q	White
AM10	20.66 ± 0.05ghij	98.16 ± 4.00f	68.50 ± 0.50jk	3.65 ± 0.06ijk	0.05 ± 0.01l-o	124.46 ± 0.20i	Dark amber
AM11	20.00 ± 0.01hij	124.53 ± 3.00e	39.85 ± 0.40n	3.69 ± 0.10ij	0.16 ± 0.03ghi	151.33 ± 0.60j	Dark amber
AM12	19.19 ± 0.08ij	78.42 ± 5.00gh	72.16 ± 1.00ijk	3.32 ± 0.20nop	0.12 ± 0.01ijk	43.38 ± 0.40n	Extra-light amber
AM13	19.73 ± 0.04hij	38.99 ± 0.30no	75.21 ± 0.50hij	3.48 ± 0.06lmn	0.03 ± 0.01mno	18.37 ± 0.80qr	White
AM14	18.93 ± 0.02ij	83.35 ± 8.00g	77.46 ± 3.00ghi	4.02 ± 0.20fg	0.20 ± 0.07efg	85.47 ± 0.40l	Amber
AM15	19.10 ± 0.01ij	81.35 ± 3.00g	92.73 ± 0.70jk	3.39 ± 0.08l-o	0.04 ± 0.01mno	33.23 ± 0.20p	White
AM16	19.44 ± 0.02hij	103.87 ± 1.00f	65.74 ± 0.40k	4.32 ± 0.40de	0.29 ± 0.10cd	87.70 ± 0.20l	Amber
Maximum	23.51	124.53	113.26	4.75	0.36	151.33	Dark amber
Minimum	12.07	23.31	< LOQ	3.32	0.02	18.37	White
Mean	19.35	62.96	69.07	3.91	0.12	68.43	Light amber

• Different letters in each column mean significant differences according to Tukey’s test (p < 0.05); data are mean ± SD of triplicate measurements; <LOQ = Limits of quantitation.
• ^*
  5-HMF = 5- hydroxymethylfurfural

Table 3
Soluble solids and sugar content (fructose, glucose and sucrose) of Meliponinae and A. mellifera honey samples.

