Functional Characterization of Scaptotrigona mexicana Honey: Physicochemical Properties, Antioxidant Capacity, and α-Amylase Inhibition for Food Process Applications

Abstract

For centuries, Scaptotrigona mexicana honey has been treasured in Mexico, where pre-Columbian cultures harvested it not only for its sweet flavor but also for its medicinal and ceremonial purposes. Today, it remains a high-value product in local markets, prized above Apis mellifera honey for its unique sensory qualities and traditional health benefits. Yet its scientific characterization and functional potential remain underexplored. In this study, twenty-four samples from diverse regions were analyzed to quantify bioactive compounds and determine physicochemical composition, α-amylase inhibition, and antioxidant activity. Non-parametric statistical tests revealed distinct compositional clusters, with samples from Cruz Blanca showing exceptional phenolic content and stronger α-amylase inhibition (5.6–49.2%). Antioxidant capacity correlated positively with phenols and flavonoids, showing moderate effect sizes for ABTS (η² = 0.49) and DPPH (η² = 0.37). Compared with Apis mellifera honey, Scaptotrigona mexicana contained more moisture, free acidity, phenols, and antioxidants, but less diastase, hydroxymethylfurfural, and reducing sugars. Importantly, natural α-amylase inhibitors can help modulate postprandial glucose, offering dietary support for type 2 diabetes management. Kinetic analyses (EC50, Vmax, and Km) suggested mixed inhibition. These findings highlight Scaptotrigona mexicana as both a heritage product and a promising functional ingredient for developing foods that merge tradition with metabolic health innovation.

1. Introduction

Honey is a natural food, primarily composed of simple sugars, that is produced by honeybees through enzymatic transformation and water evaporation from floral nectar [1]. While Apis mellifera (Hymenoptera, Apidae, and Apini) is the most commonly managed honeybee species, stingless bees (Hymenoptera, Apidae, and Meliponini) [2] are important pollinators in tropical and subtropical regions [3,4]. The tribe Meliponini has many genera, including Melipona, Scaptotrigona, and Trigona, which contribute to the production of distinctive types of honey [5,6]. There are more than 600 species of stingless bees distributed in Asia, Africa, Australia, and Latin America [4,7]. In Mexico, 46 species of stingless bees have been identified, one of which is Scaptotrigona mexicana (S. mexicana), which is traditionally used by pre-Columbian cultures in Mexico to produce honey and cerumen, and currently managed in various states, including Veracruz, San Luis Potosí, Hidalgo, Yucatán, and Puebla. Honey produced by S. mexicana is highly valued for its role in local economies, its cultural significance, and therapeutic attributes [8]. Honey from stingless bees is appreciated for its organoleptic properties, cultural relevance, and therapeutic potential. This honey is considered a functional food owing to its chemical composition, which is principally composed of carbohydrates and other components such as water, vitamins, minerals, proteins, enzymes, organic acids, amino acids, and phenolic compounds [9]. The phenolic compound content and physicochemical composition of honey from stingless bees vary between bee genera and species, but this has not been extensively studied [10]. Variables such as moisture, free acidity, diastase activity, and hydroxymethylfurfural (HMF) content are widely used to assess honey quality and freshness in Apis honey [11,12] but have not been established for the honey of stingless bees [13]. The lack of legislation about stingless bee honey and information on its nutritional value affects the producers and the commercialization of this honey in the global market [5]. Stingless bee honey contains phytochemical compounds, including phenolic compounds and flavonoids, which are known to be involved in many biological processes related to anti-inflammatory, antimicrobial, and antioxidant properties [9]. Recent studies have shown that stingless bee honey possesses considerable antioxidant activity, which is largely attributed to its phenolic content [14]. These bioactive compounds can stabilize cell membranes, scavenge free radicals, and may contribute to the prevention of oxidative stress-related diseases [15]. However, it is important to note that the antioxidant activity of these compounds in honey depends on the floral source visited by the bee to collect the nectar, as discussed in the study by Sousa et al. [16]. As noted by Kek et al. [5], stingless bee honey contains higher levels of polyphenols compared with other varieties of honey. This characteristic has been linked to the smaller body size of these bees, which enables them to forage from a broader variety of flowers, including very small blossoms, thereby increasing floral diversity in their diet [17,18].

One promising area of research is the potential role of phenolic compounds in managing postprandial hyperglycemia through the inhibition of digestive enzymes such as α-amylase [3,19]. A study by Aziz et al. [20] reported that stingless bee honey has been found to protect against the development of diabetes. Diabetes mellitus is a metabolic disorder characterized by increased blood glucose levels [6]. Over the years, several therapeutic approaches have been developed for the prevention and treatment of diabetes mellitus. A strategy for reducing postprandial hyperglycemia in type II diabetes mellitus is to decrease the absorption of carbohydrates after food uptake [21]. Some treatments have focused on key enzymes in type II diabetes mellitus, such as lipase, α-glucosidase, and α-amylase [19]. Inhibiting α-amylase, which catalyzes the breakdown of starch into glucose, can reduce the absorption of monosaccharides and help control blood glucose levels, particularly glucose that can be absorbed [22,23]. This is particularly relevant given the global increase in diabetes prevalence, projected to reach over 783.2 million people by 2045 [24]. Several studies have shown the relation between phenols and flavonoids in the α-amylase inhibitory activity; this approach recommends the use of stingless bee honey in diabetes treatment [3]. Clinical studies show that, unlike other sweeteners, honey reduces postprandial glycemia in both diabetic and non-diabetic individuals, lowering blood glucose in type 1 and type 2 diabetes. Its antidiabetic effects are linked to antioxidant activity, which is relevant since oxidative stress and radical oxidative stress are closely associated with type 2 diabetes pathogenesis [19]. In this sense, the physicochemical and bioactive composition of stingless bee honey has attracted the interest of researchers due to its potential in the prevention of diseases [25]. Given the rise in demand for natural and functional foods, understanding the bioactive and physicochemical profiles of stingless bee honey is crucial for its integration into food systems. Factors such as botanical origin, climate, geographical location, environmental and seasonal conditions, agricultural practices, extraction process, storage conditions, and bee species may influence the nutritional and functional quality of honey [26]. Setting parameters is critical for protecting consumers from fraud and ensuring product quality. Previous studies on S. mexicana honey have not systematically linked its bioactive composition with antioxidant activity or α-amylase inhibition. Our study addresses this gap by providing a detailed physicochemical characterization and demonstrating its potential as a functional ingredient for metabolic health. The objective of this study was to characterize S. mexicana honey by assessing its physicochemical parameters, quantifying bioactive compounds (total phenols and total flavonoids), and evaluating antioxidant capacity (DPPH and ABTS assays) as well as its α-amylase inhibition potential. To analyze the potential enzymatic inhibition effect, the samples of the region with the highest content of bioactive compounds (Cruz blanca, Puebla, México) were used to carry out inhibition of the enzyme α-amylase, and then the Effective Concentration (EC50), the maximum velocity (Vmax), and the Michaelis constant (Km) were calculated. The EC50 represents the compound concentration that produces half of the maximal response, expressed as a percentage of the maximum effect [27]. The Vmax indicates the enzyme’s catalytic capacity under optimal conditions [28]. The Km refers to the substrate concentration at which the enzymatic reaction reaches half of the Vmax, reflecting substrate affinity [29]. This information may contribute to future efforts to standardize quality parameters and explore the use of S. mexicana honey as a functional ingredient in food processing.

2. Materials and Methods

2.1. Honey Samples

Twenty-four samples of multifloral honey from S. mexicana bees were collected in April 2024. The samples were collected directly from hives managed by meliponicultors from eight locations in the warm–humid and temperate subhumid forest of Puebla, Mexico (Figure 1 and

Table 1. Characteristics of sampling sites, including location, number of honey samples, Latitude-Longitude, altitude (masl), temperature (°C), climate, and mean annual precipitation (mm).

