Physicochemical, Antioxidant and Antibacterial properties of two types of Honey products in EL Oued Region

 

Abdelhamid Khelef¹, Salah Eddine Laouini², Abdelkrim Rebiai¹, Mohammed Adel Mesbahi¹, Mohamed Zidane¹, Abderrhmane Bouafia²*

 

ABSTRACT:

This work contributes to the valorization of two honey samples from El-Oued region (South-East Algeria). The identification of floral sources of these two samples and the melissopalynology analysis showed that the sample of Djedaida Chamalya (A) was monofloral honey "Zygophyllum album L", while the sample of Bagouzza (B) was polyfloral honey. The physicochemical study of samples shown that they conform with international standards characterization (pH 3.03-4.15), electrical conductivity (309.5-670μs/cm), density (1.495-1.490), phenolic content (189-142mg GAE/Kg) and flavonoids (43-38mg RUE/Kg), HMF (5.54-2.55mg/Kg). The antioxidant activity was estimated by spectrophotometrically (DPPH, TAC, FRAP), and electrochemical methods, and antibacterial activityevaluatedby agar diffusion method. The results indicated that the sample of Djedaida Chamalya (A) contained high amount of phenolic, flavonoid content and exhibited high antioxidant activity than sample of Bagouzza.

 

 

 

INTRODUCTION:

Honey, this precious substance offered by nature is known and used by since the most remote times^1-3. However, honey is characterized by a certain group of substances are always present but in varying amounts depending on the source (water, carbohydrates, protins or nitrogenous substances, organic acids, lactones, minerals, trace elements, vitamins, lipids, pollutants like lead, cadmium and hydroxymethylfurfural)^4-7.

 

Indeed, the composition of the honey varies according to the floral source used by the bees, the period of the harvest and the geo-climatic conditions of the regions concerned. Honey is also valuable as a marketable commodity in both domestic and international markets and plays an important role in some cultural traditions^{1,8, 9}. It thus constitutes a significant source of income for the rural population, at the same time as it can contribute to the improvement of human nutrition^10-13.

 

The objective of the present work is to define the physicochemical characteristics, screening of melissopalynological, phenolic, flavonoid contents, and the biological activity of the two honey samples from El-Oued region.

 

RESULTS AND DISCUSSION:

Melissopalynological study: 

Microscopic analyzes of honey pollen showed that the sample of Djadaida chamalya was monofloral honey content more than 45% of "Zygophyllum albumL" pollen in the sample (Figure 1, Table 1). While Bagouzza's sample was polyfloral honey.

 

Fig. 1: Zygophyllum album L pollen in honey sample (A)

Table 1: Pollen content in the honey samples

Pollen Taxa	Family	Samples
		A	B
Zygophyllum album L	Zygophyllaceae	45	-
Retamaraetam	Fabaceae	30	-
Phoenix dactylifera L	Arecaceae	8	-
Helianthus annuus L	Compositae	-	5
Eucalyptus sp	Myrtacée	-	20
Malcolmiaaegyptiaca	Brassicaceae	-	18
Launaearesedifolia O. K	Asteraceae	-	36
Not identified		17	21
Total (%) pollen		100	100

Physicochemical determinations:

The results of the physicochemical analyzes of the honey samples are given good result according to international standards Quality Regulation¹⁴. The water found in the above honey samples ranged from 16.5% to 17.5%. Obviously, these samples are in the range of required standards (20%). Density and pH are correlated and used as a measure of adulteration in honey. The average relative density of the analyzed honey samples varied from 1.49 to 1.495 against the pH varied from 3.03 to 4.15. These values are similar to those reported elsewhere¹⁵. The results of physicochemical analyses of samples are summarized in Table 2.

 

Table 2: Physicochemical caractristicsof honey samples.

Samples Code	A	B	Limits of International Standards
Density	1.495±0.02	1.49±0.016
Water content (%)	18.275±0.025	14.875±0.125	200 g.kg-1
pH	3.03±0.048	4.15±0.02
EC (μS.cm-1)	309.5±1.22	670±3.26
Water insoluble solids content (%)	0.26±0.01	0.31±0.02	5 g.kg-1
HMF (mg.kg-1)	5.54	2.55	40 mg.kg-1
Ash (%)	0.087±0.014	0.093±0.009
Color (Pfund index)	58.53	95.89
Total sugar content (%)	81.725±0.025	85.05±0.14
Free acidity (meq.kg-1)	25.10± 0.25	45.6±0.4	50 meq.kg-1

 

Total phenolic content and flavonoids:

Total phenolic content is shown in Table 3. The values of phenolic contents are moderate compared to previous studies¹⁵. (A) sample was found to have the highest phenolic content value (189mg GAE.kg^-1), andsample B (142mg GAE.kg^-1). Phenolic compounds are one of the most important classes of compounds found in honey. The total concentration of phenols in honey is highly dependent on its plant source. The total phenolic content of the tested honey is similar to those previously reported in Algerian, Cuban, and Germany honeys¹⁶.