Samples	Soluble solid (˚Brix)	Fructose (% w/w)	Glucose (% w/w)	Sucrose (% m/m)	G + F *	F / G **
MCM1	66.63 ± 0.20s	29.30 ± 0.80jk	27.17 ± 0.20d-g	< LOQ	56.46 ± 0.90hij	1.08 ± 0.02jkl
MCM2	68.20 ± 0.01q	29.21 ± 0.80jk	26.83 ± 0.40d-g	< LOQ	56.04 ± 1.00ij	1.09 ± 0.03i-l
MCM3	67.30 ± 0.01r	29.13 ± 20jk	27.58 ± 1.00a-f	< LOQ	56.71 ± 4.00hij	1.06 ± 0.02kl
MCM4	69.00 ± 0.01p	30.30 ± 2.00h-k	27.97 ± 1.00cde	6.02 ± 0.20	58.27 ± 3.00f-j	1.08 ± 0.02jkl
MCM5	66.50 ± 0.01t	29.45 ± 0.60ijk	28.25 ± 0.90cde	< LOQ	57.70 ± 1.00f-j	1.04 ± 0.03l
MCM6	73.50 ± 0.01r	29.33 ± 1.00h-k	17.76 ± 1.00e-h	< LOQ	47.09 ± 3.00g-j	1.65 ± 0.03hij
MCM7	72.70 ± 0.01l	33.02 ± 2.00jk	28.99 ± 2.00l	< LOQ	62.01 ± 3.00k	1.14 ± 0.01d
MCM8	67.30 ± 0.01m	30.61 ± 0.90e-j	26.39 ± 0.30cd	< LOQ	57.00 ± 1.00d-g	1.16 ± 0.02h-k
MCM9	75.80 ± 0.01j	37.06 ± 2.00b-g	25.71 ± 1.00f-i	< LOQ	62.77 ± 4.00c-f	1.44 ± 0.03ef
SS1	72.70 ± 0.01m	38.58 ± 1.00b-e	8.93 ± 0.80m	< LOQ	47.50 ± 2.00k	4.34 ± 0.20a
SS2	69.00 ± 0.01p	31.93 ± 2.00f-k	21.75 ± 1.00cj	< LOQ	53.68 ± 3.00j	1.47 ± 0.02ef
SS3	66.10 ± 0.01u	26.61 ± 0.90k	20.12 ± 0.50jk	< LOQ	46.73 ± 1.00k	1.32 ± 0.01g
TA1	71.00 ± 0.01n	31.22 ± 1.00g-k	28.25 ± 0.90cde	4.14 ± 0.40	59.47 ± 2.00f-i	1.10 ± 0.01h-l
TA2	71.50 ± 0.01o	35.60 ± 0.70b-i	18.25 ± 0.40kl	< LOQ	53.86 ± 1.00j	1.95 ± 0.03c
FL	77.80 ± 0.01g	37.28 ± 1.00b-g	16.24 ± 0.40l	< LOQ	53.52 ± 1.00j	2.30 ± 0.02b
MS	80.23 ± 0.20a	40.55 ± 0.80abc	21.06 ± 0.30cd	< LOQ	61.62 ± 1.00a	1.93 ± 0.01c
Maximum	80.23	40.55	28.99	6.02	62.77	4.34
Minimum	66.10	26.61	8.93	< LOQ	46.73	1.04
Mean	70.95	32.45	23.20	5.08	55.65	1.57
AM1	79.00 ± 0.01e	40.12 ± 4.00a-d	28.47 ± 3.00cde	< LOQ	68.60 ± 7.00ab	1.41 ± 0.01fg
AM2	80.10 ± 0.01b	39.96 ± 1.00a-d	27.88 ± 0.60c-f	< LOQ	67.83 ± 2.00abc	1.43 ± 0.03ef
AM3	79.00 ± 0.01e	24.23 ± 2.00d-j	24.15 ± 1.00a	< LOQ	58.37 ± 3.00f-j	1.42 ± 0.02ef
AM4	77.00 ± 0.01h	36.29 ± 2.00b-h	24.25 ± 0.60hi	< LOQ	60.54 ± 2.00e-i	1.50 ± 0.03ef
AM5	79.00 ± 0.01e	36.27 ± 0.70b-h	25.14 ± 2.00ghi	< LOQ	61.41 ± 2.00d-h	1.45 ± 0.08ef
AM6	74.70 ± 0.01k	31.78 ± 2.00f-k	28.84 ± 2.00cd	7.63 ± 0.40	60.62 ± 4.00e-i	1.10 ± 0.02i-l
AM7	79.00 ± 0.01e	45.92 ± 2.00a	24.56 ± 0.70hi	< LOQ	70.48 ± 3.00a	1.87 ± 0.03c
AM8	79.50 ± 0.20c	37.21 ± 0.70b-g	31.24 ± 0.30ab	< LOQ	68.45 ± 0.80ab	1.19 ± 0.02h
AM9	79.00 ± 0.01e	40.69 ± 1.00ab	27.09 ± 2.00d-g	< LOQ	67.78 ± 2.00abc	1.51 ± 0.10e
AM10	77.80 ± 0.01g	33.57 ± 2.00e-j	31.22 ± 0.70ab	< LOQ	64.79 ± 2.00b-e	1.07 ± 0.03jkl
AM11	78.20 ± 0.01f	37.19 ± 2.00b-g	33.08 ± 1.00a	< LOQ	70.27 ± 3.00a	1.12 ± 0.02h-l
AM12	79.13 ± 0.20d	37.53 ± 2.00b-f	31.85 ± 1.00a	< LOQ	69.38 ± 3.00ab	1.18 ± 0.02hi
AM13	78.20 ± 0.01f	34.37 ± 0.60c-j	32.00 ± 0.50a	< LOQ	66.37 ± 1.00a-d	1.07 ± 0.01jkl
AM14	79.40 ± 0.01c	34.51 ± 3.00b-j	31.40 ± 2.00ab	< LOQ	65.91 ± 6.00a-d	1.10 ± 0.02i-l
AM15	79.40 ± 0.01c	36.15 ± 2.00b-h	32.28 ± 1.00a	< LOQ	68.43 ± 3.00ab	1.12 ± 0.02h-j
AM16	76.60 ± 0.01i	32.47 ± 4.00e-k	29.58 ± 3.00bc	< LOQ	62.04 ± 7.00d-g	1.10 ± 0.03i-l
Maximum	80.10	45.92	33.08	7.63	70.48	1.87
Minimum	74.70	31.78	24.15	< LOQ	58.37	1.07
Mean	78.44	36.77	28.94	7.63	65.70	1.29

• Different letters in each column mean significant differences according to Tukey’s test. (p < 0.05); data are mean ± SD of triplicate measurements; <LOQ = Limits of quantitation.
• ^*
  G + F = the sum of the glucose and fructose contents.
• ^**
  F / G = the ratio between the fructose and glucose contents.

Observing the results obtained for the humidity parameter, it is possible to notice that for stingless-bee honey the values vary from 18.01 to 32.64% w/w, with more than 90% of the samples showing humidity greater than 20% (value established by Codex as the maximum acceptable limit). In contrast, the values found for A. mellifera bee honey vary from 12.07 to 23.51% w/w, showing that 75% of the samples had values lower than 20%. Moreover, these results reinforce that the stingless-bee honey has as characteristics a higher moisture content when analyzed the average moisture values obtained for the stingless-bee honey (27.43%) versus the A. mellifera honey (19.35%).

The free acidity of the samples varied between 15.88 to 328.56 mEq kg^-1 for Meliponinae honey and 23.31 to 124.53 mEq kg^-1 for A. mellifera honey. For both groups of samples, it was observed that 60% of presented values were above the limits stipulated by Codex (Max. 50 mEq kg^-1). However, it is interesting to note that honey samples from Meliponinae subfamily, showed seven samples with values above 100 mEq kg^-1, and a sample with acidity above 300 mEq kg^-1.

The results of the pH analysis demonstrated that there was no discrepancy between the studied samples. Honey samples obtained from Meliponinae subfamily showed an average pH of 3.6 and A. mellifera honey samples showed an average pH of 3.9.