Location	Samples	Latitude, Longitude	Altitude (masl)	Temperature (°C)	Climate	Precipitation (mm)
La Palmilla	6	20.018477, 97.145660	147	23–32	Warm-humid	1500
Ayahualo	5	19.971886, 97.325348	603	17.5–28.5	Warm-humid	1270
Hueytamalco	4	19.940156, 97.288513	862	17.5–28.5	Warm-humid	1270
Rancho viejo	3	19.445556, 96.781389	753	17.5–28.5	temperate subhumid	1270
Cruz Blanca	3	19.893000, 97.288383	1238	17.5–28.5	temperate subhumid	1270
Limontitan grande	1	19.991056, 97.275853	543	17.5–28.5	temperate subhumid	1270
Tonalmecoyo	1	19.954604, 97.290276	737	17.5–28.5	temperate subhumid	1270
Tacuba	1	19.976806, 97.313148	533	17.5–28.5	temperate subhumid	1270

). All samples were collected in the field and stored at 4 °C in “Uline” brand amber and hermetic recipients. No endangered species were involved, and samples were collected with meliponiculturist consent.

2.2. Reagents

Chemicals and solvents used for the analysis of phenolic compounds and their antioxidant properties were methanol, ethanol (Meyer Lab., Blue Springs, MO, USA), hydrochloric acid, sodium hydroxide, aluminum trichloride, quercetin, sodium carbonate, gallic acid, potassium ferrocyanide, zinc acetate, sodium bisulfite, sulfuric acid, boric acid, potassium iodide, and sodium chloride (Sigma-Aldrich, Inc., St. Louis, MO, USA). Folin–Ciocalteu reagent (Sigma, USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Merck, Kenilworth, NJ, USA), 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich Co., Kobian, Kenya), and Fehling’s reagent A and B were all purchased from Hycel Inc., Houston, TX, USA α-amylase was sourced from porcine pancreas (Sigma-Aldrich, Inc., St. Louis, MO, USA)

2.3. Physicochemical Analyses

The samples were prepared for physicochemical analysis in uniform conditions according to the Official Methods of Analysis of AOAC International, Association of Official Analytical Chemists 920.180 [30].

2.3.1. Moisture and Ash

Moisture was measured using a refractometer at 20 °C (model Smart-1, Atago, Tokyo, Japan), according to the AOAC 969.38B method. Ash was determined by calcinating 5 g of honey in a muffle (model BF51728C-1, 2006, Thermo Fisher Scientific, Waltham, MA, USA) at 550 °C for 6 h and weighing the remaining inorganic residue using an analytical balance (model PW124, Adam Equipament Inc., Oxford, CT, USA). The percentage of original sample weight was calculated as per the reported AOAC 920.181 [30].

2.3.2. Diastase Activity

The diastase activity 958.09 [30] was determined by dissolving 5 g of honey in 10 mL of distilled water, adding 2.5 mL of buffer solution, placing it in a flask containing 1.5 mL of sodium chloride solution, and volumetrically titrating 5 mL of honey solution. After, the solution was placed in a water bath with 5 mL of starch solution at 40 °C for 15 min. The starch solution was added to the honey solution with constant stirring and allowed to stand for five minutes. An amount of 1 mL was taken, and 10 mL of dilute iodine solution was added, mixed, and measured in a UV/Vis spectrophotometer (model 6715, Bibby Scientific Ltd., 2014, Stone, UK) at 660 nm. Aliquots of 1 mL were taken until the absorbance was less than 0.235. The diastase activity was then obtained using the formula:

$$\mathrm{D}\mathrm{A}={\displaystyle \frac{300}{\mathrm{T}}}$$

DA = diastase number (this number expresses the enzyme activity in mL of 1% solution hydrolyzed by the enzyme contained in 1 g of honey). This diastase index corresponds to the Göthe scald number. T = Time.

2.3.3. Hydroxymethylfurfural

The HMF was determined according to the Official Mexican standard [31]. Briefly, 5 g of honey was dissolved in 25 mL of distilled H₂O. Later, absorbance was measured at 284 and 336 nm against a filtered solution treated with sodium bisulfite with a UV/Vis spectrophotometer (model 6715, Bibby Scientific Ltd., 2014, UK). HMF was calculated according to the following formula:

$$HMF=\left(Abs284-Abs336\right)\ast 149.7\ast 5D/W$$

D = dilution factor. W = sample weight in grams.

2.3.4. Reducing Sugars

The reducing sugars were determined according to the official Mexican standard [31], based on the method of Lane and Eynon with modifications. Briefly, 2.6 g of honey was placed in a 500 mL volumetric flask. Then, 5 mL of Fehling’s solution (A and B) was mixed with 7 mL of distilled water and 15 mL of the previously prepared honey solution. The mixture was then homogenized up to the boiling point. An amount of 1 mL of methylene blue at (1 g/L) was added for 3 min while still boiling. The honey sample was added until the indicator lost its color.

2.3.5. Free Acidity, Lactone, and Total Acidity

The free acidity, lactone, and total acidity were determined according to the official Mexican standard [31]. Free acidity was determined using the titrimetric method as follows: 10 g of honey was dissolved in 75 mL of deionized water, and then the solution was titrated with 0.1 M sodium hydroxide solution until the pH reached 8.5. The solution was then titrated with 0.05 N hydrochloric acid to a pH of 8.3. The total acidity was calculated as the sum of the free acid and the lactone. The results were expressed in milliequivalents (meq) kg⁻¹ using the following formula:

$$Freeacidity=\left(mlofsodiumhydroxide\right)-\left(mlofblank\right)\ast 50/\left(gramsofsample\right)$$

$$Lactone=\left(10mlof0.05NHCl\right)\left(50\right)/\left(gramsofsample\right)$$

$$Totalacidity=Freeacidity+Lactone$$

2.3.6. Brix

The soluble solids (total sugars) in honey were determined according to the AOAC [30], by recording the reading of the honey sample in a digital refractometer (model Smart-1, Atago, Tokyo, Japan). Distilled water was used for calibration.

2.3.7. H₂O Activity and pH

The H₂O activity was measured at 25 °C with a water activity meter (model WA—60A, 2023, Guangzhou Landtek Instruments Co. Ltd., Guangzhou, China) described by Mokaya et al. [12]. The equipment was calibrated with distilled water. The pH was determined with a pH meter (model HI-2211, 2012, Hanna instruments Romania SRL, Cluj-Napoca, Romania).

2.3.8. Protein

The protein was quantified using the Kjeldahl method according to AOAC 981.10 [30], based on the conversion of the organic nitrogen present in the sample to ammonium sulfate ((NH₄)₂SO₄), and then divided into three phases (digestion, distillation, and titration). For digestion, 5 g of the digestion mixture of anhydrous potassium sulfate (K₂SO₄) and copper sulfate pentahydrate (CuSO₄-5H₂O) wrapped in nitrogen-free paper was placed in a Kjeldahl tube. Next, 1 g of the sample was weighed, and 15 mL of sulfuric acid (H₂SO₄) was added. Then, the sample was placed in the Khjedal digester (Speed Digester K-436 and Distillation Unit K-350, BUCHI) for 3 h. Then, 50 mL of boric acid (20 g/L) and three drops of methyl red were added; 0.1 N H₂SO₄ was used for titration. The percentage of nitrogen quantified was transformed to protein content by multiplication with a conversion factor of 6.25.

2.3.9. Color

The color was measured using a colorimeter (Chroma meter, model CR-400, 2008, Konica Minolta, Tokyo, Japan) with the parameters L (lightness), a (red to green), and b (blue to yellow), based on the CIE Lab system scale (International Commission on Illumination, Vienna, Austria) [32].

2.4. Bioactive Compounds

2.4.1. Total Phenolic Compounds

Total phenolic compounds were determined according to Folin–Ciocalteu [33] with modifications. An amount of 1 g of honey was diluted in 9 mL of ethanol/water (80%) and centrifuged at 18,510× g for 15 min at 4 °C. Aliquots (1 mL) of the supernatant were mixed with 5 mL of the 0.2 N Folin–Ciocalteu reagent in a test tube and incubated for 5 min in the dark at room temperature. Under the same conditions, 2 mL of sodium carbonate (Na₂CO₃) (20g/L) was added, homogenized, and incubated for 120 min. Absorbance measurements were performed in a UV/Vis spectrophotometer (model 6715, Bibby Scientific Ltd., 2014, UK) at 765 nm. The results were expressed as mg Gallic Acid Equivalents (GAE)/100 g of honey using the gallic acid calibration curve (0–250 μg/mL).

2.4.2. Total Flavonoids

Total flavonoids were obtained using the method described by Chang et al. [34] with some modifications. An amount of 1 mL of methanol extract of the honey sample was mixed with aluminum chloride (AlCl₃) (20 g/L) and incubated at room temperature for 10 min in the dark. The absorbance was measured at 415 nm. Quercetin (QE) was used to calibrate the curve (20–200 μg/mL) and was expressed as mg QE/100 g honey.

