Influence of the type of container and traditional methods on the long-term storage of honey produced by stingless Scaptotrigona mexicana: bioactive compounds and antioxidant properties

a Universidad Veracruzana. Centro de Investigación y Desarrollo en Alimentos. Av. Dr. Luis Castelazo, s/n. 91000. Xalapa, Veracruz, México.

e Instituto de Ecología, A.C. Red de Estudios Moleculares Avanzados. Veracruz, México.

Scaptotrigona mexicana honey is characterized by its nutritional and antioxidant properties, but it has a high moisture content that affects its stability during storage. The objective of this work was to evaluate the physicochemical and antioxidant properties by UV-Visible spectroscopy, profile of phenolic compounds by ultra-high performance liquid chromatography coupled to mass spectrometry and fatty acids and volatile compounds by gas chromatography coupled to mass spectrometry, minerals by microwave plasma atomic emission spectroscopy, from honey stored in different containers that, along with traditional methods, are commonly used to increase its stability. Most physicochemical and antioxidant properties were not significantly different from those of freshly harvested honey. The results suggest that the packaging with an exhaust check valve has a significant effect on the decrease in moisture content and water activity, but not on the physicochemical and antioxidant properties for at least 2 yr of storage. These results suggest that the type of container should be considered when storing honey as it significantly (P<0.05) affects its properties and quality.

Keywords: Antioxidant activity; Container; Honey; Scaptotrigona mexicana; Meliponine stingless bees.

Stingless bee honey is highly demanded by consumers, due to its healing properties and quality(1). However, this type of honey is characterized by a higher moisture content, which increases the probability of its deterioration. In addition, it has been reported that excess water can be a negative quality attribute since it creates a high risk of inducing fermentation processes and consequently altering the organoleptic properties of honey(2). Various methodologies have been reported to maintain the nutritional and sensory properties of honey, increase its stability, and improve its handling and marketing conditions to obtain a safe product for the consumer(3), such as the use of dehydration in plastic trays using controlled-temperature ovens(4). However, exposure to high temperatures produces an increase in the content of hydroxymethylfurfural(5). It has been reported that heating to boiling temperature eliminates yeasts and reduces moisture content and that certain containers, such as unglazed clay pots, produce a reduction in moisture content of up to 20 %, increasing the shelf life of honey(6). However, clay containers have the disadvantage of being fragile, of low capacity and not functional for transport. In a traditional method used in the Totonacapan region, Veracruz, Mexico, beans are added to honey since, according to the inhabitants, these seeds absorb moisture from the honey, making it more stable. Another traditional technique is the use of vacuum packing to avoid the entry of air. Therefore, the objective of this research was to investigate the effect of storage in different plastic containers on the physicochemical properties, antioxidants, phenolic compounds and fatty acids profile, minerals and volatile compounds in honey during storage.

2,2´-Diphenyl-1-picrylhydrazyl (DPPH), Trolox (6-hydroxy- gallic acid, quercetin, Folin–Ciocalteu reagent and 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) were purchased from Sigma (St. Louis, MO, USA).

Twenty-four (24) liters of honey were provided by the company Chasseurs De Saveurs S.A. C.V. They were collected from the Zozocolco region, Veracruz, Mexico. Samples were collected from hives in rustic wooden boxes that meliponicultors keep in their homes. The extraction was carried out during the month of May 2018 using a 20 mL syringe. The 24-L batch of honey collected was divided into four batches (6 L per batch). The honey from each batch was divided into three 2-L containers (Figure 1). T0 (control) was immediately used for the analysis of the evaluated parameters and collected from rustic wooden boxes. Batches T1 to T4 were placed in plastic containers commonly used for the commercialization of honey. The description of the treatments is presented below: T1: honey stored in an opaque plastic tray (high density polyethylene), T2: honey stored in an opaque plastic tray added with five bean seeds, T3: honey stored in an opaque plastic tray with an exhaust check valve (ZAZOLYNE, China) used in the fermentation of wines, T4: honey stored in a transparent plastic container (polyethylene terephthalate, 1). The honey samples were placed in a room with a temperature of 25 ºC and were analyzed at the beginning and after 2 yr of storage. All the determinations were made by triplicate.

T0= control; T1= honey stored in an opaque plastic tray; T2= honey stored in an opaque plastic tray added with five bean seeds; T3= honey stored in an opaque plastic tray with an exhaust check valve; T4= honey stored in a transparent plastic container.

The moisture, electrical conductivity, pH, and titratable acidity of the honey samples were determined, using the appropriate analytical standard procedures(7). Electrical conductivity was determined using a conductivity meter (Mettler Toledo, ME 226 model, Pittsburgh, USA) and water activity was measured using a water activity meter (AquaLab, Model 4TE, Meter group, Inc, USA). The color was measured with a colorimeter (ColorFlex V1–72 SNHCX 1115, Hunter Lab, USA) using parameters CIE L*,a*, b* and total color change, browning index and Chroma were calculated.