In the analyzes performed to identify and quantify 5- HMF in stingless-bee honey, it was observed that the values ranged from < LOQ to 195.43 mg kg^-1 and that more than 50% of the analyzed samples were with values higher than those acceptable (Max. 80 mg kg^-1- (Codex Alimentarius Commission, 2001). On the other hand, the results found for A. millifera bee honey showed variable values from < LOQ to 113.26 mg kg^-1, with more than 80% of the samples within the values proposed by Codex, that is, only two samples will show disagreement. The highest values of 5-HMF found in stingless bee honey are from samples purchased directly from the local market (samples MCM1, MCM2, and MS). These results suggest that this type of honey could be commercialized under inadequate temperature conditions, favoring the formation of this compound.

For stingless-bee honey, the ash content ranged from 0.01 to 0.49%, and for bee honey A. mellifera the results range from 0.02 to 0.36%.

Meliponinae subfamily honey samples tended to have darker colors (Table 2, Figure 2). The samples show colors that range from extra-white to dark amber, however, the dark amber has been the predominant color of the analyzed samples (56.25%). It was also verified that all the samples of honey from Scaptotrigona sp. presented a dark amber color, even though they were collected in different regions. Regarding the A. mellifera honey, the samples showed the same color range as stingless-bee honey (extra-white to dark amber), but a predominant color was not observed in the analyzed samples (Figure 2). The color is an important indication of the multiflora or uniflora origin of the honey samples (Wen et al., 2017). Honey samples obtained from native flora as those collected in the present study tend to show a broad spectrum of colors that tend to the dark range. However, it is important to highlight that the inappropriate storage of honey samples could also lead to the Maillard reaction that produces the contaminant 5-hydroxymethylfurfural which is also involved with the presence of the dark color of some investigated samples (Shapla et al., 2018).

Figure 2
Percentage of honey color categories of Apis mellifera and Meliponinae from eastern Amazon, Brazil (this figure is in color in the electronic version).

Table 3 shows the results for °Brix and sugars quantified in honey samples. In °Brix analysis, the values found in stingless-bee honey ranged from 66.10 to 80.23 °Brix. While for A. mellifera bee honey the values ranged from 74.70 to 80.10.

The sugar content varied greatly among samples of both A. mellifera and stingless-bee samples (Table 3). Considering the stingless bee honey, the values ranged from 28.99 to 40.55% for fructose and 8.93 to 26.61% for glucose. Regarding the A. Mellifera samples, the values were higher, ranging from 33.08 to 45.92% for fructose and 24.15 to 31.78% for glucose. When the sum of glucose and fructose is observed, it is even clearer than the samples obtained from the species of Meliponinae subfamily honey has lower reducing sugar values. About 80% of the stingless bee honey samples have reduced sugar values (G + F), which are below the minimum values required by Codex Alimentarius (min. 60%) (Codex Alimentarius Commission, 2001). In addition, the F/G rate of the samples analyzed was 1.04 to 4.34 for stingless-bee honey and 1.7 to 1.87 for A. mellifera honey. Among the total samples analyzed, only three were quantified with sucrose (MCM4 = 6.02%; TA1 = 4.14% e TA1 = 7.63%).

The results for the evaluation of total phenolic contents and DPPH free radical scavenging activity honey are shown in Table 4. The values for the Folin-Ciocalteu reagent (FCR) indicate the total phenolics contents ranged from 18.00 to 266.96 mg GAE 100g^-1 in stingless-bee honey and, 31.06 to 106.50 mg GAE 100g^-1 in A. mellifera honey.

Table 4
Folin-Ciocalteu Reagent (FCR) reducing capacity and DPPH free radical scavenging activity of Meliponinae and A. mellifera honey samples.

Samples	FCR (mg EAG 100 g-1)	DPPH (%)
MCM1	22.97 ± 1.00s	30.02 ± 4.00n
MCM2	27.85 ± 0.60qr	35.92 ± 15.00mn
MCM3	27.10 ± 1.00r	20.20 ± 1.00o
MCM4	102.64 ± 3.00t	96.53 ± 0.50p
MCM5	22.43 ± 0.30p	11.17 ± 1.00n
MCM6	18.00 ± 0.50n	10.63 ± 2.00n
MCM7	62.87 ± 0.70f	60.80 ± 1.00e-i
MCM8	36.90 ± 1.00kl	32.43 ± 3.00efg
MCM9	68.10 ± 1.00f	53.60 ± 1.00c
SS1	129.61 ± 5.00d	88.83 ± 0.60a
SS2	65.96 ± 4.00ij	60.73 ± 0.40ef
SS3	52.50 ± 0.40hi	31.00 ± 5.00ef
TA1	76.26 ± 4.00s	56.13 ± 1.00p
TA2	58.89 ± 1.00gh	57.97 ± 2.00g-k
FL	74.74 ± 4.00b	78.73 ± 7.00b
MS	266.96 ± 4.00a	95.77 ± 0.80a
Maximum	266.96	96.53
Minimum	18.00	10.63
Mean	69.61	51.28
AM1	38.19 ± 0.20p	50.23 ± 4.00i-l
AM2	54.95 ± 2.00mn	76.50 ± 4.00cd
AM3	56.16 ± 0.30lm	71.39 ± 2.00c
AM4	57.40 ± 0.01klm	56.92 ± 3.00e-h
AM5	60.56 ± 0.60jk	87.57 ± 0.90b
AM6	31.06 ± 2.00q	14.69 ± 2.00op
AM7	77.14 ± 2.00f	39.86 ± 0.60m
AM8	58.07 ± 0.80klm	51.25 ± 1.00h-l
AM9	106.50 ± 2.00c	47.95 ± 3.00kl
AM10	69.73 ± 2.00g	51.70 ± 1.00g-l
AM11	77.26 ± 1.00f	61.06 ± 1.00e
AM12	86.09 ± 4.00e	57.05 ± 3.00e-h
AM13	44.71 ± 2.00o	46.52 ± 0.70l
AM14	59.10 ± 2.00kl	49.39 ± 4.00jkl
AM15	51.49 ± 1.00n	54.71 ± 1.00f-j
AM16	52.15 ± 1.00n	50.80 ± 8.00h-l
Maximum	106.50	87.57
Minimum	31.06	14.69
Mean	61.28	54.22

