Conventional and Modern Analytical Methods used for Algerian Honey Authentication

 

Laid Zeghoud^1,2, Bachir Ben Seghir^1,2,3*, Hadia Hemmami^1,2, Soumeia Zeghoud^1,2,

Ilham Ben Amor^1,2, Imane Kouadri^2,4*, Abdelkrim Rebiai², Ali Tliba⁵, Meriem Dia¹, Soumeia Aiba¹

¹Department of Process Engineering, Faculty of Technology,

University Hamma Lakhdar El Oued, BP 789, El-Oued 39000, Algeria.

²Renewable Energy Development unit in Arid Zones (UDERZA),

University of El Oued, El Oued 39000, Algeria.

³Laboratory of Industrial Analysis and Materials Engineering (LAGIM),

University 8, May 1945, BP 401, Guelma 24000, Algeria.

⁴Department of Process Engineering, Faculty of Sciences and Technology,

University May 8, 1945, Guelma 24000, Algeria.

⁵Laboratory Valorisation and Technology of Saharan Resources (VTRS).

University of El Oued, BP 789 El-Oued (39000) Algeria.

*Corresponding Author E-mail: bbachir39@gmail.com

In terms of food safety and quality, the authenticity of food products is a major concern. The authenticity of honey in regard to its botanical or geographic origin and adulteration has drawn more attention in recent years. Honey is a natural, ready-to-eat product that has a high level of nutrition and offers a number of health advantages. In order to counteract frequent honey frauds including mislabeling honey's origin and adulteration with sugar or syrups, companies and consumers must prioritize the authentication of honey. Different analytical techniques are employed to identify genuine honey. The traditional analytical procedures are still employed in conjunction with cutting-edge approaches as part of preliminary screening, processing, and product standards with a wide variety of tools and methodologies. The majority of analytical techniques offer pollen distribution information, physicochemical data, and profiles of phenolic, flavonoid, carbohydrate, amino acid, fragrance, and individual marker components. In this study, the sugar profile, physicochemical characteristics, and honey quality of ten samples of Algerian honey from various areas were examined. High-performance liquid chromatography was used to identify the sugars (fructose, glucose, sucrose, maltose, and lactose) as well as the physicochemical characteristics (moisture, water activity, pH, acidity, conductivity, and color) using the Harmonized Method of the International Honey Commission. The quality of honey throughout storage and processing was not appropriately described by the moisture content alone.

 

 

INTRODUCTION:

A natural sweetener created by honey bees from plant nectars is called honey¹. The sugar content varies substantially depending on the habitat and floral origin².

 

Carbohydrates are the most common chemicals found in nature and are the primary source of heat and energy for the human body³. "Carbohydrate" is a general term that refers to many biomacromolecules⁴. A biomolecule called a carbohydrate is made up of the elements carbon (C), hydrogen (H), and oxygen (O)⁵. Simple carbohydrates like sugars are essential for biological processes that occur every day, such as giving the body the energy it needs to perform essential functions. Most naturally occurring sugars have 6 or 12 carbon atoms in each of their molecules. Sugars taste sweet, crystalline, and soluble in water⁶.

 

Sugar is a component of carbohydrates and is naturally present in various meals, including fruit, milk, vegetables, cereals, and honey, amongst other things. A common element in the industry is added sugar, which can also be discovered in flavored yogurt, sweetened beverages, baked products, and cereals. So, added sugar is a typical component in industrial products⁷. The honey's sugar content is responsible for its nutritional, physicochemical, and sensory properties, such as energy value, crystallization, viscosity, hygroscopicity, humectant, miscibility, spreadability, sweetness, flavor, and color. These properties include energy value, crystallization, viscosity, hygroscopicity, humectant, miscibility, spreadability, and sweetness¹.

 

Since ancient times, people have used honey as a tasty sweetener and a folk medicine component ⁸. Fructose, glucose, and water make up the three main ingredients of floral honey⁹, making up 38.2, 31.3, and 17.2 percent of the total. Honey only contains two monosaccharides: glucose and fructose. 85 to 95% of all sugars and 95% to 100% of the dry matter in honey are made up of sugar. These simple sugars in honey are responsible for their sweetness, hygroscopicity, energy content, and other physical characteristics, along with trace amounts of at least 22 other more complicated sugars. Minor components like flavorings, colors, organic acids, and minerals are also found in honey¹⁰.