2.5. Antioxidant Assays

2.5.1. DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Free Radical-Scavenging Assay

The antioxidant activity was determined via inhibition of the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical, which was measured according to the method reported by Brand-Williams et al. [35] with modifications. The radical scavenging assay is based on the measurement of the antioxidant scavenging ability of 2,2-diphenyl-1-picrylhydrazyl. A solution of DPPH 6.5 × 10⁻⁵ M in 80% methanol was prepared and stirred for 2 h in the dark. One gram of honey was dissolved in methanol (80% v/v), and 0.5 mL of this solution was mixed with 2.5 mL of a DPPH solution. The mixture was kept in the dark at room temperature for 30 min. The absorbance of the solutions was then measured at 515 nm using a UV/Vis spectrophotometer (model 6715, Bibby Scientific Ltd., Stone, Staffordshire, UK). The concentration of the honey required to quench the DPPH was calculated from a standard curve of gallic acid, and the results were expressed in mg 100/g⁻¹ gallic acid.

2.5.2. ABTS (2,2-Azino-Bis(3-Ethylbenzthiazoline-6-Sulfonic Acid)) Free Radical Scavenging Assay

The determination of antioxidant activity via inhibition of the ABTS (2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) radical was performed as described by Re et al. [36] with some modifications. A stock solution of 7 mM ABTS (Sigma-Aldrich) was prepared and reacted with 10 mL of 2.45 mM potassium Persulfate (K₂S₂O₈) for 16 h in the dark. The absorbance was adjusted with 20% ethanol to obtain a value of 0.7 ± 0.1 at 734 nm. Aliquots of 0.2 mL of honey in ethanol/water (80% w/v) were mixed with 3 mL of ABTS methanolic solution. Absorbance was measured at 734 nm after 2 h of incubation in darkness. A calibration curve was developed using gallic acid as a standard. The results were expressed in mg equivalents of gallic acid per 100 g (GAE/100 g DM).

2.6. α-Amylase Inhibition In Vitro Assay

The α-amylase inhibition assay was carried out in aqueous extracts of S. mexicana honey, based on the method of Abirami et al. [37] with minor adjustments. The reaction contained 100 µL of concentrations of honey extract (20, 40, 60, 80, and 100 µg/mL) mixed with 100 µL of 0.02 mol/L sodium phosphate buffer (pH of 6.9) and 100 µL of buffer solution of porcine pancreatic α-amylase (1 U/mL), which was pre-incubated at 37 °C for 10 min. After the pre-incubation time, 100 µL of aqueous starch solution (1%) was added and incubated at 37 °C for 60 min. The reaction was stopped with 1 mL of dinitrosalicylic acid reagent (DNS) (1 g of 3,5-dinitrosalicylic acid, 20 mL of a solution of NaOH 2 mol/L, 50 mL of distilled water, and 30 g Rochelle salt). The assay tubes were incubated in a water bath set at 90 °C for 5 min and immediately cooled to room temperature in an ice bath. The reaction mixture was then diluted by adding 5 mL of distilled water, and absorbance was measured spectrophotometrically at 540 nm (spectrophotometer Jenway 6715, Staffordshire, ST15 OSA, UK). Acarbose was used as a positive control. The results were expressed in the inhibition percentage using the following equation:

$$Inhibition\left(\%\right)=\left(1-{\displaystyle \frac{Absorbance\left(sample\right)}{Absorbance\left(blank\right)}}\right)\ast 100$$

The percentage of α-amylase inhibition was translated to the Effective Concentration at 50% (EC50), which is the sample concentration for 50% α-amylase inhibition.

Kinetics of α-Amylase Inhibition

The kinetic experiments were performed as described by Thummajitsakul et al. [38] with slight modifications involving concentration-independent inhibition; the inhibitor/extract was taken at its EC50 value and incubated with α-amylase, while the concentration of starch (substrate) was varied from 0.2 to 1 mM/mL, and the reaction allowed to proceed as highlighted above. The 1/V (O.D.540nm/min) 1 and 1/[substrate] were used to generate the Lineweaver–Burk graph. The Km (Michaelis–Menten constant) and Vmax (maximum velocity) values were then calculated.

2.7. Data Analysis

All experiments were conducted in triplicate, and the results are reported as mean ± standard deviation (SD). Due to the non-normal distribution of the physicochemical parameters (assessed via the Shapiro–Wilk test), non-parametric statistical methods were employed. The Kruskal–Wallis test (α = 0.05) was used to detect significant differences among the 24 samples, followed by Dunn’s post hoc test with Bonferroni correction to identify pairwise differences. Bioactive compound concentrations (total phenols and total flavonoids) and antioxidant activity (DPPH and ABTS assays) were analyzed by grouping samples according to their geographical region. Spearman’s rank correlation coefficient (p) was used to evaluate associations between selected variables (p < 0.05). To classify the honey samples based on their physicochemical characteristics, an unsupervised multivariate approach was applied using hierarchical cluster analysis. Euclidean distances were computed using the complete linkage method. Prior to hierarchical cluster analysis, the physicochemical property data matrix was auto-scaled (mean-centered and divided by standard deviation) to standardize the influence of all variables. All statistical analyses and data visualizations were performed in R (version 2024.12.0+467) using the following packages: dplyr, tidyverse, ggplot2, FSA, rstatix, psych, corrplot, and stats.

3. Results and Discussion

3.1. Physicochemical Analysis

3.1.1. Moisture

Moisture is one of the most critical physicochemical parameters influencing honey’s stability, viscosity, maturity, specific weight, crystallization behavior, shelf life, and sensory properties [39]. In this study, the mean moisture content of S. mexicana honey was 28.13%, which is significantly higher than the limit of ≤20% established for A. mellifera honey by the Codex Alimentarius Commission [40]. These findings are consistent with previous reports showing that stingless bee honey varieties typically have higher moisture levels due to their intrinsic production characteristics [13,41]. Statistical analysis revealed significant differences in moisture content among the 24 honey samples (p < 0.05;

Table 2. Mean comparison of physicochemical parameters of Scaptotrigona mexicana samples collected from different locations of the state of Puebla, Mexico, compared to Apis mellifera honey standards, including ash, diastase activity, hydroxymethylfurfural (HMF), reducing sugars, and free acidity.