• Different letters in each column mean significant differences according to Tukey’s test. (p < 0.05); data are mean ± SD of triplicate measurements.

The results of the DDPH radical scavenging activity (%) varied between 10.63 and 96.53% for honey samples obtained from species of Meliponinae subfamily. In contrast, the DPPH scavenging activity (%) varied from 14.69 to 87.57% for A. mellifera honey. A significant difference was observed between the two kinds of honey studied, regardless of the species or collected regions. On the other hand, when analyzing the global averages, it is possible to observe that stingless bees honey and A. mellifera honey kinds of honey present show close percentages (51.28% and 54.22%, respectively).

Principal component analysis (PCA) was performed aiming to evaluate the data of the physicochemical, FCR, and DPPH for the discrimination of honey samples according to their bee species origin. The chemometric evaluation shows that PC1 explained up to 39.7% of the total variance, and PC2 explained 22.6%. Thus, the first two PCs explained 62.3% of the variability in the data (Figure 3).

Figure 3
PCA scatter plot of honey samples and the descriptors: physicochemical and bioactive characteristics. Considering Apis mellifera (▲), Melipona comprensipes manaosenseis (●), Scaptotrigona sp. (■), Tetragonisca angustula (♦), Melipona sp. (◄) and Frisiomelitta longipes (►) species of bee (this figure is in color in the electronic version).

4 Discussion

4.1 Physicochemical properties

In the present study, we noticed differences in the moisture content between the honey obtained from the species of the Meliponinae subfamily and the honey from A. mellifera. Some studies have also reported that honey produced by stingless bees has a high moisture content, reaching values greater than 40% (w/w) (Nordin et al., 2018; Biluca et al., 2016). These results can be attributed to different factors, such as the humidity of environments, nectar collection from undergrowth flowers, ripe fruits that are rich in water, or even different species of stingless bees (Ávila et al., 2018). It is also important to note that high moisture content in stingless-bee honey influences the sensorial characteristics, due to its lower viscosity and reduced shelf life due to its accelerated fermentation.

Free acidity is a parameter of quality-related mainly to the presence of organic acids obtained from the nectar, derived from the fermentation process of sugars – producing acetic acid – as well as from some enzymatic mechanisms, such as the transformation of glucose into gluconic acid by glucose oxidase (Ávila et al., 2018). Interestingly, some studies carried out with stingless-bee honey have shown higher free acidity content than those reported for A. mellifera honey. For example, Duarte et al. (2018) reported a free acidity for stingless bee honey ranged ranging from 17 mEq kg^-1 to 125 mEq kg^-1(Duarte et al., 2018). In contrast, Chuttong et al. (2016), exhibited high acidity for these honey samples, ranging from 440.0 to 592.0 mEq kg^-1.

The pH results corroborate with previous findings reported for honey samples from some species of Meliponinae subfamily collected from the Amazon region (Silva et al., 2013a) that ranged from 3.10 to 5.04 mEq kg^-1. Similarly, another study reported for A. mellifera honey samples showed acidity ranging from 3.86 to 4.41 mEq kg^-1 (Silva et al., 2020).

It is worth mentioning that regions with a hot and humid climate as the Amazon region, favor the formation of 5-HMF (Bandeira et al., 2018) even during storage at room temperature (Khalil et al., 2010). Studies with Brazilian stingless bee honey from other climate conditions observed the absence of 5-HMF for fresh honey (Biluca et al., 2016). Thus, the climatic conditions used to store the A. mellifera and Meliponinae honey samples produced in the Amazon eastern region in general favor the formation of 5-HMF.

The ash content represents the total mineral residue present in honey and depends mainly on the composition of the collected nectar, due to the absorption of nutrients from the soil by the plant (Silva et al., 2016); Nordin et al., 2018).

Although the Codex Alimentarius (Codex Alimentarius Commission, 2001) does not provide a standard value for this parameter, studies with A. mellifera honey have shown the average ash content in honey is 0.17% (w/w), ranging between 0.02% and1.03% (w/w) (Chakir et al., 2011). However, Gomes et al. (2017) and Silva et al., 2013a found ash values for stingless-bee honey in the state of Pará, varying from 0.03 to 0.33% and 0.09 to 1.11%, respectively. Mineral content may have a relation to the color and flavor of honey, with a higher mineral content leading to a darker and stronger flavor, which are attractive features for its consumption (Silva et al., 2016).