The content of total phenolic compounds and the antioxidant activity: DPPH (2,2-Diphenyl-1-picrylhydrazyl), FRAP (Ferric reducing ability of plasma) and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) assays were determined using methanolic honey extract with a 1:100 dilution(8).

The total phenolic compounds were determined by the Folin-Ciocalteu method with some modifications(9). Thirty microliters of each sample and 30 μL of Folin-Ciocalteu were mixed and incubated for 2 min (40 °C). After 240 μL of Na2CO3 (5%) were added, they were incubated for 20 min (40 °C). After that time, the absorbance was read (λ= 765 nm). Vitamin C content was determined using a standard curve made with L-ascorbic acid (99% purity; Sigma Rec. 84272, St. Louis, Missouri, USA) at a concentration of 0–50 mg and the results were expressed as mg equivalents of ascorbic acid (AAE) per gram.

The percentage of inhibition of the DPPH radical was determined by mixing 30 µL of each sample with 270 µL of DPPH reagent, then incubated for 30 min, subsequently the absorbance was read (λ= 517 nm, Multiskan FC, model IVD, Finland). ABTS was determined, 30 μL of extract and 270 μL of ABTS reagent were mixed and then incubated for 30 min (25 °C). Then the absorbance (λ= 734 nm, Multiskan FC, model IVD, Finland) was measured. Finally, FRAP was determined, 30 μL of extract and 270 μL of FRAP reagent were mixed, then incubated for 30 min (37 °C) and absorbance was measured (λ= 593 nm, Multiskan FC, model IVD, Finland). For the two technics 0.1–1 mg/mL Trolox calibration curve was performed(9). The results are expressed in miligrams of Trolox equivalents per gram of dry weight of each sample.

For the honey extracts, one gram of honey was weighed, 10 mL of methanol was added, and it was subjected to ultrasonication (Sonics Materials VCX 750 ultrasonic microprocessor, Connecticut, USA) for 10 min, this process was repeated until exhaustion. Subsequently, the solvent was evaporated to dryness in a rotary evaporator (Rotavapor R-100, Büchi, Flawil, Switzerland). Then, the honey extract (10 mg) was re-dissolved in 1.0 mL of MeOH with 0.1% of formic acid (Both MS grade, Sigma-Aldrich), filtered and placed in a 1.5 mL Ultra High-Performance Liquid Chromatography (UPLC) vial. The identification and quantitation of individual phenolic compounds was performed with an UPLC coupled to a triple quadrupole mass spectrometer (Agilent Technologies 1290-6460, Santa Clara, California, USA). Chromatographic conditions were: flow 0.3 mL/min, injection volume 2 µL, and column temperature 40 °C. The gradient started at 1% B, then changed to 50% B in 30 min, then 99 % B in 4 min followed by an isocratic step to 99 % B for 4 min. Subsequently, a gradient to 1% B in 1 min followed by an isocratic step for 5 min. The mass spectrometry conditions were electrospray ionization in positive and negative modes, temperature (T) of the gas 300 °C and T of the sheath gas 250 °C with flows of 5 and 11 L/min, respectively. The nebulizer pressure was 45 Psi and the capillary and nozzle voltages were 3,500 and 500 V, respectively. Forty-eight (48) compounds were searched: shikimic acid, gallic acid, L-phenylalanine, protocatechuic acid, 4-hydroxybenzoic acid, gentisic acid, 4-hydroxyphenylacetic acid, (-)-epigallocatechin, (+)-catechin, vanillic acid, scopoline, chlorogenic acid, caffeic acid, malvin, kuromanin, procyanidin B2, vanillin, keracyanin, (-)-epicatechin, 4-coumaric acid, mangiferin, umbelliferone, (-)-gallocatechin gallate, scopoletin, ferulic acid, quercetin 3,4-di-O-glucoside, 3-coumaric acid, salicylic acid, sinapic acid, epicatechin gallate, ellagic acid, myricitrin, pelargonidin, quercetin 3-D-galactoside, rutin, p-anisic acid, quercetin 3-glucoside, luteolin 7-O-glucoside, malvidin, 2,4-dimethoxy-6-methylbenzoic acid, penta-O-galloyl-B-D-glucose, kaempferol 3-O-glucoside, quercitrin, naringin, rosmarinic acid, trans-cinnamic acid, luteolin, and kaempferide. Each compound was identified using a dynamic multiple reaction monitoring method and quantified using calibration curves from 0.25 to 19 µM, with a coefficient of determination greater than 0.99(9).

Volatile compounds (esters, aldehydes, ketones and terpenes that are characteristic of this type of sample) were determined in 3.0 g of honey stored. The honey was placed in a vial sealed with a PTFE / Teflon cap and heated to 100 °C, then the sample was injected using an Agilent Technologies Head-space model 7694E and a gas chromatograph (Agilent Technologies™, model 6890 N, Net Work GC system, Santa Clara, California, USA) equipped with a DB-5 capillary column (60 m × 0.25mm id × 0.25 µm film thickness) was used. The GC conditions were initial temperature: 45 °C for 5 min, heating ramp: 15 °C/min up to 280 °C, for 1 min. Helium at a flow of 1 mL/min, injector temperature of 250 °C. Identification of volatile compounds was performed by mass spectrometry using the Agilent Technologies™ Model 5975 inert XL mass spectrometer, mass spectra were obtained by electron impact ionization at 70 eV. For identification, the mass spectra obtained for each compound were compared with a database (HP Chemstation-NIST 05 Mass Spectral search program, version 2.0d).