 

Studies on the sugar profile and Physico-chemical properties of honey have been conducted in several countries, including Tabasco, Mexico¹¹, India¹², and Romania¹³. The primary difference in honey's qualities was its botanical source. Similarly, the flower origin determines how each monofloral honey behaves differently from the others ¹⁴. Therefore, this study aimed to investigate the qualities of a selection of Algerian honey, with a particular focus on the sugar profile of monosaccharide (fructose and glucose) and disaccharide (sucrose, maltose, and lactose) sugars using HPLC-Refractive, and Physico-chemical properties (Fig. 1).

 

MATERIALS AND METHODS:

Chemicals:

Sigma Aldrich supplied sodium hydroxide (NaOH) and sodium carbonate (Na₂CO₃). All studies were performed with ultrapure water. The analytical quality of all other reagents used was ensured.

 

Instrumentation:

A high-performance liquid chromatography system called a Shimadzu LC 20 AL was employed. It was equipped with a universal injector and a UV-VIS detector. A UV-(1800 Shimadzu) Spectrophotometer was used to carry out spectrophotometric measurements (double-beam). pH meter (model number: PM 210), and an electrical conductivity meter (CDM 210). Digital Abbe refractometer (WAY-2S). Sonicator (Branso Ultrasonics).

 

Honey Samples:

Unifloral and Multifloral types of typical Apis mellifera honey samples were obtained from the 2012 crop by going straight to 10 provinces in Algeria: Laghouat (33°48′00″N 2°51′54″E,) Blida (36°28′20″N 2°50′00″E), Tippasa (36°35′39″N 2°26′35″E), El Oued (33°22′06″N 6°52′03″E), Mssila (35°42′21″N 4°32′31″E), Bidjaia (36° 45′04″N 05° 03′51E), Djelfa (34°40′00″N 3°15′00″E), and Boumerdase (33° 19′ 59″N 6° 52′ 59″E) (Fig. 2).

 

Fig. 1. Abstract graphic showing the steps of work.

Before being put through the testing process, the samples were stored in a refrigerator in airtight plastic containers. Table 1 lists the geographical areas from which the honey samples were obtained.

 

Table 1: Samples code and their botanical origin of honey.

 

Fig. 2. The samples of honey

 

METHODS:

Quantitative Sugar analysis by HPLC:

Reagents and equipment:

D (-) fructose, D-(+)-glucose, sucrose, D-(+)-maltose monohydrate, and lactose standards; All the solvents were used in this study (i.e., methanol, acetonitrile, and acetic acid). They were of HPLC grade and were purchased from Sigma Aldrich. Sonicator (Branso Ultrasonics), membrane filter (Whatman nylon 0.45 µm membrane filter), syringe (HSW Henk), vials (Agilent), HPLC with RI-detector thermo-stated at 30°C, column (Zorbax Carbohydrate analysis column 4.6 x 250mm, 5 microns), and sensitive balance were the pieces of equipment that were utilized. In the mobile phase, acetonitrile and water were combined at a ratio of 75:25 by volume, and the mixture was sonicated.

 

Preparation of standards:

Sugar standards (fructose, glucose, sucrose, maltose, and lactose) were prepared using five-level serial dilutions, based on the International Honey Commission (IHC) determination of sugars by HPLC, With a few changes. The water of HPLC-grade quality was used to dissolve each standard solution of fructose (25mM), glucose (50 mM), sucrose (50mM), maltose (75mM), and lactose (75 mM). Methanol in 12.5mL was placed into a 50mL calibrated flask. Then, a methanol-containing flask was filled to the mark with HPLC-grade water, and a standard solution was added. The filtrate from this solution was placed into a sample vial for injection after passing through a 0.45µm nylon membrane filter.

 

HPLC conditions:

For the HPLC separation, the following parameters were utilized: flow rate of 1.0mL/min, a mobile phase of acetonitrile: water (80:20, v/v), column temperature, and detector temperature of 35°C. The sample volume was 20μL. Peaks were identified based on their retention times.

 

Sample preparation:

A 2g sample of honey was weighed and dissolved in 20 mL of HPLC-compatible water in a beaker. The honey solution was quantitatively put into a 50ml volumetric flask after 12.5mL of methanol and then filled to the mark with HPLC-grade water. After using a nylon membrane filter syringe to filter the honey solution, it was then transferred to the sample vial.