The prevalence of dark color was also found in a previous study that analyzed samples of stingless-bee honey from the same studied region (Gomes et al., 2017). Similarly, another study analyzed honey samples of the species of the Meliponinae family from the Brazilian semi-arid region, and they also observed colors ranging from white to amber (Sant’ana et al., 2020). The range of colors from A. mellifera honey samples was also observed in a previous study that analyzed honey samples from southern Brazil, but the authors identified a predominance of dark colors (Rizelio et al., 2020). Studies have also suggested that stingless-bees from the Amazon eastern region tend to produce darker honey (Silva et al., 2013; Silva et al., 2013a; Gomes et al., 2017).

Regarding soluble solids, similar results are found in other studies, with values between 55.2 and 76.1 °Brix (Biluca et al., 2016) and 70.0 and 85.0 °Brix (Alqarni et al., 2016), for Meliponinae and A. mellifera honey, respectively.

Sugars are the major components in the composition of honey, where fructose and glucose are the reducing sugars present in the greatest amounts. Some studies have also reported lower reducing sugar values than those found in the A. mellifera samples, thus highlighting this characteristic of stingless-bee honey (Ávila et al., 2018; Biluca et al., 2016). The F / G ratio points to the time required for honey to crystallize, i.e. honey with a ratio greater than 1.3 may remain fluid for longer (Escuredo et al., 2014). Considering the honey samples analyzed in the present study, showed slow crystallization.

High values of sucrose usually indicate a premature harvest of honey, since the enzyme still invertase did not completely dissociate sucrose into glucose and fructose (Silva et al., 2016). Biluca et al. (2016) reported the absence of sucrose in thirty-three samples of stingless-bee honey (Biluca et al., 2016). However, Chuttong et al. (2016) reported an average content of 19% (w/w) of sucrose in stingless bee honey (Chuttong et al., 2016).

4.2 Reducing activity and free radical scavenging activity

The differences in the phenolic contents of different types of honey are largely related to the floral origin of the honey. Moreover, the constituents of the floral source could be influenced by the nectar and pollen, the geographical origins, and the honey-producing bee species (Shamsudin et al., 2019).

Thus, the values of radical scavenging activity found varied for both analyzed honey types. However, it is important to highlight that for stingless-bees honey, this variation has already been reported in previous studies performed in Malaysia, which showed values of 27.33 and 55.86 mg GAE 100g^-1 for honey samples obtained from different regions (Shamsudin et al., 2019). In contrast, another study performed in Brazil presented values from 10.3 to 98 mg GAE 100g^-1 for Brazilian honey (Biluca et al., 2016). Similarly, a previous study reported values between 18.20 and 148.62 mg GAE 100 g^-1, respectively, for A. mellifera honey samples collected from the southern region of Brazil (Rizelio et al., 2020).

Currently, different methods have been applied to determine the antioxidant activity of foods (Amorati & Valgimigli, 2015; Nascimento et al., 2020). In the present study, the antioxidant activity was determined by the DPPH radical scavenging activity. Other author shave found similar results for antioxidant activity. For honey samples of the same geographical origin, the DPPH radical scavenging activity (%) ranged from 11.98 to 70.51% for stingless-bees honey (Gomes et al., 2017) and from 49.18 to 61.62% for A. mellifera honey (Bandeira et al., 2018).

4.3 Chemometric analysis

The scatter plot showed that honey samples of AM were the most homogeneous in the measured analyzed parameters. According to the scatter plot, the weights of these parameters do not have relevance to separate differentiate these samples. The diametrical opposition of pH and ashes from the quadrant of those samples shows these parameters as the most different regarding values found for TA, MCM, and SS samples. One AM sample has moisture content similar to TA and MCM samples and was grouped with these clusters.

The contents of soluble solids (ºBrix), glucose, 5-HMF, fructose, free acidity, pH, and ashes were similar between MCM samples clustering them, one TA sample was grouped with MCM samples by moisture content while another one was grouped with one MCM and one SS sample by ashes contents. One SS sample was grouped into an MCM sample by pH content, these samples coded as SS presents an outlier, and one sample was separated from others by FCR values. Other samples do not show the formation of groups. According to the scatter plot, the values of DPPH and FCR have a diametrical opposition to the majority of parameters, therefore the increase of one of these parameters is followed by the decrease of the other.

Considering these findings, we can affirm that the physicochemical composition between the samples of stingless-bee honey is variable, which impairs the correct discrimination of the samples at the species level. However, we noted that their characteristics are different when compared with A. mellifera honey which enables their discrimination in different clusters in the PCA plot.

5 Conclusion

In the present study, honey samples obtained from different species of Meliponinae subfamily showed a greater free acidity, high moisture content, lower content of reducing sugars, and darker color, when compared to A. mellifera honey samples collected from the same Brazilian Amazon Eastern region. These differences contributed to the discrimination of these honey samples through their physicochemical properties analyzed by the PCA plot. In addition, the honey samples of different species from the Meliponinae subfamily showed antioxidant activity comparable to that reported for A. mellifera honey samples. Thus, our results reinforce that the stingless-bee honey samples have unique characteristics, requiring a specific Brazilian regularization for them.