Oily material was extracted from honey using a Soxhlet extractor with hexane (60–80 °C) for 6 h. The oily extract was filtered and concentrated under vacuum (Büchi, Flawil, Switzerland) to get crude extracts. Methyl Esters of Fatty Acids (FAMEs) were obtained through an esterification process and analyzed by gas chromatography coupled to mass spectrometry (GC-MS)(10). A gas chromatograph (Agilent Technologies™, model 6890 N, Net Work GC system, Santa Clara, California, USA) equipped with a DB-5 column (5% methylpolysiloxane, cat-1225082, J&W Scientific, USA) was used. The GC conditions were: initial temperature: 150 °C for 5 min, heating ramp: 30 °C/min to a temperature of 210 °C, 1 °C/min to 213 °C for 40 min, finally 20 °C/min up to 280 °C for 40 min. Helium at a flow of 1 mL/min, injector temperature of 250 °C. Identification of fatty acids was performed by mass spectrometry using the Agilent Technologies™ Model 5975 XL Inert Mass Spectrometer. The identity of each fatty acid was assigned using an external standard (FAMEs mix, C8:C22, cat no. 18920-1AMP, Sigma-Aldrich) which contained: octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, 9-hexadecenoic acid, hexadecanoic acid, cis-9,12, heptadecanoic acid, octadecadienoic acid, cis-9-octadecaenoic acid, heptadecanoic acid, eicosanoic acid, 11-eicosenoic acid.

For mineral extraction, honey (1 g) was digested in digester tubes with a nitric acid solution (5%) in a 1:10 (p:v) ratio. The tubes were placed in a Kjeldahl digester (Speed Digester K-439, Büchi, Flawil, Switzerland) and digested at 170 °C for 2 h until an almost clear solution was obtained. This solution was filtered and later transferred to a 50 mL volumetric flask and diluted with 5% HNO3 to finally be injected. The determination was performed with an Agilent MP 4100 MP-AES (Santa Clara, California, USA) consisting of a One Neb inert nebulizer, a double-pass glass cyclonic spray chamber, and a charge-coupled detector (CCD) of solid state. The plasma gas flow was 20 L/min and the makeup gas flow 1.5 L/min. A calibration curve was made from a mixture of 27 elements (Ag, Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Si, Sr, Ti, Tl, V, Zn) with eight points (concentrations: 100, 75, 50, 25, 10, 7.5, 5.0 and 1 ppm). The coefficient of determination was greater than 0.98 for each element. The conditions of the equipment for the analysis were: capture time 13 sec, plasma stabilization time with sample aspiration 15 sec, reading time 3 sec (reading in triplicate) and washing time 20 sec.

The count of total mesophilic aerobic bacteria and molds and yeasts was carried out by weighing 1 g of honey that was mixed with 9 mL of PBS buffer. Subsequently, serial dilutions were made until the 10-9 dilution was obtained. Finally, 1 mL of each dilution was seeded in Petri dishes and plate count agar (Difco™, BD Detroit US) and PDA agar (potato dextrose agar, BD™ Difco™ plate count agar) for aerobics were poured bacteria and molds and yeasts, respectively. Finally, they were incubated for 48 h and 5 d to 35 °C, and the colonies were counted.

Treatment and analysis were performed in triplicate, and the values were expressed as mean ± SD (standard deviation). All data were analyzed using one way of variance (ANOVA), followed by a Tukey’s test with a significance level of 5% (P<0.05) using Minitab 16 statistical software (Minitab Inc. State College, PA, USA)(6).

Table 1 shows the physicochemical properties of honey stored in different packages. The moisture content of the analyzed samples varied from 20.60 to 23.40 %, while the water activity varied from 0.663 to 0.675 after 2 yr of storage in the different treatments. Honey stored in a container with an anaerobic valve showed the greatest decrease in moisture content (20.60 %) and in water activity (0.667) compared to the control treatment, followed by honey stored in a transparent plastic container (moisture content: 22.80 %, aw= 0.675). High moisture content is related to the environment in which the flowers from which the bees collect nectar are found. In addition, it must be considered that honey obtained from stingless bees contains a greater amount of water, so the effect that the type of container and its permeability has a greater effect on its stability compared to commercial honey obtained from Apis mellifera(3,5).

The color parameters of the honey exhibited slight changes during storage; these changes were reflected in the chroma values and in the total color change. The sample stored in transparent containers (T4) exhibited a greater total color change (8.08).