 

Analysis of physicochemical properties:

Based on the International Honey Commission's Harmonized Methods, the following physicochemical analyses of the honey samples were performed, including those of conductivity, pH, and water content ¹⁵:

 

Specific gravity (Density):

Specific gravity is a substance's weight ratio to the same water volume at a given temperature. Density is a substance's weight per unit volume¹⁶.

 

Determination of pH:

10% (w/v) of honey produced in distilled water had its pH tested using a pH meter¹².

 

Determination of free acidity:

The titrimetric approach was used to determine the acidity level. In a 250ml beaker, 10g of the material was dissolved in 75ml of carbon dioxide-free water. The mixture was agitated, and then a persistent pink tint lingered for ten seconds after titration with 0.05N sodium hydroxide (NaOH) to pH=8.3. The amount of soda was adjusted after a blank test with pure water was conducted. During titration, undissociated acids generated free acids and hydrogen ions¹⁷. The acidity of the honey was measured in m_Eq/kg.

 

Determination of electrical conductivity:

The dry matter of honey was used to evaluate electrical conductivity at 20°C in a 20% (w/v) solution (dry matter basis) in deionized water¹⁵. mS/cm is the unit of measurement used to express results¹⁶.

 

Determination of moisture content:

A refractometric technique was used to determine the moisture content. As a sample's solid composition rises, so does its refractive index. Using a refractometer, the refractive indices of honey samples were determined at room temperature. 0.00023°C then adjusted the readings to account for the reference temperature of 20°C. Wedmore's table computed the moisture content percentage corresponding to the corrected refractive index after the moisture content was measured in triplicate³.

 

Determination of insoluble solidsm:

Water-insoluble solids were measured according to the instructions and harmonized methods of the international honey commission¹⁸. "The measurement of the insoluble matter is an important means to detect honey impurities higher than the permitted maximum. However, the interlaboratory coefficient of variation, lying between 26 and 85%, is very high. This should be borne in mind when interpreting results"¹⁹.

 

Pfund and Honey color:

In order to determine the Pfund value, 8 mL of distilled water was added to 4g of honey to dissolve it completely, depending on the method²⁰ with some modification. This mixture was then heated to 50°C and stirred until the total solvation of sugar crystals was achieved. The resulting solution's absorbance was measured with a UV-Vis spectrophotometer (NanoDrop 2000c; Thermo Fisher Scientific, Waltham, MA, USA) operating at wavelength = 635 nm. Pfund scale was used to determine the hue of the honey. The formula was used to determine the Pfund value (1) ²¹.

Where  is the intensity of honey color in the Pfund scale;  is the absorption of honey solution at 635 nm.

 

Protein content:

Using Lowry's method, the protein content of honey was determined. Briefly, 5 mL of a stock BSA solution (1 mg/mL) was diluted to create BSA solutions. The levels of BSA varied from 0.05 to 1.00 mg/mL. Based on these various dilutions, 0.2 mL of protein solution and 2 mL of alkaline copper sulfate (analytical reagent) were added to several test tubes. The resulting mixture was thoroughly mixed and then incubated for 10 minutes at room temperature. After that, each tube received 0.2 mL of the reagent Folin-Ciocalteau solution, which was then incubated for 30 minutes. At 660 nm, the absorbance was measured after the colorimeter had been calibrated with a blank ²².

 

Fig. 3. Standard curve for sugars.

 

Fig. 4. Standard curves for each sugar (a. D-Fructose, b. Glucose, c. Sucrose, d. Maltose, e. Lactose).

 

Fig. 5. HPLC of honey samples

 

Statistical Analysis:

The one-way analysis of variance (ANOVA) was used in the statistical analysis of all outcomes. For (p 0.05), the difference was deemed statistically significant.

 

RESULT AND DISCUSSION:

Analysis of sugars by chromatographic methods:

The emergence of peaks in the chromatographic curves obtained for samples of honey that correspond to the same time of exit for each sugar establishes the presence of the examined sugars (Fig. 3). It establishes that the sample contains the same sugar. Fig. 4 represented the standard curves for each sugar. All samples, except for samples (HS₅), (HS₆), (HS₈), and (HS₁₀) for honey, do not contain lactose, as can be seen in Figure 5.

 

 

Table 2: The proportions of various sugars in honey samples

 

Quantitative analysis:

Once the types of sugars contained in the samples are determined, the ratio of sugars present in the samples can be computed using standard curves. The findings showed no appreciable variations in the fructose and glucose contents of the analyzed samples (Table 2, Fig. 6).