Acknowledgements

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, grant number: 88881.159143/2017-01) and Fundação Paraense de Amparo à Pesquisa (FAPESPA). The authors also thank the beekeepers of western of Pará, Brazil.

• Practical Application: Apis mellifera and Meliponinae are the main groups of bees found in Brazil. The honey produced by these groups has different physical, chemical, sensory and bioactive properties. In addition, the composition of these honeys can also be influenced by other factors, such as geographic regions. Considering also that most studies focus only on honey produced by a group of bees, having the comparison between the composition of these honeys, coming from the same geographic regions, can help in standardization and regulation of these products, in addition to the valorization in the beekeeping market.

References

• Alqarni, A. S., Owayss, A. A., & Mahmoud, A. A. (2016). Physicochemical characteristics, total phenols and pigments of national and international honeys in Saudi Arabia. Arabian Journal of Chemistry, 9(1), 114-120. http://dx.doi.org/10.1016/j.arabjc.2012.11.013
  » http://dx.doi.org/10.1016/j.arabjc.2012.11.013
• Amorati, R., & Valgimigli, L. (2015). Advantages and limitations of common testing methods for antioxidants. Free Radical Research, 49(5), 633-649. http://dx.doi.org/10.3109/10715762.2014.996146 PMid:25511471.
  » http://dx.doi.org/10.3109/10715762.2014.996146
• Association of Official Analytical Chemists – AOAC. (2005). Official methods of analysis of AOAC International (18th ed.). Washington: AOAC.
• Ávila, S., Beux, M. R., Ribani, R. H., & Zambiazi, R. C. (2018). Stingless bee honey: quality parameters, bioactive compounds, health-promotion properties and modification detection strategies. Trends in Food Science & Technology, 81, 37-50. http://dx.doi.org/10.1016/j.tifs.2018.09.002
  » http://dx.doi.org/10.1016/j.tifs.2018.09.002
• Ávila, S., Lazzarotto, M., Hornung, P. S., Teixeira, G. L., Ito, V. C., Bellettini, M. B., Beux, M. R., Beta, T., & Ribani, R. H. (2019). Influence of stingless bee genus (Scaptotrigona and Melipona) on the mineral content, physicochemical and microbiological properties of honey. Journal of Food Science and Technology, 56(10), 4742-4748. http://dx.doi.org/10.1007/s13197-019-03939-8 PMid:31686706.
  » http://dx.doi.org/10.1007/s13197-019-03939-8
• Bandeira, A. M. P., Gomes, V. V., Vasconcelos, A. A., Taube, P. S., Barros, E. C., Costa, S. C., Lima, A. K. O., Boligon, A. A., Waczuk, E. P., & Rocha, J. B. T. (2018). Antioxidant activity and physicochemical characteristics of honeys from the eastern Amazon region, Brazil. Acta Amazonica, 48(2), 158-167. http://dx.doi.org/10.1590/1809-4392201702721
  » http://dx.doi.org/10.1590/1809-4392201702721
• Biluca, F. C., Betta, F. D., Oliveira, G. P., Pereira, L. M., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2014). 5-HMF and carbohydrates content in stingless bee honey by CE before and after thermal treatment. Food Chemistry, 159, 244-249. http://dx.doi.org/10.1016/j.foodchem.2014.03.016 PMid:24767051.
  » http://dx.doi.org/10.1016/j.foodchem.2014.03.016
• Biluca, F. C., Braghini, F., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2016). Physicochemical profiles, minerals and bioactive compounds of stingless bee honey (Meliponinae). Journal of Food Composition and Analysis, 50, 61-69. http://dx.doi.org/10.1016/j.jfca.2016.05.007
  » http://dx.doi.org/10.1016/j.jfca.2016.05.007
• Chakir, A., Romane, A., Barbagianni, N., & Bartoli, D. (2011). Major and trace elements in different types of Moroccan honeys. Australian Journal of Basic and Applied Sciences, 5, 223-231.
• Chuttong, B., Chanbang, Y., Sringarm, K., & Burgett, M. (2016). Physicochemical profiles of stingless bee (Apidae: Meliponini) honey from south east Asia (Thailand). Food Chemistry, 192, 149-155. http://dx.doi.org/10.1016/j.foodchem.2015.06.089 PMid:26304332.
  » http://dx.doi.org/10.1016/j.foodchem.2015.06.089
• Codex Alimentarius Commission. (2001). Revised codex standard for honey, standards and standard methods Rome: Codex Alimentarius Commission.
• Crăciun, M. E., Pârvulescu, O. C., Donise, A. C., Dobre, T., & Stanciu, D. R. (2020). Characterization and classification of Romanian acacia honey based on its physicochemical parameters and chemometrics. Scientific Reports, 10(1), 20690. http://dx.doi.org/10.1038/s41598-020-77685-9 PMid:33244024.
  » http://dx.doi.org/10.1038/s41598-020-77685-9