The pH of the samples varied slightly from 3.23 to 3.66. The values of pH obtained in the samples were in the range reported for this type of honey(11). The total acidity of the samples stored in different containers (T1-T4) ranged from 85.66 to 87.33 meq/kg. The samples stored in the different types of containers (T2–T4) were not significantly different from each other but were different from the control sample (73.66 meq/kg). The acidity values for treatments T1–T4 were similar to those reported for stingless bee honey of 85 meq/100 g(12). The hydroxymethylfurfural (HMF) values of the samples stored in different containers (4.00–4.78 mg/kg) were not significantly different from those for the control treatment (4.09 mg/kg).

Table 2 shows that the content of total phenolic compounds, vitamin C and antioxidant compounds of honey stored in different containers were not significantly different (P>0.05) from that of the control treatment, except for vitamin C and DPPH radical inhibition. The content of total phenolic compounds varied from 12.55 to 14.31 mg GAE/100 g honey and that of vitamin C from 86.86 to 114.17 mg AA/g. Consistent with these values, the antioxidant activity determined by the DPPH radical scavenging activity presented high inhibition values (75.57–94.70 %). Similarly, the range of values determined by the FRAP (2.23-3.70 mg TE/g) and ABTS (0.61-1.13 mg TE/g) tests for the different types of containers show that honey contains compounds with a high capacity to reduce ferric ions and that they are stable during storage. These results are consistent with those reported for other types of honey(13) and the opposite to those reported for honey subjected to a temperature of 22–40 °C after 90 d of storage(14).

Table 2: Antioxidant properties of the honey recently harvested (T0-control), stored after two years in different containers (T1-T4)

Property	T0	T1	T2	T3	T4
Vitamin C, mg AAE/g	91.40 ± 5.24a,b	87.50±8.26a	86.86±1.02a	114.17±2.16d	106.64±1.35c
Total phenolic compounds, mg GAE /g	12.55 ± 3.24a	13.46±3.19a	14.06±3.70a	11.82±3.50a	14.31± 2.63a
DPPH inhibition, %	87.25 ± 2.65b	94.05±2.69c	75.57±5.65a	94.70±1.88c	84.71±4.65b
FRAP, mg TE/g	2.97 ± 0.95a	3.70±1.87a	2.94±1.05a	2.23±0.98a	2.94±1.60a
ABTS, mg TE/g	0.68 ± 0.18a	0.61±0.22a	0.72±0.31a	0.69±0.24a	1.13±0.76a

The values are shown as the mean ± SD (n=3). AAE: Ascorbic acid equivalents. GAE: Gallic acid equivalents, TE: Trolox equivalents.

Table 3 shows the analysis of phenolic compounds present in freshly harvested honey and honey stored in different containers. A total of 17 phenolics plus two precursors (shikimic acid and L-phenylalanine) were identified in the honey stored in the different containers. Shikimic acid (35511–38504.90 µg/g dry extract), 4-hydroxybenzoic acid (2781.36–2996.87 µg/g dry extract), 4-hydroxyphenylacetic acid (1685.49–2294.62 µg/g dry extract) and L-phenylalanine (2917.68–3004.45 µg/g dry extract) were the major compounds in the samples. No significant differences (P>0.05) were found in most of the phenolics and precursors of the samples stored in the different containers after 2 yr of storage. The phenolics gentisic acid, 4-hydroxyphenylacetic acid, p-anisic acid and the precursor shikimic acid exhibited significant differences (P<0.05) in the samples stored in different containers, mainly in T4 (honey stored in a transparent plastic container). The profile of phenolic compounds found was similar to that reported for stingless honey by other authors(8), however, variations were found in relation to concentration, these differences in concentration have been attributed to floral and geographical variation and a collection time(8).

Table 4 shows that 18 volatile compounds were found in honey in the different treatments, ethyl acetate (20.20–30.24 %), cis-linalool oxide (30.05–34.73 %), trans-linalool oxide (12.97–15.75 %), and 1,5,7-octatrien-3-ol, 3,7-dimethyl (12.55-14.67 %) were the major compounds, representing approximately 50 % of the volatile compounds present in the samples. Alcohol derivatives were the predominant ones found in honey during storage.

Table 4: Volatile compounds (%) determined in the honey recently harvested (T0-control), stored after two years in different containers (T1-T4)