 

Fig. 6. Representation of the proportions of different sugars in honey samples.

 

These results were used to compute the fructose to glucose ratio, which is a crucial statistic for honey authentication and a predictor of a honey's propensity to crystallize (Table 3).

 

Table 3: Fructose to glucose ratio of honeys

 

Fructose and glucose are the main sugars found in honey. The data obtained demonstrate that the proportions of monosaccharides are essentially in honey sugars because insects produce enzyme invertase activity on the nectar, which converts saccharide sucrose into glucose and fructose. The samples under study had a higher proportion of fructose than glucose, ranging from 25,040 to 49,724 %. In contrast, the glucose percentage was between 19,392 and 43,126 %. These findings supported earlier research on various honey varieties^23,24.

 

For disaccharides like sucrose, the quantities varied between samples and typically ranged from 00,123 to 16,593 %. Maltose content has ranged from 00,054 to 10,742 %. The sample (HS₁) was significant, but the lactose values were reliably calculated to range from 00,294 to 09,143 %. We were unable to determine how much lactose was present in samples (HS₅), (HS₆), (HS₈), and (HS₁₀). The sample's lowest percentage was recorded (HS₇).

 

Physical analysis:

Density:

Table 4 shows the density ranges for the honey samples that were evaluated between (1.477-1.522). Because the density alters with changes in water content, indicating that they have an inverse proportion, the density varies with poor storage, especially in humid environments. As a result, we can state that all honey samples are 1.39 to 1.41 up to 1.522 g/cm³ and meet the norms advised by the French Association for Standardization. Its density ranges from 1.4009 to 1.4505 g/cm³, the same as that reported by ²⁵ for Algerian honey varieties. The temperature and changes in the water content of the dry matter (chemical makeup) of honey are the leading causes of variations in the density of honey.

 

The pH :

The pH values of the honey samples we analyzed ranged from (3.52-4.74), as indicated in Table 5. Different honey varieties, maturity times, bee nutrition, and honey's chemical composition in terms of the proportion of minerals and acids in it all contribute to the variation in pH values. All of the examined honey samples had varying pH levels and were acidic.

 

 

Table 4: The density of honey samples.

 

Table 5: pH of honey samples.

 

Table 6: Electrical conductivity of honey samples.

 

Table 7: Quantification of insoluble materials in honey samples

 

These numbers matched those previously reported for other honey samples from Turkey, Spain, Brazil, and India, which were said to have pH values ranging from 3.49 to 4.70^26,27.

 

Samples of honey from Algeria were previously shown to have pH values between 3.5 and 4.5²². A sample of highly acidic honey suggests that organic acids may have been produced during the fermentation of carbohydrates. No examined sample exceeded the permitted limit, indicating that all the honey samples were fresh.

 

Electrical conductivity) EC):

Except for sample (HS₉), whose conductivity value was more significant than 0.8 mS.cm^-1, we learned that the conductivity values of honey samples ranged between (0.142-0.907 mS.cm^-1), as indicated in Table 6, and that they correspond to the proposed international requirements. One of the most critical honey quality measures is electrical conductivity readings, which reveal the presence of proteins and negative and positive salt ions. Ash, organic acids, proteins, some complex sugars, and polyol concentration all affect electrical conductivity, which varies depending on the source of the material. The distinction between honeydew and blossom honey, as well as the characterization of unifloral honey, are frequently made using electrical conductivity²⁶.

 

According to research published in²⁸, the electrical conductivity of honey from the Harenna forest ranged from 0.63 to 0.79 mS.cm^-1 and that of Algerian honey from 0.417 to 0.806 mS/cm ²². Honeydew has electrical conductivity values greater than 0.8 mS/cm. In contrast, blossom honey and honeydew blends have values less than 0.8 mS/cm²⁸.

 

 

Insoluble materials (IM):

As demonstrated in Table 7, the percentage of insoluble materials in honey samples varied throughout the investigation, ranging from (0.51-0.12%). One key criterion for measuring the purity of honey is the measurement of solids that are insoluble in water. Since wax is one of the primary components of honey's solid components and all samples surpassed the proportion stated in the CODEX standards, the method used to extract and filter the honey can be blamed for this rise. These results confirmed the findings of previous research¹⁸.