• Dourado, G. S., Gomes, V. V., Maia, M. T. V., Vasconcelos, A. A., Costa, K. S., Faial, K. C. F., Carneiro, B. S., Vasconcelos, N. T. Jr., & Taube, P. S. P. S. (2019). Determination of macro and trace element levels in honey from the lower Amazonian region, Brazil. Brazilian Journal of Analytical Chemistry, 6(23), 29-44.
• Duarte, A. W. F., Vasconcelos, M. R. S., Oda-Souza, M., Oliveira, F. F., & López, A. M. Q. (2018). Honey and bee pollen produced by meliponini (Apidae) in Alagoas, Brazil: Multivariate analysis of physicochemical and antioxidant profiles. Food Science and Technology, 38(3), 493-503. http://dx.doi.org/10.1590/fst.09317
  » http://dx.doi.org/10.1590/fst.09317
• Escuredo, O., Dobre, I., Fernández-González, M., & Seijo, M. C. (2014). Contribution of botanical origin and sugar composition of honeys on the crystallization phenomenon. Food Chemistry, 149, 84-90. http://dx.doi.org/10.1016/j.foodchem.2013.10.097 PMid:24295680.
  » http://dx.doi.org/10.1016/j.foodchem.2013.10.097
• Farias, S. A. S., Costa, K. S., & Martins, J. B. L. (2021). Analysis of conformational, structural, magnetic, and electronic properties related to antioxidant activity: revisiting flavan, anthocyanidin, flavanone, flavonol, isoflavone, flavone, and flavan-3-ol. ACS Omega, 6(13), 8908-8918. http://dx.doi.org/10.1021/acsomega.0c06156 PMid:33842761.
  » http://dx.doi.org/10.1021/acsomega.0c06156
• Gomes, V. V., Dourado, G. S., Costa, S. C., Lima, A. K. O., Silva, D. S., Bandeira, A. M. P., Vasconcelos, A. A., & Taube, P. S. (2017). Evaluation of the quality of honey commercialized in western Pará, Brazil. Revista Virtual de Química, 9, 815-826. http://dx.doi.org/10.21577/1984-6835.20170050
  » http://dx.doi.org/10.21577/1984-6835.20170050
• Guerrini, A., Bruni, R., Maietti, S., Poli, F., Rossi, D., Paganetto, G., Muzzoli, M., Scalvenzi, L., & Sacchetti, G. (2009). Ecuadorian stingless bee (Meliponinae) honey: a chemical and functional profile of an ancient health product. Food Chemistry, 114(4), 1413-1420. http://dx.doi.org/10.1016/j.foodchem.2008.11.023
  » http://dx.doi.org/10.1016/j.foodchem.2008.11.023
• Khalil, M. I., Sulaiman, S. A., & Gan, S. H. (2010). High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than one year. Food and Chemical Toxicology, 48(8-9), 2388-2392. http://dx.doi.org/10.1016/j.fct.2010.05.076 PMid:20595027.
  » http://dx.doi.org/10.1016/j.fct.2010.05.076
• Maione, C., Barbosa, F. Jr., & Barbosa, R. M. (2019). Predicting the botanical and geographical origin of honey with multivariate data analysis and machine learning techniques: a review. Computers and Electronics in Agriculture, 157, 436-446. http://dx.doi.org/10.1016/j.compag.2019.01.020
  » http://dx.doi.org/10.1016/j.compag.2019.01.020
• Miłek, M., Bocian, A., Kleczyńska, E., Sowa, P., & Dżugan, M. (2021). The comparison of physicochemical parameters, antioxidant activity and proteins for the raw local Polish honeys and imported honey blends. Molecules, 26(9), 2423. http://dx.doi.org/10.3390/molecules26092423 PMid:33919361.
  » http://dx.doi.org/10.3390/molecules26092423
• Nascimento, L. D., Moraes, A. A. B., Costa, K. S., Galúcio, J. M. P., Taube, P. S., Costa, C. M. L., Cruz, J. N., Andrade, E. H. A., & Faria, L. J. G. (2020). Bioactive natural compounds and antioxidant activity of essential oils from spice plants: new findings and potential applications. Biomolecules, 10(7), 988. http://dx.doi.org/10.3390/biom10070988 PMid:32630297.
  » http://dx.doi.org/10.3390/biom10070988
• Nordin, A., Sainik, N. Q. A. V., Chowdhury, S. R., Saim, A., & Idrus, R. B. H. (2018). Physicochemical properties of stingless bee honey from around the globe: a comprehensive review. Journal of Food Composition and Analysis, 73, 91-102. http://dx.doi.org/10.1016/j.jfca.2018.06.002
  » http://dx.doi.org/10.1016/j.jfca.2018.06.002
• Rizelio, V. M., Gonzaga, L. V., Borges, G. S. C., Micke, G. A., Fett, R., & Costa, A. C. O. (2012). Development of a fast MECK method for determination of 5-HMF in honey samples. Food Chemistry, 133(4), 1640-1645. http://dx.doi.org/10.1016/j.foodchem.2011.11.058
  » http://dx.doi.org/10.1016/j.foodchem.2011.11.058
• Rizelio, V. M., Tenfen, L., Gonzaga, L. V., Borges, G. S. C., Biluca, F. C., Schulz, M., Costa, A. C. O., & Fett, R. (2020). Physicochemical and bioactive properties of southern Brazilian Apis mellifera L. honeys. Journal of Apicultural Research, 59(5), 910-916. http://dx.doi.org/10.1080/00218839.2020.1735760