N	Compound name	RT (min)	T0	T1	T2	T3	T4
1	Ethyl acetate	5.46	-	20.20b	22.25b	30.24c	28.67c
2	3-Methyl butanal	5.66	-	-	-	-	-
3	Hexane, 3 methyl	7.46	-	1.79a	1.77a	2.18b	1.91b
4	2-Hexene, 3 methyl	8.64	-	0.240b	0.236b	0.272c	0.16a
5	Propanoic acid, 2-hydroxy-, ethyl ester	8.83	8.85c	1.49a	1.54a	1.78b	1.66b
6	Furfural	9.60	-	1.71b	1.87b	0.96a	0.68a
7	D-Limonene	10.90	0.37	-	-	-	-
8	2-Heptanal acetate	10.93	1.38	-	-	-	-
9	Lilac alcohol B	11.05	-	0.14b	0.15b	0.110a	0.112a
10	Lilac alcohol C	11.19	0.09a	0.11a	0.10a	0.09a	0.09a
11	Benzaldehyde	11.68	0.87a	1.76b	1.98b	1.99b	2.47c
12	trans-γ-Caryophyllene	11.89	18.72	-	-	-	-
13	Benzeneacetaldehyde	12.53	-	6.93b	6.19b	3.24a	3.70a
14	cis-Linalool oxide	12.71	48.07c	34.09b	34.73b	30.05b	31.51b
15	trans-Linalool oxide	12.90	21.648b	15.75a	14.81a	12.97a	13.44a
16	1,5,7-Octatrien-3-ol, 3,7-dimethyl	13.11	-	13.67a	12.55a	14.67a	13.44a
17	Nerol oxide	13.67	-	1.52c	1.25b	0.85a	1.51c
18	Linalool oxide	14.00	-	0.61a	0.62a	0.69a	0.69a

ab Different letters in the same row are significantly different (P<0.05). --Not present.

Analysis of the hexane extract of the honey samples revealed the presence of eight fatty acids (Table 5). Hexadecanoic acid (31.12–49.65 %), octadecanoic acid (21.48–26.86 %) and cis-9-octadecadienoic acid (14.31–40.04 %) were the major compounds found in the different stored samples.

Table 5: Relative area (%) of fatty acids in the hexane extract in the honey recently harvested (T0-control) and stored after two years in different containers (T1-T4)

Compound name	RT (min)	T0	T1	T2	T3	T4
Decanoic acid	6.42	-	0.47d	0.25b	0.22b	0.19a
Dodecanoic acid	8.34	29.72c	1.96a	7.02b	2.27a	1.90a
Tetradecanoic acid	10.49	1.91c	2.07b	1.15a	2.48b	1.19a
9-Hexadecenoic acid	12.21	-	0.65a	0.75a	0.55a	0.68a
Hexadecanoic acid	12.41	22.27a	43.08c	34.33b	49.65c	31.12b
cis-9,12, Octadecadienoic acid	13.79	1.41a	2.96b	3.24b	3.71b	3.41b
cis-9-Octadecaenoid acid	13.83	15.90a	22.36b	32.23c	14.31a	40.04d
Octadecanoic acid	13.98	28.80b	26.47b	21.44a	26.86b	21.48a

abcd Different letters in the same row are significantly different (P<0.05). -Not present.

The mineral content remained constant during storage, and it descended in the following order: K > Mg > Ca > Na > Si, for honey stored in the different containers (Table 6). The concentration of As, Be, Cd, Mo, Ni, Pb, Sb, Ti, Tl and V was similar to that reported by Villacrés-Granda et al(2). K (109.36–125.68 mg/100 g DW) and Mg (31.60–100.49 mg/100 g DW) were found in higher concentrations compared to the other minerals present. Potassium was the majority mineral, representing a third of the total content and exceeding that of other minerals by approximately 10 times. No statistically significant differences were found for the minerals investigated in most samples evaluated during storage.

Table 6: Mineral and trace elements (mg / 100 g DW) in the honey recently harvested (T0-control) and stored after two years in different containers (T1-T4)

Mineral	T0	T1	T2	T3	T4
Al	1.378±0.01a	1.352±0.04a	1.451±0.10a	1.221±0.07a	1.235±0.00a
As	-	-	-	-	-
B	1.670±0.02b	1.208±0.03a	1.456±0.10ª,b	1.765±0.09b	2.089±0.00c
Ba	-	-	-	-	-
Be	-	-	-	-	-
Ca	76.090±0.10b	43.172±0.5a	95.441±0.90c	96.662±0.95c	61.210±1.00b
Cd	-	-	-	-	-
Co	-	-	-	-	-
Cr	-	-	-	-	-
Cu	0.578±0.07b	0.476±0.03b	0.554±0.03b	0.753±0.02c	0.159±0.01a
Fe	0.970±0.08b	0.740±0.02b	1.179±0.09c	0.815±0.03b	0.613±0.06a
K	115.90±3.00a	125.686±2.76b	109.97±2.00a	109.361±3.09a	122.840±2.67b
Mg	84.32±1.00b	100.490±1.98b	31.608±1.65a	96.608±1.49b	87.096±1.34b
Mn	0.11±0.00a	0.123±0.00a	0.14±0.00a	0.113±0.00a	0.189±0.00b
Mo	-	-	-	-	-
Na	28.65±1.02b	35.794±1.17c	20.562±1.07a	26.655±0.45b	21.470±0.98a
Ni	-	-	-	-	-
Pb	-	-	-	-	-
Sb	-	-	-	-	-
Se	4.870±0.98a	4.589±0.76a	4.892±0.49a	4.891±0.29a	5.494±0.57a
Si	45.88±1.00a	46.798±1.06a	40.195±0.30a	53.352±0.69a	51.485±0.70a
Sr	-	-	-	-	-
Ti	-	-	-	-	-
Tl	-	-	-	-	-
V	-	-	-	-	-
Zn	0.678±0.05a	0.814±0.06b	0.972±0.05b	0.349±0.06a	0.995±0.04b

ab Different letters in the same row are significantly different (P<0.05). -: no detectable.