Sample Identification	Moisture (%)	Ash	Diastase Activity (Göthe Units)	HMF (mg/kg)	Reducing Sugars	Free Acidity (meq/kg)
1 P	27.07 ± 0.05 abc	0.54 ± 0.02 abc	1.66 ± 0.01 ab	1.61 ± 0.14 ab	50.42 ± 1.91 a	13.67 ± 0.58 abc
2 P	24.76 ± 0.08 a	0.36 ± 0.01 abc	28.33 ± 2.89 a	1.58 ± 0.01 ab	58.33 ± 0.72 abc	3.67 ± 0.58 c
3 P	26.4 ± 0.08 abc	0.33 ± 0.01 abc	1.42 ± 0.01 ab	1.57 ± 0.01 ab	55 ± 5.73 abc	20.33 ± 1.53 abc
4 H	27.29 ± 0.02 abc	0.85 ± 0.05 b	1.02 ± 0.02 ab	1.95 ± 0.5 ab	68.75 ± 1.25 c	34 ± 1 abc
5 H	25.2 ± 0.04 ac	0.55 ± 0.05 abc	6.44 ± 0.38 ab	1.53 ± 0.06 ab	62.08 ± 0.72 abc	38.5 ± 0.5 abc
6 A	28.28 ± 0.07 abc	0.4 ± 0.01 abc	1.07 ± 0.02 ab	1.57 ± 0.01 ab	62.92 ±0.72 abc	44 ± 1 abc
7 L	30.55 ± 0.17 bc	0.55 ± 0.05 abc	3.40 ± 0.11 ab	1.61 ± 0.14 ab	58.58 ± 0.29 abc	67.67 ± 0.58 abc
8 H	28.57 ± 0.05 abc	0.46 ± 0.05 abc	1.61 ± 0.05 ab	1.78 ± 0.05 b	58.29 ± 0.59 abc	44.33 ± 1.53 abc
9 T	28.59 ± 0.1 abc	0.38 ± 0.03 abc	1.39 ± 0.04 ab	1.64 ± 0.01 ab	63.75 ± 1.25 abc	50.33 ± 0.58 abc
10 A	29.05 ± 0.06 abc	0.19 ± 0.01 abc	1.01 ± 0.01 ab	1.72 + 0.04 ab	58.33 ± 0.72 abc	66.67 ± 0.58 abc
11 T	28.46 ± 0.08 abc	0.6 ± 0.1 abc	1.54 ± 0.04 ab	0.05 + 0.01 a	67.08 ± 1.91 bc	36.67 ± 1.53 abc
12 H	28.39 ± 0.04 abc	0.55 ± 0.05 abc	7.22 ± 0.48 ab	1.57 ± 0.06 ab	51.67 ± 0.72 ab	47.33 ± 0.58 abc
13 A	26.04 ± 0.11 abc	0.65 ± 0.05 abc	1.08 ± 0.02 ab	1.64 ± 0.04 ab	53.58 ± 3.61 abc	29.83 ± 0.76 abc
14 A	27.61 ± 0.09 abc	0.29 ± 0.01 abc	1.05 ± 0.02 ab	1.62 ± 0.01 ab	63.33 ± 1.44 abc	55.67 ± 1.33 abc
15 C	28.82± 0.03 abc	0.16 ± 0.01 abc	3.22 ± 0.1 ab	1.68 ± 0.01 ab	57.92 ± 1.44 abc	76 ± 1 a
16 C	27.33 ± 0.02 abc	0.37 ± 0.05 abc	1.22 ± 0.01 ab	1.58 ± 0.01 ab	59.58 ± 1.44 abc	33.67 ± 0.58 abc
17 A	27.11 ± 0.07 abc	0.42 ± 0.01 abc	1.62 ± 0.04 ab	1.56 ± 0.01 ab	60.83 ± 1.44 abc	49.33 ± 1.53 abc
18 R	29.34 ± 0.01 abc	0.5 ± 0.01 abc	9.05 ± 0.82 a	1.75 ± 0.05 ab	57.50 ± 1.25 abc	69 ± 2.65 ab
19 R	29.54 ± 0.19 abc	0.12± 0.01 c	ND	1.69 ± 0.01 ab	57.92 ± 0.72 abc	49.33 ± 1.15 abc
20 P	28.09 ± 0.06 abc	0.19 ± 0.01 abc	1 ± 0.01 ab	1.68 ± 0.01 ab	55 ± 3.3 abc	15 ± 1 abc
21 P	32.64 ± 0.87 b	0.32 ± 0.01 abc	1 ± 0.01 ab	1.62 ± 0.01 ab	57.08 ± 1.44 abc	9 ± 1 bc
22 R	26.34 ± 0.11 abc	0.19 ± 0.01 abc	ND	1.55 ± 0.01 ab	54.58 ± 0.72 abc	40 ± 1 abc
23 C	29.60 ± 0.07 abc	0.15 ± 0.01 ac	1.43 ± 0.01 ab	1.77 ± 0.08 ab	55.83 ± 3.15 abc	48.67 ± 1.53 abc
24 P	30.10 ± 0.81 abc	0.17 ± 0.01 abc	1.25 ± 0.01 ab	1.67 ± 0.05 ab	57.08 ± 0.72 abc	13.67 ± 0.58 abc
Overall mean	28.13 ± 1.75	0.39 ± 0.19	3.25 ± 5.74	1.58 ± 0.35	58.60 ± 4.64	39.85 ± 19.74
A. mellifera Standards *	<20%	0.60 g/100g	>3 Schade Units	40–80 mg/kg	>60 g/100g	50 meq/kg
Min–max	24.76–32.64	0.12–0.85	1–28.33	0.05–1.95	50.42–68.75	3.67–76
Kurtosis	3.58	2.7	16.5	16.8	3.03	2.21
Asymmetry	0.34	0.49	3.66	−3.27	0.21	−0.08
Variation coefficient	6.25	48.42	176.55	21.97	7.92	49.54

* Standards based on Codex Standard for Honey, 2001: NMX-F-036-981; NOM-004-SAG/GAN-2018. ND = No detected. As shown in the data, different letters indicate statistically significant differences (Dunn’s post hoc test with Bonferroni correction, p < 0.05).

). Samples 20P, 21P, and 24P from the Palmilla region exhibited the highest moisture levels. These elevated levels may be attributed to the region’s low latitude, higher temperatures, and environmental humidity, which could increase the moisture percentage of honey samples, whereas lower temperatures are likely to result in honey samples with lower moisture percentages [42]. Environmental and geographical factors, including vegetation, climatic conditions, soil, harvesting, processing, bee species, and geographical origin, can markedly affect honey moisture content [9]. In tropical regions, with abundant polyfloral vegetation and high annual precipitation, nectar tends to have high water content [2,12]. In this sense, environmental conditions such as the temperature and high humidity may influence the flowering plant to produce nectar with high moisture but low sugar content [43]. The low altitude of this region, with its high temperature and high relative humidity, could potentially explain this. Moisture contents above 20% promote fermentation using osmotolerant yeasts, leading to the production of ethanol and carbon dioxide, which may degrade into acetic acid and water, giving the honey a sour or “off-taste” and a runny texture with small bubbles [44,45,46]. Therefore, the high moisture content observed in S. mexicana honey confirms earlier findings and underscores the importance of defining species-specific moisture standards and implementing adequate post-harvest practices to preserve product quality and extend shelf life. These results agree with previous studies that reported a moisture content > 20% [9,47]. For example, in the Tetragonula laeviceps-pagdeni complex, the average moisture content was 28 ± 1.8% [13]. Similarly, Sousa et al. [41] reported moisture values ranging from 17.2% to 35.4% with a mean of 29.12% in honey varieties produced by Melipona, Scaptotrigona, and Nannotrigona species.

3.1.2. Ash

The ash content in S. mexicana honey samples ranged from 0.12 to 0.85 g/100 g (Table 2), with statistically significant differences observed among different locations (p ≤ 0.05). Ash represents the total mineral concentration in honey and is commonly influenced by the botanical source of the nectar as well as the geographical area and climatic conditions of the collection site [47,48]. In general, light-colored honey tends to exhibit lower ash content compared with darker varieties, which are typically richer in mineral elements [49]. Mineral content contributes to the nutritional and functional value of honey [50,51,52]. The range of ash content observed in this study is comparable to that reported for Heterotrigona itama honey from Malaysia, which varied between 0.15 and 0.90 g/100 g [25]. These findings support the importance of ash content for standardization in food process applications, particularly when evaluating honey as a mineral-rich ingredient in functional food formulations.

3.1.3. Diastase Activity

Diastase is a natural enzyme secreted by honeybees and is widely recognized as a marker of honey freshness and quality, particularly in A. mellifera honey [11]. According to the Codex Alimentarius Commission [40], honey produced in tropical regions must exhibit a minimum diastase activity of 3.00 Göthe units, with acceptable values up to 8 Göthe units. In the present study, S. mexicana honey samples showed diastase activity ranging from 1 to 28.33 Göthe units, with a mean value of 3.25 ± 5.74 Göthe units (Table 2). Although some samples exceeded the minimum threshold, the majority fell below the threshold established by the Codex Alimentarius Commission [40]. This observation aligns with the previous studies on stingless bee honey. The presence of diastase in honey is primarily attributed to bee salivary secretions during nectar collection and transformation [53]. The low enzymatic activity observed may be influenced by bee species and does not necessarily indicate poor quality in stingless bee honey. However, these results underscore the need for redefining diastase activity thresholds specifically for S. mexicana honey. For instance, Biluca et al. [54] reported a mean diastase activity of 4.34 in S. bicuntata honey, reflecting relatively low enzymatic activity.

3.1.4. Hydroxymethylfurfural

Hydroxymethylfurfural (HMF) is a key chemical marker used to evaluate the establishment of the freshness, thermal history, and overall quality of honey [12]. In this study, HMF concentrations in S. mexicana honey samples ranged from 0.05 to 1.95 mg/kg, as shown in Table 2, indicating exceptionally low levels of thermal degradation. HMF is formed through the dehydration of hexoses, particularly fructose, and also via the Maillard reaction between reducing sugars and amino acids [9]. Related HMF levels are commonly associated with excessive heating, prolonged storage under non-optimal conditions, or advanced aging of honey [55]. Importantly, high concentrations of HMF have been linked to adverse health effects, including DNA damage, mutagenicity, organotoxicity, genotoxicity, and carcinogenicity [56]. The low HMF values observed in S. mexicana honey suggest minimal processing and high product freshness, which is desirable from both nutritional and technological standpoints. Interestingly, Biluca et al. [57] reported that stingless bee honey could show resistance to HMF formation even after exposure to high temperatures, probably because of high acidity, high moisture, and predominance of fructose. These results are consistent with previous studies on stingless bee honey varieties, which have reported HMF levels between 0.80 and 3.42 mg/kg in Trigona honey [58].