  » http://dx.doi.org/10.1080/00218839.2020.1735760
• Sant’ana, R. S., Carvalho, C. A. L., Oda-Souza, M., Souza, B. A., & Dias, F. S. (2020). Characterization of honey of stingless bees from the Brazilian semi-arid region. Food Chemistry, 327, 127041. http://dx.doi.org/10.1016/j.foodchem.2020.127041 PMid:32454276.
  » http://dx.doi.org/10.1016/j.foodchem.2020.127041
• Scholz, M. B. S., Quinhone, A. Jr., Delamuta, B. H., Nakamura, J. M., Baudraz, M. C., Reis, M. O., Kato, T., Pedrão, M. R., Dias, L. F., Santos, D. T. R., Kitzberger, C. S. G., & Bianchini, F. P. (2020). Indication of the geographical origin of honey using its physicochemical characteristics and multivariate analysis. Journal of Food Science and Technology, 57(5), 1896-1903. http://dx.doi.org/10.1007/s13197-019-04225-3 PMid:32327800.
  » http://dx.doi.org/10.1007/s13197-019-04225-3
• Shamsudin, S., Selamat, J., Sanny, M., Razak, S.-B. A., Jambari, N. N., Mian, Z., & Khatib, A. (2019). Influence of origins and bee species on physicochemical, antioxidant properties and botanical discrimination of stingless bee honey. International Journal of Food Properties, 22, 238-263. http://dx.doi.org/10.1080/10942912.2019.1576730
  » http://dx.doi.org/10.1080/10942912.2019.1576730
• Shapla, U. M., Solayman, M., Alam, N., Khalil, M. I., & Gan, S. H. (2018). 5-Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on bees and human health. Chemistry Central Journal, 12(1), 35. http://dx.doi.org/10.1186/s13065-018-0408-3 PMid:29619623.
  » http://dx.doi.org/10.1186/s13065-018-0408-3
• Silva, A. S., Alves, C. N., Fernandes, K. G., & Müller, R. C. S. (2013a). Classification of honeys from Pará state (Amazon region, Brazil) produced by three different species of bees using chemometric methods. Journal of the Brazilian Chemical Society http://dx.doi.org/10.5935/0103-5053.20130147
  » http://dx.doi.org/10.5935/0103-5053.20130147
• Silva, I. A. A., Silva, T. M. S., Camara, C. A., Queiroz, N., Magnani, M., Novais, J. S., Soledade, L. E., Lima, E. O., Souza, A. L., & Souza, A. G. (2013b). Phenolic profile, antioxidant activity and palynological analysis of stingless bee honey from Amazonas, northern Brazil. Food Chemistry, 141(4), 3552-3558. http://dx.doi.org/10.1016/j.foodchem.2013.06.072 PMid:23993520.
  » http://dx.doi.org/10.1016/j.foodchem.2013.06.072
• Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2016). Honey: chemical composition, stability and authenticity. Food Chemistry, 196, 309-323. http://dx.doi.org/10.1016/j.foodchem.2015.09.051 PMid:26593496.
  » http://dx.doi.org/10.1016/j.foodchem.2015.09.051
• Silva, P. M., Gonzaga, L. V., Biluca, F. C., Schulz, M., Vitali, L., Micke, G. A., Costa, A. C. O., & Fett, R. (2020). Stability of Brazilian Apis mellifera L. honey during prolonged storage: physicochemical parameters and bioactive compounds. Lwt, 129, 109521. http://dx.doi.org/10.1016/j.lwt.2020.109521
  » http://dx.doi.org/10.1016/j.lwt.2020.109521
• Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16(3), 144-158.
• Vásquez-Espinal, A., Yañez, O., Osorio, E., Areche, C., García-Beltrán, O., Ruiz, L. M., Cassels, B. K., & Tiznado, W. (2019). Theoretical study of the antioxidant activity of quercetin oxidation products. Frontiers in Chemistry, 7, 818. http://dx.doi.org/10.3389/fchem.2019.00818 PMid:31828060.
  » http://dx.doi.org/10.3389/fchem.2019.00818
• Wen, Y. Q., Zhang, J., Li, Y., Chen, L., Zhao, W., Zhou, J., & Jin, Y. (2017). Characterization of Chinese unifloral honeys based on proline and phenolic content as markers of botanical origin, using multivariate analysis. Molecules, 22(5), 735. http://dx.doi.org/10.3390/molecules22050735 PMid:28513535.
  » http://dx.doi.org/10.3390/molecules22050735
• Zhang, H.-Y. (2005). Structure-activity relationships and rational design strategies for radical- scavenging antioxidants. Current Computer-Aided Drug Design, 1(3), 257-273. http://dx.doi.org/10.2174/1573409054367691
  » http://dx.doi.org/10.2174/1573409054367691
• Zhou, X., Taylor, M. P., Salouros, H., & Prasad, S. (2018). Authenticity and geographic origin of global honeys determined using carbon isotope ratios and trace elements. Scientific Reports, 8(1), 14639. http://dx.doi.org/10.1038/s41598-018-32764-w PMid:30279546.
  » http://dx.doi.org/10.1038/s41598-018-32764-w

Publication Dates

• Publication in this collection
  13 July 2022
• Date of issue
  2022

History

• Received
  13 Dec 2021
• Accepted
  07 June 2022