The results showed a higher number of microorganisms in the initial samples for total aerobic mesophilic bacteria (1.50 x 102 CFU/g) and molds, and yeast (2.30 x 102 CFU/g), compared to the samples in different containers stored for two years (Table 7). The results obtained for the analysis of microorganisms for the samples at the beginning of storage ranged from 0.78 x 102 - 0.98 x 102 CFU/g of sample for aerobic mesophiles and 0.03 x 102- 0.32 x 102 CFU/g of sample for molds, and yeasts.

Table 7: Total aerobic mesophilic bacteria, and molds, and yeast present in samples at the beginning and after two years of storage in different containers

Determination of the physicochemical properties, such as moisture, pH, acidity, and Brix degrees allow the evaluation of honey quality. The moisture of honey favors the growth of bacteria and fungi present in honey. The range of moisture values obtained for the different treatments was consistent with those reported for the honey from Ecuadorian stingless bees(2). It was found that the sample stored in the container with an escape check valve showed a significant reduction in moisture content compared to the initial treatment, which it is possibly due to the gases produced by fermentation dragging the moisture present into the headspace of the container, preventing the moisture from returning. At the same time, color changes in honey are related to its botanical origin, mineral content, the content of phenolic compounds, antioxidant properties, room temperature, and storage time. The change in honey stored in the transparent container is possibly because light could affect the components of the honey such as carotenoids and flavonoids(15)..

The values of pH and the total acidity, play an important role in the quality of the honey(11). The acidity values show that this honey stored for 2 yr has a higher acidity. The increase in acidity value during storage may be related to the fermentation of honey and to its antimicrobial properties, but it may also result in an undesirable vinegar taste because of acetic acid production. The acidity of honey is related to the glucose content. Glucose is converted by the action of the enzyme D-glucose oxidase into gluconic acid. This process produces hydrogen peroxide which is a component of the antimicrobial action of honey(16). The production of acids occurs not only by enzymatic action but also by fermentation of the microorganisms present in the matrix. Increased acidity may also be associated with the transformation of sugars from honey into alcohols and then into organic acids by osmophilic yeasts. It is also necessary to consider that when the moisture content is high, the bacteria grow and ferment the sugars, producing compounds such as acetic acid that can affect the taste of honey(17).

A very important quality factor in honey is the HMF concentration, since it is an indicator of the quality, freshness, and aging of honey. Under conditions such as processing or aging, mainly influenced by temperature fluctuation, pH, storage conditions, and floral origin, may help bring about its presence(18).

The antioxidant activity of honey depends on its floral origin and the processing conditions and is closely related to the chemical compounds it possesses. The components of honey – phenolic compounds, flavonoids, and phenolic acids, as well as chlorophyll, carotenoids, and vitamin C – contribute to its antioxidant activity(15), coupled with the fact that the antioxidant properties are related to its color and the moisture content.

The antioxidant activity of honey is due, among other factors, to the presence of phenolic compounds, which are produced in plants as a protection system and are entrained in the nectar extracted by bees. UPLC analysis revealed the presence of shikimic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid and L-phenylalanine, among others. These compounds confer antioxidant activity to honey since they possess delocalized electrons, which cause free radical scavenging activity(19). The radical scavenging activity of phenolics mainly depends on the number and position of hydroxyl groups in the molecules. The presence of these compounds is explained by the fact that shikimic acid is a precursor of aromatic metabolic intermediates, within which are flavonoids such as luteolin(20). The presence of these phenolic compounds may suggest possible anti-inflammatory and antimicrobial activity, among other properties. Consistent with the analysis of total phenolic compounds, the concentration of most of the quantified phenolic compounds decreased in the sample subjected to heat treatment, compared to the control. This helps to explain the decrease in antioxidant activity.

Identifying volatile compounds plays a crucial role in assessing the quality of honey. These compounds, linked to flower nectar, geographical origin, and overall stability, offer insights into the honey's unique characteristics(21). When honey is stored in various containers, there is an increase in volatile compounds over the initial storage day. Notably, esters, aldehydes, ketones, and alcohols become predominant after 2 yr, contributing significantly to the honey's odor and flavor. The appearance and rise of specific volatile compounds may be linked to the fermentation process, with packaging type influencing oxygen availability and anaerobic respiration enhancement. Moisture content further affects fermentation, favoring the production of alcohol, carbon dioxide, and acetic acid, all influencing the concentration of volatile compounds in honey(17).