3.1.5. Reducing Sugars

Reducing sugars (RSs), primarily fructose and glucose, are the main monosaccharides in honey and serve as an immediate source of energy while significantly influencing flavor, hygroscopicity, viscosity, and granulation [10,43,59,60]. In this study, the reducing sugar content in S. mexicana honey samples ranged from 50.42 to 68.75 with a mean of 58.60 ± 4.64, as shown in Table 2. These values are slightly lower than those reported in A. mellifera honey and are consistent with previous findings in stingless honeybees, which typically exhibit reduced monosaccharide (glucose and fructose) content [13]. For instance, Silva et al. [61] reported an RS value of 56.14% in Melipona species from Brazil, which is consistent with the range observed in the present study. The lower RS levels observed in S. mexicana honey may be due to a higher proportion of alternative disaccharides such as trehalose, a sucrose isomer, as suggested by Fletcher et al. [62]. In this study, the observed variability is likely influenced by the diversity of floral sources visited by bees, geographical origin, environmental conditions (temperature and humidity), post-harvest honey processing (e.g., heating, weather exposure, and manipulation), storage time, and bee species [8,52,54]. In the sampled regions, dominant nectar-producing species include Pimenta dioica and Coffee arabica, as well as native plants, such as Heliocarpus sp and Miconia spp [63]. According to Clearwater et al. [64], nectar yield per flower, hexose ratios, and sucrose content can vary among plant species and throughout the flowering season, contributing to the compositional heterogeneity of honey.

3.1.6. Free Acidity, Lactone, and Total Acidity

Free acidity is one of the most important parameters for quality control of honey, as it contributes to its organoleptic profile, enhances antioxidant activity, and provides a natural defense against microbial growth [46]. In the samples analyzed in this study, free acidity values ranged from 3.67 to 76 meq/kg with an overall mean of 39.85 ± 19.74 meq/kg (Table 2). Although most samples fell within acceptable ranges, some exceeded the maximum limit of 50 meq/kg established by the Codex Alimentarius Commission for A. mellifera honey [40]. The elevated acidity observed in some samples could be attributed to the ongoing fermentation process, in which, due to the naturally occurring yeast present in this type of honey, the sugars and alcohol are transformed into acids [65]. In the presence of oxygen, alcohol may be transformed into acetic acid and water, imparting a sour taste and possible sensory degradation [60,66]. The variation in the total acid content in the different honey samples could be due to the geographical zone and the floral source [9]. In this regard, the stingless bees live in perennial colonies and pollinate a broad array of native and cultivated plants, including global commodities such as coffee [67]. Free acidity in honey primarily results from the presence of organic acids, particularly gluconic acid, which is in equilibrium with the lactone [46]. The mean lactone was 24.24 ± 5.49 meq/kg with values ranging from 8.67 to 31 meq/kg, as shown in

Table 3. Physicochemical parameters of Scaptotrigona mexicana samples in the warm humid forest of Puebla, Mexico.

Sample	Lactone (meq/kg)	Total Acidity (meq/kg)	Brix	Activity Water (Aw)	pH	Protein (g/100 g)
1 P	22.33 ± 1.53 a	36 ± 2 ab	72.92 ± 0.06 abc	0.88 ± 0.006 ab	3.56 ± 0.02 a	0.34 ± 0.01 abc
2 P	17.33 ± 1.04 a	21 ± 1.5 b	75.23 ± 0.09 a	0.89 ± 0.005 b	4.63 ± 0.06 b	0.47 ± 0.05 cde
3 P	8.67 ± 1.53 a	29 ± 2.65 ab	73.59 ± 0.08 abc	0.88 ± 0.01 ab	3.74 ± 0.01 ab	0.49 ± 0.05 def
4 H	29.17 ± 1.04 a	63.16 ± 1.60 ab	72.71 ± 0.02 abc	0.79 ± 0.01 ab	3.57 ± 0.06 ab	0.25 ± 0.01 a
5 H	28.33 ± 1.53 a	66.83 ± 1.44 ab	74.83 ± 0.04 ac	0.77 ± 0.00 ab	3.48 ± 0.01 ab	0.61 ± 0.01 fg
6 A	31 ± 2.65 a	75 ± 1.73 ab	71.72 ± 0.07 abc	0.78 ± 0.01 ab	3.16 ± 0.01 ab	0.32 ± 0.02 ab
7 L	27 ± 2 a	94.67 ± 2.52 ab	69.45 ± 0.17 bc	0.89 ± 0.01 ab	3.49 ± 0.01 ab	0.56 ± 0.05 ef
8 H	27.33 ± 1.53 a	71.67 ± 3.06 ab	71.43 ± 0.05 abc	0.78 ± 0.0 ab	3.19 ± 0.01 ab	0.34 ± 0.02 abc
9 T	25.83 ± 0.7 a	76.17 ± 0.29 ab	71.10 ± 0.1 abc	0.76 ± 0.0 ab	3.16 ± 0.01 ab	0.25 ± 0.01 a
10 A	24.83 ± 0.29 a	91.50 ± 0.87 ab	70.94 ± 0.06 abc	0.77 ± 0.0 ab	2.95 ± 0.04 ab	0.33 ± 0.03 ab
11 T	24.33 ± 0.58 a	61 ± 2 ab	71.54 ± 0.08 abc	0.77 ± 0.0 ab	3.25 ± 0.01 ab	0.57 ± 0.03 ef
12 H	26.67 ± 0.58 a	74 ± 1 ab	71.61 + 0.04 abc	0.78 ± 0.00 ab	3.41 ± 0.02 ab	0.84 ± 0.04 h
13 A	28.67 ± 0.58 a	58.5 ± 0.87 ab	73.96 ± 0.11 abc	0.75 ± 0.00 ab	3.55 ± 0.01 ab	0.71 ± 0.03 gh
14 A	26.50 ± 0.5 a	82.17 ± 6.53 ab	72.39 ± 0.09 abc	0.77 ± 0.00 ab	3.43 ± 0.03 ab	0.73 ± 0.04 gh
15 C	29.33 ± 0.58 a	105.33 ± 0.58 a	71.18 ± 0.03 abc	0.78 ± 0.00 ab	2.95 ± 0.01 a	0.46 ± 0.02 bcde
16 C	28 ± 1 a	61.67 ± 1.15 ab	72.67 ± 0.02 abc	0.75 ± 0.00 a	3.17 ± 0.02 ab	0.73 ± 0.1 gh
17 A	27.50 ± 0.5 a	76.83 ± 1.76 ab	72.89 + 0.07 abc	0.75 ± 0.003 ab	3.16 ± 0.01 ab	0.33 ± 0.02 ab
18 R	29.67 ± 4.73 a	98.67 ± 6.43 a	70.66 ± 0.01 abc	0.77 ±0.001 ab	3.14 ± 0.02 ab	0.25 ± 0.01 a
19 R	25.67 ± 0.58 a	75 ± 1 ab	70.45 ± 0.2 abc	0.75 ± 0.001 ab	3.11 ± 0.11 ab	0.39 ± 0.04 bcd
20 P	18.67 ± 3.79 a	33.67 ± 4.62 ab	71.18 ± 0.03 abc	0.76 ± 0.002 ab	3.81 ± 0.03 ab	0.4 ± 0.09 bcd
21 P	20 ± 1 a	29 ± 2 ab	67.35 ± 0.88 b	0.76 ± 0.001 ab	3.83 ± 0.02 ab	0.4 ± 0.04 bcd
22 R	17.67 ± 1.15 a	57.67 ± 0.58 ab	73.66 + 0.12 abc	0.75 ± 0.001 ab	3.87 ± 0.03 ab	0.6 ± 0.01 fg
23 C	18.67 ± 2.08 a	67.33 ± 3.21 ab	70.39 ± 0.07 abc	0.77 ± 0.002 ab	3.77 ± 0.04 ab	1.15 ± 0.06 i
24 P	18.57 ± 0.58 a	32.33 ± 0.58 ab	69.89 ± 0.81 abc	0.75 ± 0.003 a	3.92 ± 0.02 b	0.33 ± 0.03 ab
Overall mean	24.24 ± 5.49	64.09 ± 23.32	71.87 ± 1.76	0.79 ± 0.05	3.47 ± 0.38	0.08 ± 0.04
Min–Max	8.67–31	21–105.33	67.35–75.23	0.75–0.9	2.95–4.63	0.24–1.22
Kurtosis	3.85	2.23	3.58	3.9	4.39	4.37
Asymmetry	−0.09	−0.24	−0.35	1.59	1.02	1.23
Variation coefficient	22.66	36.39	2.44	6.03	11.01	44.27

As shown in the data, different letters indicate statistically significant differences (Dunn’s post hoc test with Bonferroni correction, p < 0.05).