 

Total flavonoid content is shown in Table 3. The flavonoid contents values are convergent. However, the highest value of flavonoid content was determined in (A) sample (43mgREE.kg-1), and in (B) sample (38 mgREE.kg-1). These values are similar to the average values found for some Burkina Fasan and Australian honeys^17,18.

 

Antioxidant activity:

The antioxidant properties of the samples was estimated by the chemical methods (DPPH, TAC, FRAP) and electrochemical method, The results are presentedin Table 3. The results of the two methods TAC and FRAP show that the sample A has a high activity compared to the sample B.

 

The almost stable organic radical DPPH has been generally used for the determination of antioxidant activity of various bee products¹⁹. From Table 3, free radical scavenging capacity of the tested honey was found to be as follows (IC50, 116.9 and 146.9mg. mL^-1) for sample A and B respectively, which were consistent with FRAP and TAC assays.

 

Figure 2. shows the cyclic voltammograms of the tested samples. The determination of the anodic current peak at 460 mV, was used as a measure of the quantity of antioxidants present in the tested samples. Djedaida Chamalya honey sample (A) exhibited the highest anodic current peak value at 460mV, therefore this sample had the highest antioxidant capacity.

 

Antioxidant capacity was expressed as mg ascorbic acid (AA) per kg of honey based on the ascorbic acid calibration standards. The antioxidant capacity of the tested honey was found to be as follows (AC, 110 and 70 mg.kg^-1) for sample A and B respectively

 

 

The results obtained for cyclic voltammetry are generally consistent with total phenolic and flavonoid content and with spectrophotometric antioxidant assays including DPPH, FRAP and total antioxidant capacity assays.

 

Fig.2: Cyclicvoltammogramsofa: Djedaida Chamalya and b: Bagouzzahoney samples. Experimental conditions: pH=7.0, scanrates 100 mv/s.

 

 

Table 3: Antioxidant properties of honey samples.

Samples	A	B
Total Phenolic Content (mg GAE.kg-1)	189	142
Total Flavonoids (mgREE.kg-1)	43	38
DPPH, IC50 (mg.mL-1)	116.9	146.9
FRAP (μM AA.kg-1)	330	80
TAC (mgGAE.kg-1)	233	163
Electrochemical method AC (mg.kg-1)	110	70

 

Antibacterial activity:

In this study two different types of honey samples collected from different localities from El-Ouedregionwere evaluated for their antibacterial activity against some gram-positive Staphylococcus aureus ATCC 6816 and gram-negative Escherichia coli ATCC 25922and Pseudomonas aerigunosa ATCC 27853bacteria. As a result, antibacterial activity was determined in different values against different microorganisms in the honey samples and the inhibition zones were varied from 05 mm to 14 mm in diameters.

 

The honey samples used in our study showedmore antibacterial activity than the antibiotics used in some species, even higher than the strains of Escherichia coliand Pseudomonas aeruginosa. Monofloral honey samplefrom Zygophyllum album L(sample A) forms the maximum inhibition zone 14 mm in diameters against Escherichia coli. The results of antimicrobial activity were given in Table 3 according to the inhibition zone diameters formed around the wells. In generally, honey samples (A and B) have antibacterial activity against three bacteria and they are more effective than the AMX.

 

Fig. 3: Test of inhibition of Staphylococcus aureus ATCC 6816 by Zygophyllum album L honey (0.1, 0.3, 0.5 mg/mL)

Table 4: Antibacterial activity ofhoney.

Bacteria	Diameter of inhibition zone (mm)
	A	B	CL	AMX
E.coliATCC 25922	14	10	11	7
P.aeruginosa ATCC 27853	9	10	4	8
S.aureus ATCC 6816	7	5	9	2

 

EXPERIMENTAL:

Collection of Honey Samples:

Two honey samples were collected for analysis over the 2017 period. They came from the DjadaidaChamalia and Bagouzza areas of El-Oued region (South-East Algeria). The samples were stored in glass containers at room temperature.

 

Apparatus and Instruments:

physicochemical properties measurements were performed on a Electrical conductivity meter CDM 210 (Radiometry, FRANCE), pH meter PHM 210 (Radiometry, FRANCE), and refractometer HAND-HELD REF106B (Mettler-Toledo, Switzerland), polarimeter (3B Scientific, FRANCE). UV-Visible spectrophotometer (Shimadzu UV-1800, Japan) used for the spectrophotometric measurements. PGP301 potentiostat/galvanostat (voltamaster 4 version 7.08 software radiometer analytical SAS). A light microscope (Optika B350, ITALIA) attached to a digital camera with Optika Vision Pro image analysis software was used.