Free fatty acids, akin to volatile compounds, serve as lipid markers reflecting the floral origin of honey and can be crucial authenticity indicators(22). Changes in the concentration of certain volatile compounds in honey stored in containers are likely due to the container material's permeability. Plastics, in particular, may retain some volatile compounds, facilitating their transfer between honey and the container material. This involves the adsorption or retention of volatile compounds in honey, causing shifts in their concentration. Additionally, some volatile compounds might be lost or absorbed, affecting the honey's aromatic profile and consequently altering its taste and aroma. Fatty acids such as hexadecanoic acid increased in proportion, while the proportion of dodecanoic acid decreased, and others like decanoic acid and 9-hexadecenoic acid emerged during storage. These changes may be related to variations in water activity and the permeability of different containers used for storage. Another factor is that honey crystallization can impact the mobility and availability of fatty acids, influencing their proportion.

Mineral content is another quality factor for honey. The analysis shows that the type of minerals and their concentration does not vary during storage in the evaluated containers, and it was similar to those reported by other authors regarding other types of honey; Consistent with other reports, potassium was the most abundant mineral, this is considered the most quantitatively important mineral in the honey, accounting for around 50 % of the total mineral content. The presence of Al, Ba, Si, and Co is mainly since these minerals are naturally present in the environment, demonstrated that the honey is a very good environmental indicator so reflects the content of toxic elements in the surrounding water, soil, and air(23). Honey can contribute to the diet with elements such as Mg, Ca, and K. Mg and K are important micronutrients for the human body since they are involved in many physiological processes and are essential for the maintenance of the normal function of cells and organs, by which they make an important contribution to health(24). These results are also consistent with those reported in a study on honey from stingless bees from Brazil, where it was found that these minerals are the most important quantitatively(8).

Honey can contain microorganisms from different resources, such as pollen digestive tracts dust, air soil, and nectar, or due to handling and processing. The presence of these microorganisms can affect the quality of honey during storage, so an analysis of the total count of aerobic microorganisms and molds, and yeasts were performed at the beginning and end of storage in different containers. These values were below the limit reported by other authors and that established for Apis mellifera honey, which may be due to proper handling in harvesting and the presence of phenolic compounds, organic acids, and other bioactive compounds present in the honey that has an inhibitory effect on this type of microorganism. The concentration of total aerobic microorganisms and molds and yeasts decreased in honey during storage, which is consistent with the reduction in water activity and moisture. This could be attributed to various factors such as sugar crystallization or water evaporation due to plastic permeability. The decrease in microorganism concentration is a positive factor that ensures the quality of honey during its storage.

In this study, a comparison was made between plastic containers used commercially, since the use of other types of containers, such as glass or metal, are more expensive for the producer. The study demonstrated that storing honey in traditional plastic containers (high-density polyethylene and polyethylene terephthalate) and using certain traditional methodologies provide significant differences in the moisture content of honey during storage, with the moisture content being minor in honey stored in the container with an escape check valve (T3). It was also found that, in general, storage for 2 yr does not produce major changes in the physicochemical properties and in the content of phenolic compounds, which are associated with a decrease in antioxidant properties and volatile compounds that together can affect the honey quality. Furthermore, storage had a positive effect on the microbiological analysis of the honey. Finally, the evaluation of these parameters suggests that treatment T3 would be the most suitable for storing honey since it presented a total color change of less than 3, an important quality parameter for consumers.

The Authors would like to thank Company Chasseurs De Saveurs S.A. C.V. from Coatepec Veracruz for providing the honey. Also, we are grateful to the Mexican Association of Edible Forests.

Table 1: Physicochemical properties of the honey recently harvested (T0-control), stored after two years in different containers (T1-T4)

Properties
	T0	T1	T2	T3	T4
Moisture, gH20/ 100 g d.m.	25.70 ± 0.47b	23.23 ± 0.28ab	23.40 ± 0.09b	20.60 ± 1.33a	22.80 ± 0.39a
Water activity (25 °C)	0.733 ± 0.006b	0.663 ± 0.001a	0.671 ± 0.007a	0.667 ± 0.006a	0.6759 ± 0.003a
L*	18.33 ± 0.37a	20.80 ± 1.85a	21.67 ± 3.59a	18.27 ± 0.43a	25.64 ± 2.55b
a*	2.84 ± 0.75a	6.66 ± 0.64b,c	6.27 ± 0.87b,c	5.40 ± 0.64b	8.91 ± 1.82c
b*	5.39 ± 0.23a	7.69 ± 2.34b	8.41 ± 4.58b	4.63 ± 0.08a	7.26 ± 3.45b
Chroma	6.11 ± 1.53a	10.17 ± 2.21a	10.49 ± 4.22a	7.11 ± 0.51a	10.02 ± 3.54a
Total color change	-	4.81 ± 0.54b	5.32 ± 0.38b	2.69 ± 0.45a	8.08 ± 0.37c
Browning index	-	0.38 ± 0.09a	0.42 ± 0.05a	0.42 ± 0.03a	0.39 ± 0.02a
pH	3.66 ± 0.05a	3.23 ± 0.05a	3.26 ± 0.05a	3.40 ± 0.06a	3.43 ± 0.05a
Brix (º)	71.66 ± 0.57a	71.90 ± 0.55a	72.03 ± 0.55a	72.10 ± 0.26a	72.76 ± 0.37a
Electric conductivity, mS/cm	293.33 ± 11.54b	320.25 ± 20.00 a	293.33 ± 15.27b	303.33 ± 5.77b	296.00 ± 15.16b
Density, g/mL	1.40 ± 0.02b	1.36 ± 0.01a	1.36 ± 0.00a	1.36 ± 0.02a	1.36 ± 0.01a
Hydroxymethylfurfural, mg /kg	4.09 ± 0.53a	4.33 ± 0.20a	4.00 ± 0.39a	4.23 ± 0.22a	4.78 ± 0.52a
Titratable acidity, meq/kg d.m.	73.66 ± 0.57a	87.33 ± 6.65b	87.33 ± 0.57b	87.33 ± 2.08b	85.66 ± 1.15b