. The determination of lactone is particularly relevant as its hydrolysis contributes directly to an increase in free acidity [62]. These results are consistent with those reported by Lopez-Garay et al. [68], who analyzed honey of S. mexicana from Veracruz, Mexico, and the values were 35.33 ± 0.58 meq/kg.

3.1.7. Brix

The soluble solid contents were expressed as Brix values, ranging from 67.35 to 75.23 in S. mexicana honey samples (Table 3). This parameter reflects the concentration of dissolved compounds, primarily sugars, along with organic acids and minerals [10]. Lower Brix values in stingless bee honey compared with A. mellifera honey are commonly observed and are often attributed to their higher moisture content, which results in a dilution of nectar during its transformation into honey [69]. Environmental conditions also play a critical role; for example, increased rainfall during flowering and nectar collection has been shown to reduce nectar concentration, thereby decreasing the final soluble solids content in honey [70]. The Brix values obtained in this study are consistent with those previously reported for stingless bee honey, such as Melipona beecheii honey, from Yucatán, which ranged from 72.8% to 77.3% as reported by Moo-Huchin et al. [46]. This similarity supports the idea that neotropical stingless bee honey varieties share comparable compositional traits, which are likely influenced by the bee species and the climate conditions of its native habitats.

3.1.8. Water Activity (Aw)

Water is the second most abundant component of honey after sugars. Honey samples from S. mexicana exhibited Aw values ranging from 0.75 to 0.9 with a mean of 0.79 ± 0.05, as shown in Table 3. Water activity plays a critical role in determining the microbial stability of honey [52]. Although no official thresholds for Aw are specified in the Codex Alimentarius Commission [40], high Aw could lead to honey fermentation during storage, caused by the action of osmotolerant yeasts on the sugars fructose and glucose and resulting in the formation of ethyl alcohol and carbon dioxide [66]. Several factors may influence the Aw of honey, including botanical origin, maturity, processing techniques, and storage conditions [52]. These results are similar to those of Kek et al. [71], who reported a value of 0.76 ± 0.03 in Heterotrigona itama honey from the forest in Teluk Intan, Perak.

3.1.9. pH

The pH value is used as a reference for the acidity of the product and is essential for assessing its quality since this parameter indicates whether the medium is suitable for safe AIM consumption. The pH values obtained in the current study ranged from 2.95 to 4.63, with a mean of 3.47 ± 0.38 (Table 3). The variation among the stingless bee honey samples could be affected by the type of flora and influenced by high rainfall, which can alter the nectar composition collected by bees, which in turn affects the pH of the honey [64]. All the samples analyzed had low pH values and high levels of total acid. As mentioned above, the presence of organic acids, such as gluconic acid and lactone, impacts the pH value of honey, as a high level of total acidity lowers the pH [9,46]. Therefore, the low pH improves the shelf life, texture, and stability in honey [60]. The acidic nature of stingless bee honey helps prevent the presence and growth of microorganisms; it is an antimicrobial factor, hence its use to treat infectious diseases such as cough and wounds [40]. Xolalpa-Aroche et al. [8] conducted a study with M. beecheii and S. mexicana honey samples from Veracruz and Puebla and reported pH values ranging from 3.37 and 4.18.

3.1.10. Protein

The protein content in S. mexicana honey samples analyzed in this study ranged from 0.24 to 1.22 g/100 g with a mean of 0.08 ± 0.04 g/100 g (Table 3). Proteins present in honey may originate from the honeybee itself, the nectar, or the pollen of the visited plants [26]. The principal proteins in honey are enzymes, which are incorporated during the honey ripening process. Among them, diastase (amylase) catalyzes the hydrolysis of starch into maltose and is relatively heat- and storage-stable. Invertase (α-glucosidase) facilitates the breakdown of sucrose into glucose and fructose. Glucose oxidase and catalase are also important, as they participate in the formation and regulation of the production of hydrogen peroxide, a compound contributing to the antibacterial properties in honey [72]. The variability in the protein content among S. mexicana honey samples may be attributed to the differences in enzyme composition derived from nectar sources and the wide botanical diversity available to these bees [67]. Notably, there is no official regulation or standard defining acceptable protein content in honey [26]. These results are consistent with those reported by Lim et al. [25], who observed protein levels between 0.2 and 0.8 g/100 g in stingless bee honey from Malaysia.

3.1.11. Color

Color is the first sensory attribute perceived by consumers and often influences their preference and perception of quality [9,71]. In this study, the color parameters of S. mexicana honey are presented in

Table 4. Color of Scaptotrigona mexicana honey samples in the warm humid forest of Puebla, Mexico.

Sample		Color
	L (Lightness)	a (Red+/Green − Axis)	b (Yellow+/Blue − Axis)	C (Chroma)	H (Hue)
1 P	24.76 ± 0.6 ef	1.8 ± 0.08 a	12.67 ± 0.25 e	24.83 ± 0.61 d	8.12 ± 0.52 a
2 P	20.52 ± 0.45 b	4.37 ± 0.06 de	28.66 ± 0.13 o	20.98 ± 0.45 ab	8.68 ± 0.13 a
3 P	19.6 ± 0.23 a	5.28 ± 0.06 fg	26.21 ± 0.38 n	20.3 ± 0.24 a	11.38 ± 0.16 b
4 H	22.96 ± 0.17 d	2.68 ± 0.06 b	8.09 ± 0.11 ab	23.12 ± 0.17 c	18.36 ± 0.36 gh
5 H	32.28 ± 0.1 l	6.25 ± 0.12 hi	20.44 ± 0.24 k	32.88 ± 0.11 i	17.01 ± 0.12 fg
6 A	29.34 ± 0.21 hi	5.02 ± 0.23 f	15.2 ± 0.73 gh	29.77 ± 0.23 g	18.29 ± 0.51 gh
7 L	38.87 ± 0.37 q	4.99 ± 0.15 f	29.19 ± 0.43 o	39.19 ± 0.35 n	9.71 ± 0.41 ab
8 H	30.48 ± 0.18 k	5.77 ± 0.03 gh	16.18 ± 0.17 hi	31.02 ± 0.18 h	19.63 ± 0.19 h
9 T	29.55 ± 0.39 ij	8.47 ± 0.03 k	16.82 ± 0.08 i	30.74 ± 0.38 h	26.73 ± 0.14 k
10 A	37.63 ± 0.41 p	4.88 ± 0.05 ef	24.15 ± 0.5 m	37.94 ± 0.4 m	11.42 ± 0.27 b
11 T	26.29 ± 0.13 g	5.8 ± 0.13 gh	10.36 ± 0.29 c	26.93 ± 0.15 f	29.23 ± 0.31 l
12 H	29.45 ± 0.17 hi	5.02 ± 0.2 f	20.07 ± 0.25 k	29.87 ± 0.14 g	14.04 ± 0.5 cd
13 A	33.49 ± 0.25 m	6.37 ± 0.11 i	22.42 ± 0.54 l	34.09 ± 0.23 jk	15.87 ± 0.16 ef
14 A	27.09 ± 0.7 g	2.21 ± 0.09 ab	9.1 ± 0.75 b	27.18 ± 0.71 f	13.69 ± 0.78 c
15 C	35.93 ± 0.02 o	6.03 ± 0.02 hi	23.78 ± 0.02 m	36.44 ± 0.22 l	14.23 ± 0.05 cde
16 C	30.31 ± 0.4 jk	7.11 ± 0.13 j	18.10 ± 0.63 j	31.13 ± 0.42 h	21.47 ± 0.65 i
17 A	26.8 ± 0.03 g	5.21 ± 0.05 f	11.49 ± 0.07 d	27.34 ± 0.03 f	24.4 ± 0.17 j
18 R	25.43 ± 0.34 f	3.91 ± 0.54 cd	8.01 ± 0.59 a	25.73 ± 0.27 e	25.92 ± 1.42 jk
19 R	21.51 ± 0.4 c	3.41 ± 0.48 c	14.39 ± 0.31 fg	21.78 ± 0.4 b	13.32 ± 1.86 c
20 P	23.19 ± 0.41 d	3.76 ± 0.14 c	13.50 ± 0.32 ef	23.49 ± 0.4 c	15.59 ± 0.83 def
21 P	34.51 ± 0.41 n	3.79 ± 0.15 c	15.74 ± 0.3 h	34.72 ± 0.41 k	13.56 ± 0.48 c
22 R	31.54 ± 0.31 l	11.32 ± 0.4 m	31.24 ± 0.64 p	33.52 ± 0.33 ij	19.93 ± 0.88 hi
23 C	24.31 ± 0.39 e	9.35 ± 0.34 l	16.94 ± 0.67 i	26.05 ± 0.31 e	28.93 ± 1.43 l
24 P	28.64 ± 0.29 h	6.53 ± 0.40 i	19.53 ± 0.5 k	29.37 ± 0.28 g	18.5 ± 1.36 gh
Overall mean	28.52 ± 5.2	5.39 ± 2.16	18.01 ± 6.63	29.1 ± 5.24	17.42 ± 6.11
Min–Max	19.33–39.44	1.74–11.99	7.5–32.05	20.02–29.73	7.72–31.2