 

Determination of total phenolic content:

The total phenolic content in different floral honey evaluated using by Folin-Ciocalteus method^20,21. Briefly, 20µL of the sample and the standard (gallic acid) were mixed with 1.57mL of water and followed by the addition of 100µL Folin-Ciocalteus reagent. After 10 min 300µL of sodium carbonate aqueous solution (7%, w/v) was added to the mixture and was completed the reaction for 2 hours in darkness at room temperature. The absorbance determinated at 765nm using a UV-visible spectrophotometer. The result was expressed in equivalent milligrams of gallic acid (mg GAE/g).

 

Determination of total flavonoids content:

The determination of flavonoid was counted according to the colorimetric method²². 5mL of honey was mixed with 5mL of sodium acetate 100mg/mL, 3mL aluminium chloride (10%) and completed to 25mL in calibrated flask. The mixture was thoroughly vortexed and the absorbance of the pink colour developed was determined at 430nm. The result was expressed as mg catechin equivalents (CE/g).

 

DPPH radical scavenging assay:

The radical scavenging activity using free-radical DPPH assay was carried out using the colorimetric method²³. 0.2mL of sample was added to 1.8mL of DPPH solution in ethanol (0.04mg/ml in ethanol). The mixture was saved in the dark for 30 min at room temperature. The antioxidant activity was read at 517nm. The Anti-radical activity was expressed as IC₅₀ (μL/mL) i.e. the Anti-radical percentage inhibition calculated by the following equation:

 

Where A_o is the absorbance of control test after 30 min. A₁ is the absorbance of the sample extract after 30 min. All results are means ±SD.

 

Measurement of reducing ferric iron:

The reducing power was determined by using FRAP assay²⁴. FRAP reagent controlled 10 mMtripyridyltriazine (TPTZ) in 40mM HCl, 20mM of FeCl₃ and 300mM soium acetate buffer (pH=3.6) with the ratio of 1:1:10. After, 0.2mL of different floral honey was mixed with 3mL of FRAP reagent prepared previously. The absorbance was then measured at 595 nm, FeSO₄ was prepared in same solvent extraction in the range of 100–700µM as standards. The results were expressed as mg/ml of Fe(II).

 

Total antioxidant capacity:

The total antioxidant capacity of the different floral honey estimated based on the reduction of Mo (VI) to Mo (V) by formation of the green phosphate/M(V)²⁵. 0.1 mL of different floral honey was added to 3mL of the reaction solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The mixture was incubated in a boiling water bath (Mammert D-91126 Schwabach FRG, Germany) at 95°C for 90 min. The absorbance was read at 695nm. The antioxidant capacity was expressed as mg of gallic acid equivalent (GAE/g). All determinations were performed in triplicate.

 

Voltammetric determination of antioxidant capacity:

The electrochemical cyclic voltammetry technique was used in order to evaluate the antioxidant capacity of the studied samples. For this, a potentionstatgalvanostat apparatus of the type (Voltalab PGZ 301) was used equipped with Volta Master 4 software was used for voltammetric experiments. The measurements were carried out in a 12ml volume electrochemical cell equipped with three electrodes. The first electrode is the working one (a glassy carbon electrode GCE), the second is a counter electrode which consists of a Pt wire and the third is a KCl saturated Hg/Hg₂Cl₂ as a reference. The potential was varied in an inverse scanning mode in the range (0 to +1000 mV) at a rate of 100mV/s. Before each test, the sample is placed in the electrochemical cell which is de-aerated by passing through it high purity nitrogen gas and its flow is maintained during the electrochemical measurement. The antioxidant capacity of the studied samples was obtained using calibration curves where ascorbic acidwas used as standards²⁶.

 

The antioxidant capacity was calculated using the following relation: AC (g/mg) = Ceq/C, where AC: antioxidant capacity, Ceq: equivalent concentration of ascorbic acid and C: initial concentration of the extracts.

 

Antibacterial activity:

Antibacterial activity of honeysamples contrary to: Escherichia coli, Staphylococcus aureusand Pseudomonas aeruginosa. Compassion of different bacterial strains to the honey was measured in rapports of zone of inhibition by agar-diffusion assay.

 

The bacterial suspension was accustomed with sterile saline to a concentration of approximately 1.0 10⁷ cells. mL^-1. Mueller–Hinton agar was prepared with 0.2mL of the inoculums. Wells (6mm diameter) were cut out from agar plates using a sterilized stainless steel borer^27,28. After, samples dissolved ina minorvolume of ethanol and were then prepared (0.1mg/mL). Each well was completed with 100µL of solution and the plates inoculated with different bacteria were incubated at 37 ̊C for 24 h and diameter of resultant zone of inhibition was measured.

 

CONCLUSION:

The honey samples analysed in this study possessed significant quantities of sugars, phenolic compounds and antioxidant activity. The honey sample from Djadaida Chamalia showed the highest phenolic compounds concentration and hence is most indicated for the development of therapeutic products. The analysis of honey from South-East Algeria is rare, especially with the above-mentioned properties. This study can serve as an important reference for future studies.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 10.07.2020                    Modified on 17.08.2020

Accepted on 12.09.2020                   ©AJRC All right reserved