Data represent the average of three replicates or measurements ± standard deviation.

abcd Different letters in the same row indicate significant differences (P<0.05).

Table 3: Phenolic compounds (µg/g dry extract) of the honey recently harvested (T0-control), stored after two years in different containers (T1-T4)

Phenolic compound	T0	T1	T2	T3	T4
Gallic acid	126.78 ± 10.00a	136.17 ± 16.20a	137.88 ± 4.91a	143.32 ± 5.97ª	135.25 ± 8.16a
4-Hydroxybenzoic acid	2996.87 ± 135.76a	2847.66 ± 207.18a	2906.04 ± 118.78a	2934.22 ± 105.08a	2781.36 ± 146.05a
Protocatechuic acid	637.87 ± 21.79a	660.19 ± 22.55a	666.90 ± 13.40a	648.85 ± 24.07a	634.87 ± 34.48a
Vanillic acid	654.89 ± 38.33a	612.57 ± 59.27a	645.12 ± 39.07a	677.20 ± 29.77a	633.24 ± 27.82a
Gentisic acid	312.90 ± 89.87a	312.82 ± 36.07a	280.94 ± 29.34a	294.31 ± 10.82a	541.69 ± 48.62b
4-Hydroxyphenylacetic acid	2198 ± 129.88a	2294.62 ± 115.55b	1702.48 ± 195.65a	2036.27 ± 133.77b	1685.49 ± 103.27a
Sinapic acid	1677.33 ± 87.99a	1677.70 ± 81.08a	1636.52 ± 23.01a	1663.18 ± 57.32a	1565.35 ± 80.53a
Salicylic acid	1244.11 ± 199.55a	1240.45 ± 409.54a	1053.87 ± 54.18a	1074.40 ± 30.10a	1098.68 ± 32.97a
p-Anisic acid	399.97 ± 29.73b	317.57 ± 26.29ª	455.27 ± 39.95c	384.97 ± 38.77b	456.20 ± 52.69c
Rosmarinic acid	297.45 ± 12.95a	275.61 ± 20.76a	220.68 ± 58.40a	248.44 ± 16.14a	276.69 ± 67.38a
4-Coumaric acid	301.86± 38.26a	291.65 ± 37.60a	320.81 ± 13.12a	323.82 ± 7.57a	271.41 ± 53.92a
Trans-cinnamic acid	139.87 ± 9.41a	124.02 ± 7.43a	123.18 ± 4.77a	122.89 ± 3.74a	127.99 ± 3.83a
Luteolin	289.56 ± 29.44a	273.83 ± 38.24a	325.69 ± 104.80a	315.66 ± 45.43a	320.41 ± 62.01a
Scopoletin	689.85 ± 58.33ª	673.76 ±72.47ª	720.55 ± 26.69ª	740.75 ± 17.46ª	619.54 ± 120.49ª
Ferulic acid	132.63 ± 27.82a	127.52 ± 21.48a	136.69 ± 20.29a	150.77 ± 40.86a	111.18 ± 27.34a
Caffeic acid	478.86 ± 96.43a	410.22 ± 122.14a	570.36 ± 24.02a	590.65 ± 27.56a	452.59 ± 120.71a
Shikimic acid	36986.87±3999.20a	38200.95±3974.40a	37735.48±2688.39a	38504.90±2817.73a	35511.01±5741.62a
Vanillin	97.45 ± 6.98a	96.17±5.16a	97.36 ± 16.74a	85.01 ± 12.63a	98.26 ± 12.83a
L-phenylalanine	2930.99 ± 120.89a	2934.82±100.09a	2886.78 ±115.55a	3004.45 ± 130.22a	2917.68 ± 118.89a

Treatment code	Total Aerobic mesophilic count (CFU/g)	Total molds and yeast count (CFU/g)
T0 (initial storage)	1.50 x 102a	2.30 x 102a
T1	0.89 x 102b	0.32 x 102b
T2	0.98 x 102b	0.15 x 102b
T3	0.95 x 102b	0.08 x 102b
T4	0.78 x 102b	0.03 x 102b