The different letters in the same column indicate significant differences (p ≤ 0.05) between honeys from the same genera and produced in different geographical areas, as determined by Tukey’s test.

. The lightness (L) values range from 19.33 to 39.44, indicating that all samples fall within the dark honey category, as values ≤ 50 on the L scale are classified as dark-colored honey [9]. Dark honey varieties have been associated with higher mineral content, as reported by Chua et al. [73]. The chromatic coordinates “a” and “b” also showed positive values across all samples. The “a” ranged from 1.74 to 11.99, which indicates varying intensities of red pigmentation, while “b” ranged from 7.5 to 32.05, corresponding to a yellow hue. Honey color is influenced by its pigment composition and is closely linked to nectar source, botanical and geographical origin, mineral and phenolic contents, as well as environmental and processing factors such as storage duration and temperature [74,75]. Color changes may also result from beekeeping practices, including the use of aged honeycombs, metal contact, or suboptimal storage conditions [76]. These results are comparable to those reported by Kek et al. [71] for Heterotrigona itama honey (L = 24.9 ± 1.38, a = 1.90 ± 0.49, and b = 2.52 ± 1.05).

3.2. Bioactive Compounds

3.2.1. Phenols

The total phenolic content of S. mexicana honey from different regions is presented in Figure 2. The samples were grouped by region due to the differences in local vegetation and geographical characteristics. Honey from the Cruz Blanca region exhibited the highest phenolic content, ranging from 45.39 to 100.39 mg GAE/100 g, while the samples from Tacuba showed the lowest values, with a minimum of 29.69 mg GAE/100 g. Statistical analyses revealed significant differences among regions (p < 0.005), and the effect size (η² = 0.38) confirmed that geographic origin has a considerable impact on the phenolic content levels. Phenolic compounds are secondary plant metabolites derived from nectar, pollen, or propolis [77,78], and they present one of the main functional honey bioactives [15]. Their relevance lies in their demonstrated role in preventing oxidative stress-related conditions, including atherosclerosis, cancer, infections, and inflammatory diseases [48]. Moreover, phenolic compounds contribute to the antibacterial and antioxidant properties of honey, which are potentially enhanced by other components such as vitamins C and E [9]. Stingless bee honey has been reported to contain a wide variety of phenolics, such as kaempferol, p-coumaric acid, hesperidin, ferulic acid, ellagic acid, trans-cinnamic acid, rutin, catechin, chrysin, and hesperidin [16,79]. These compounds also influence organoleptic characteristics (color, taste, and flavor) and serve as chemical indicators to determine different botanical and geographical origins of honey [80,81,82]. The variability observed in the phenolic content among S. mexicana honey samples may depend on the botanical source of nectar, season, storage conditions, geographical area, environmental conditions, beekeeping management, foraging area, and bee species [5,54,83]. Notably, altitude has been identified as a critical factor influencing the concentration and diversity of bioactive compounds in plants, which in turn affects the phenolic content of the honey derived from their nectar [84]. These results align with those reported by Jimenez et al. [9], who found phenolic concentrations ranging from 25.85 to 40.1 mg GAE/100 g in S. mexicana honey from Veracruz, Mexico.

3.2.2. Flavonoids

The flavonoid contents of S. mexicana honey from different regions are presented in Figure 2. The samples from the Ayahualo region showed the highest flavonoid levels, ranging from 0.70 to 10.80 mg QE/100 g, while those from the Tonalmecoyo region showed the lowest concentration, ranging from 3.2 to 3.27 mg QE/100 g. Statistically significant differences were observed among regions (p < 0.005), and the effect size (η² = 0.26) indicated that botanical origin plays an important role in determining the flavonoid content in honey. Flavonoids are a major subclass of polyphenolic compounds in honey, known for their antioxidant, anti-inflammatory, and antimicrobial activities. Their presence and concentration vary depending on the floral source, and the variability in S. mexicana honey can be attributed to regional differences in vegetation and environmental conditions. Common flavonoids reported in honey include apigenin, pinocembrin, quercetin, galangin, chrysin, and hesperetin [85]. The content of flavonoids in honey is strongly influenced by several factors such as the botanical source of nectar, season, geographical area (altitude and latitude), environmental conditions, foraging area, and bee species [5,54,83]. Altitude has a key influence on plant metabolism, as higher elevations are associated with lower temperatures and increased UV-B radiation, both of which stimulate the synthesis of secondary metabolites such as flavonoids [86,87]. Comparable results were reported by Sousa and colleagues [16], who noticed a range of flavonoids from 1.9 to 4.4 mg QE/100 g in Melipona honey from Brazil. These values fall within the range observed in the present study, supporting the idea that stingless bee honey generally contains appreciable quantities of flavonoids, which contribute to its bioactive potential.

3.2.3. Antioxidants (DPPH and ABTS)

These results of the in vitro antioxidant assays showed considerable variability among S. mexicana honey samples. DPPH values ranged from 3.5 to 18.27 mg GAE/100 g, while ABTS values ranged from 5.83 to 112.11 mg GAE/100 g (Figure 3). Statistically significant differences were observed between regions, p < 0.05. The effect size analysis confirmed that S. mexicana honey had a considerable impact on antioxidant activity with η² = 0.49 for the ABTS assay and η² = 0.37 for the DPPH assay, indicating a strong association with regional origin. The samples from the regions of Hueytamalco and Limontitan Grande exhibited the highest antioxidant activity. These areas are characterized by the cultivation of crops such as apple, pear, avocado, coffee, and orange, suggesting that the local flora may influence the antioxidant profile of the honey. This observation aligns with previous findings by Shakoori et al. [88], who emphasized the role of botanical origin and geographical location in determining honey’s antioxidant capacity. Antioxidants in honey neutralize free radicals, thereby contributing to the prevention of chronic diseases [89,90]. The elevated antioxidant capacity of honey varieties from Limontitan Grande and Rancho viejo (Figure 3) reflects their higher phenolic and flavonoid contents, which is consistent with prior studies that reported a strong correlation between phenols, flavonoids, and antioxidant activity [14,15,82,91]. The antioxidant activity of honey is closely related to the chemical structure of phenols, specifically the number of hydroxyl groups and the position of hydroxyl groups on the aromatic rings [90]. In addition to phenols and flavonoids, the antioxidant potential of honey may also be influenced by other components such as vitamins E and C, and carotenoids [9,70]. These compounds may originate from multiple sources, including nectar (e.g., vitamins and phenolic compounds), pollen (e.g., proteins and amino acids), the bee (e.g., enzymes), and processing conditions (e.g., Maillard reaction products) [92], highlighting the complex and multifactorial nature of antioxidant mechanisms in S. Mexicana honey.