 

Color intensity (Pfund):

As reported during the color examination, the color intensity values of the honey samples ranged from 23.693-266.58mm, as shown in Table 8. As indicated in Table 8, the honey's hue ranges from white to yellow to light yellow to dark yellow, with the bulk of the samples being dark yellow. The color of the samples was categorized as white²⁹ based on their absorbance, with a mean value of 23.07mm Pfund. Dark honeys typically have higher ash concentrations than light ones^30,31. Light honeys typically have low ash contents. In some foods, the concentration of melanoidin molecules (Maillard reaction), which have a brown color, can also be significant³². The nectar source, coloration, pollen color, sugar reaction, and storage time all affected how the honey tasted, smelled, and looked. Darker honey has a more robust flavor than lighter honey, which has a milder flavor.

 

Chemical composition:

Water content and total sugars:

Water content:

The amount of water in honey determines its maturation and storage. During the honey sample analysis, we found that the water content in the various samples ranged between (15.4-17.33%), except for sample (HS₁₀), which had a lower amount of water. The results are shown in Table 9. Its absence of water can be attributed to the area's dryness and high temperature. In contrast, the results of the other samples were to CODEX specifications. These results were almost identical to many previous types of research^33,34.

 

Table 8: Estimate the color intensity of honey samples.

 

Table 9: The water content of honey samples.

 

Table 10: The ratio of the sugar content of honey samples.

 

Table 11: The number of acids in the honey samples.

 

Table 12: The protein content of honey samples.

 

We can explain these results by stating that the samples were taken when they were mature and prepared to be stored for a long time. Environmental factors, the amount of moisture already present in the nectar, the honey's level of maturity, and the storage conditions after picking all influence the concentration of water in honey.

 

 

Total sugars:

The number of sugars in the honey samples ranged from 80.66 to 87%, as shown in Table 10. International standards took all samples, and the discrepancy is caused by the type of flower from which the bees obtain nectar. Polysaccharides are responsible for qualities that set bee honey apart, including sweetness, viscosity, water association, crystallinity, and energy. The examined honey's total sugar content was comparable to those of other Algerian honeys previously undergoing research²⁵.

 

Free acidity:

According to our findings, the acids in the honey samples ranged from 17.73 to 73.21m_eq/kg, as shown in Table 11.

 

The botanical diversity, in addition to organic acids and amino acids, is crucial in determining the final pH of honey.

 

The results met CODEX requirements, except for samples (HS₁), (HS₅), and (HS₉). By contrasting the samples, we can observe that sample (HS₉), which had a very significant number of acids, the samples (HS₇) and (HS₁₀) had a far lower concentration of acids.

 

Free acidity of North Indian honey was previously reported to range from 14.57 to 32.65³⁵, and Moroccan Carob honey's ranged from 17 to 42m_eq/kg³⁶. These results can be interpreted differently depending on the honey or plant species utilized. The phases of collection and storage can also have an impact on the amount of acid in honey.

 

Proteins:

The protein composition of the honey samples ranged from 2920.4 to 7502.8mg/Kg, as shown in Table 12. It had the most significant (HS₈) and the lowest sample value (HS₄), respectively. Egyptian honey had a protein value of 1690±0.015mg/kg, while Kashmiri honey had a protein content of 4670±0.171mg/kg²⁴. High protein content (4,097.00±3.54mg/kg) was found in²². Since nectar and pollen are the primary sources of protein in honey, depending on their botanical or geographic origin and the length of storage, different honeys have different quantities of proteins and amino acids. Furthermore, its protein concentration increases honey's quality, nutritional worth, and general attributes.

 

CONCLUSION:

This paper examined and obtained the physicochemical characteristics of 10 Algerian honeys. The honey samples came from diverse regions, and they were all collected and examined (beehives located at Laghouat, Blida, Tippasa, El Oued, Mssila, Bidjaia, Djelfa, and Boumerdase). We investigated and reported on density, water content, electrical conductivity, pH, free acidity, and color. The bulk of the Algerian honey specimens under investigation was deemed to be of acceptable quality by global standards. This study can aid in selecting honey products with higher physicochemical quality. Though expectations, requirements, and preferences differ significantly from person to person. Therefore, teaching them more about the honeys offered in Algeria made sense. There will be more research on the features of honey according to their color and trace element concentration about their origin. Overall, considering the physicochemical characteristics, Algerian honey has been proven valuable globally.

 

CONFLICT OF INTEREST:

The authors have no relevant financial or non-financial interests to disclose.

 

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Received on 24.08.2022                    Modified on 09.09.2022

Accepted on 22.09.2022                   ©AJRC All right reserved