3.2.4. Scatter Plot Matrix and Spearman’s Correlation

The scatter plot matrix (Figure 4) illustrated significant correlations between phenolic content, flavonoids, color, and antioxidant activity, supporting the role of bioactive compounds in shaping functional properties. The red color parameter “a” and the total phenolic content in S. mexicana honey samples (r = 0.43, p < 0.001) indicate that phenol content is associated with more reddish tones. However, the coefficient of determination (ρ² = 0.19) suggests that 19% of the variability was in the red hue, implying that other pigments may also contribute to color. As reported by Otmani et al. [93], honey color reflects the presence of natural pigments, such as flavonoids and carotenoids. In a related study, Jimenez and coworkers [9] noticed a strong correlation (r = 0.827) between the phenols and the yellow parameter “b” in honey, reinforcing the idea that coloration can serve as a proxy for bioactive content. In our study, the yellow color “b” also showed a significant positive correlation (r = 0.43, p < 0.001) with antioxidant activity as measured using the DPPH assay. Additionally, both total phenols and flavonoids were significantly correlated with antioxidant activity evaluated through ABTS (r = 0.49, p < 0.001, and r = 0.43, p < 0.001, respectively), corroborating findings by Duarte et al. [77], who reported similar correlations between the total phenolic content and flavonoid content (r = 0.53 and r = 0.534, p < 0.01, respectively) in relation to the DPPH assay in Melipona honey varieties from Brazil. These findings support the widely established association between phenols and antioxidant potential in stingless bee honey [14,94]. Turkmen et al. [85] also noted that flavonoids contribute significantly to antioxidant properties, with the radical scavenging activity depending on molecular structure and the number of hydroxyl groups [15]. Nevertheless, honey is a complex matrix composed of multiple bioactive substances. In addition to phenols and flavonoids, minor components such as minerals, amino acids, peptides, proteins, organic acids, and enzymes also contribute to its antioxidant activity, albeit to a lesser extent [90].

3.2.5. Hierarchical Cluster Analysis

The hierarchical cluster analysis was performed to examine the similarities and differences in the physicochemical parameters of S. mexicana honey from various localities. This multivariate approach allowed for the grouping of honey samples based on their compositional profiles. As shown in Figure 5, the analysis identified two main clusters (highlighted in red and blue), suggesting distinct physicochemical patterns among the samples. These groupings likely reflect differences in geographical origin or climatic and local factors, such as rainfall. Notably, honey samples from La Palmilla showed higher moisture content, which may be associated with the elevated precipitation levels typical of that region. Such environmental influences can affect nectar composition and, consequently, the final properties of honey.

Vegetation is strongly influenced by environmental and geographical factors, which in turn affect the physicochemical characteristics of honey. The cluster plot with confidence ellipses based on physicochemical properties exposed the presence of two distinct groups (%) of different S. mexicana honey samples (Figure 6). This analysis reduces the dimensionality of the dataset into two principal components: Dimension 1 (Dim 1), which explains 26.6% of the variance, and Dimension 2 (Dim 2), which accounts for 17.4%. The clustering pattern indicates that the samples are grouped into two well-differentiated clusters based on their physicochemical parameters. Cluster 1 is characterized by higher values in the following physicochemical parameters: Brix degrees (sugar concentration), reducing sugars, free acidity, lactone, pH, moisture, Aw, protein, diastase, HMF, and minerals. The samples in this cluster were sourced from a region of higher latitude (20.018477), suggesting that the climate, temperature, and vegetation may influence the physicochemical characteristics of honey in this area. In contrast, Cluster 2 comprises samples with lower values for the physicochemical parameters. Interestingly, these samples showed higher moisture content that could be related to high precipitation compared with the other regions. The samples of this cluster were sourced from a region of lower latitude, which could be related to higher values of the above parameters, due to differences in climate or local conditions. Previous studies have demonstrated that the physicochemical properties of honey are closely linked to the geographical area, specifically the environmental and climatic conditions in which the plants grow, which determine their metabolism, and directly affect the composition of the nectar offered to the bee for honey production [12,16].

3.2.6. α-Amylase Inhibition In Vitro Assay

In the current study, the aqueous extracts of S. mexicana honey significantly inhibited α-amylase activity (p < 0.05). The selected honey samples originated from Cruz Blanca, which exhibited the highest phenol content. Tree honey extract samples (15 C, 16 C, and 23 C) were evaluated at concentrations ranging from 20 to 100 μg/mL. As shown in Figure 7, a dose-dependent increase in α-amylase inhibition was observed, indicating that higher concentrations of honey extract resulted in stronger α-amylase inhibition. This suggests a reduction in the breakdown of α-(1–4) glycosidic bonds, effectively delaying glucose release from starch. Effect size analysis revealed a strong influence of the extract concentration on enzyme inhibition (η² > 0.9), which means that more than 90% of the variability in enzyme inhibition was attributable to extract concentration. At 100 μg/mL, the highest inhibitory effect was observed in sample 23 C (49.20% ± 2.74%), followed by samples 16 C (34.04% ± 2.12%) and 15 C (23.89% ± 1.08 %). Although the inhibitory activity was lower than that of the reference drug acarbose, the results clearly demonstrate the potential of S. mexicana honey as a natural inhibitor of α-amylase. The results are consistent with those of Ali et al. [6], who reported 46.76% of inhibition using Heterotrigona itama multifloral honey from Malaysia. The inhibitory effects are likely attributed to phenols, which have been implicated in modulating enzymatic activity and delaying carbohydrate digestion [94,95]. Inhibition of intestinal α-amylase has been proposed as an effective strategy for managing postprandial hyperglycemia [21]. While synthetic α-amylase inhibitors are effective, they are often associated with undesirable side effects [22]. In this context, S. mexicana honey represents a promising natural alternative for decreasing serum blood glucose levels. Potential functional food applications of S. mexicana honey include its incorporation into yogurt-based products targeting glycemic control, as shown by Prokisch et al. [96], and its use in antioxidant-rich bakery items such as antioxidant syrups or diabetic friendly baked goods, leveraging honey’s moisture retention and sensory enhancement properties as reviewed by Bogdanov [97].

3.2.7. Kinetics of α-Amylase Inhibition

The kinetics of α-amylase inhibition in the honey samples were further evaluated using Lineweaver–Burk plots. The results showed that all of the samples (15 C, 16 C, and 23 C) exhibited mixed non-competitive inhibition, as evidenced by the decrease in both the Vmax and Km values compared with the control (Figure 8 and

Table 5. Kinetics of α- amylase inhibition.

Sample	Equation	Vmax (O.D.540/min)	Km (mM)	Inhibition Type
Control	y = 6.4842x + 17.202	0.058	0.37	No inhibition
Honey 15 C	y = 33.699x + 32.943	0.030	1.02	Mixed non-competitive inhibition
Honey 16 C	y = 32.112x + 25.043	0.039	1.28	Mixed non-competitive inhibition
Honey 23 C	y = 39.161x + 23.48	0.042	1.66	Mixed non-competitive inhibition

). This suggests that the inhibitory compounds present in S. mexicana honey interact with the enzyme at or near the active site, potentially altering both enzyme affinity and catalytic efficiency. The variations observed in the Vmax and Km values may be attributed to the differences in their bioactive compound profiles, particularly phenols with known enzyme-modulating properties.

4. Conclusions

The findings from this study demonstrate that S. mexicana honey presents distinct physicochemical characteristics compared with conventional A. mellifera honey, particularly in parameters such as moisture content, free acidity, diastase activity, pH, and HMF levels. These differences highlight the need to establish species-specific quality standards considering the influence of botanical origin, geographical conditions (altitude, climate, and precipitation), and production process. A positive correlation was observed between total phenolic content, flavonoids, and antioxidant capacity, supporting the classification of S. mexicana honey as a potential source of functional bioactive compounds. Furthermore, samples with higher phenolic content exhibited significant α-amylase inhibitory activity, suggesting potential applications in the development of food ingredients aimed at glycemic control. This study contributes valuable data for the functional and technological characterization of S. Mexicana honey and underscores its relevance in food process development, particularly in the formulation of natural sweeteners with added health benefits. These findings support ongoing efforts to integrate stingless bee honey into regulated markets and functional food systems. Further research is required to explore its biological activity in vivo, standardize processing methods, and promote sustainable meliponiculture as a strategy for ecological preservation and rural development. Overall, S. mexicana represents a promising bioresource that aligns with current trends in sustainable food systems and functional product innovation. This study is limited by the sample size, the absence of long-term stability analysis, and the lack of in vivo validation. Therefore, future research should include clinical trials and comparative studies with other stingless bee species to identify unique bioactive profiles and the development of standardized quality guidelines tailored to stingless bee honey. Additionally, incorporating artificial intelligence tools, such as machine learning models for honey authentication, could support the standardization of stingless bee honey quality.