Analysis of Honey

 

Introduction :

Honey is a complex mixture of 82.0% carbohydrates (sucrose, fructose, and maltose), 0.3% protein, 17.0% water and 0.7% minerals, vitamins and antioxidants. A part from sugars, honey also contains several vitamins, especially- B complex and vitamin C, together with a lot of minerals. Some of the vitamins found in honey include ascorbic acid, pantothenic acid, niacin and riboflavin; while minerals such as calcium, copper, iron, magnesium, manganese, phosphorus, potassium and zinc are also present. Honey contains at least 181 constituents. The other constituents of honey are amino acids, antibiotic-rich in hibine, proteins and phenol antioxidants. It also contains other bioactive substances such asphenolic constituents, flavonoids, organic acids, carotenoid-derived compounds, nitric oxide (NO) metabolites, amino acids and proteins. Evidence indicates that some varieties of honey contain kynurenicacid (a tryptophan metabolite with neuro active activity) which may contribute to its antimicrobial properties. The presence of enzymes such as glucose oxidase, diastase, invertase, phosphatase, catalase and peroxidasehas also been documented in honey. High levels of ascorbic acid, catalase, peroxidase, flavonoids, phenolicacids, and carotenoids ensure a high level of anti oxidantsin honey. Honey consumption by humans has been reported to increase total plasma antioxidant and reducing capacity, which can be protective to human health. In Bangladesh, honey is produced and consumed on a large scale. Sundarbons, which is the largest mangrove forest in the world, consists of 334 plant species and is ideal for giant honey bees (Apisdorsa) and honey collectors. However, there is still a lack of information on the comparative physicochemical and biochemical properties of different types of Bangladeshi honeys.

Chemical composition:

 Honey is composed mainly from carbohydrates, lesser amounts of water and a great number of minor components.

i.                   PH

 A 10% (w/v) solution of honey was prepared in mill water for pH measurement using a pH meter. The moisture content wasdetermined using refracting metric method. In general, the refractive index increases with an increase in the solidcontent of a sample. The refractive indices of honeysamples were measured at ambient temperature using a handheld refractometer and the measurements were further correctedfor a standard temperature of 20°C by adding thecorrection factor 0.00023/C

Multiresidual Analysis Antibiotics
Chemical analysis that allows to detect the presence of, for example, sulfonamides, tetracyclines and tylosin and other classes of antibiotics.

ii.                  Pesticide / Acaricide analysis

Chemical analysis that detects the presence of active ingredients in the most widespread treatments in agriculture and beekeeping: Acrinatrin, Amitraz and Metabolites, Bromopropylate, Cimiazole, Clorfenvinfos, Cumafos, Fluvalinate, Rotenone, Tetradifon.

HPLC Analysis for Pesticide Residues

HPLC Machine and Parameters.

 After the aforementioned sample cleanup, the samples were quantified using an LC-10 ADvp HPLC (Shimadzu) equipped with a photodiode array (PDA) detector (Shimadzu SPD-M 10 Avp, Japan) (200–800nm). An All tech C18 Reverse Phase (5𝜇m, 250×4.6mm) analytical column was used and maintained at 30∘Cinacolumnoven.Themobilephasewasacombination of 70% acetonitrile and 30% water. The mobile phase was filtered fresh daily using a cellulose filter (0.45𝜇m) and ran at1.0mL/min through the HPLC machine.

iii.                Melissopalinological Orientation Analysis

Microscopic analysis consisting in the study of honey sediment in order to obtain a botanical indication of the product. In the test report are reported the pollen types identified and divided by frequency classes and the sensory analysis to confirm what was found at the microscopic level. Photographic documentation and geographical indication of honey are not reported. The analysis is carried out only on honey samples declared Italian.

iv.                Qualitative Melissopalinological Analysis

Microscopic analysis of honey sediment that - through the recognition of pollen grains, the study of their relative frequency and the presence of other microscopic elements - allows to trace the botanical and geographical origin of the honey in question. The list is shown in the test report complete of the identified pollen spectrum, the presence of any indicators of honeydew, yeasts, carbonaceous particles and other elements of the sediment, the photographic documentation of the analyzed sample and the descriptive sensory analysis.

v.                  Sugar analysis

Chemical analysis that detects the amount of fructose, glucose, F / G ratio, F + G, sucrose, maltose.

v Sugar Adulteration Screening

·        Analysis with a combination of pollen (quantitative + qualitative) and analysis of sugars.

·        NMR analysis

·        Isotopic analysis

Analysis of the most important constituents

v Moisture

The water content is an important factor that contributes to honey stability against granulation and fermentation during storage.

i.                    Refractometric method

This is the method proposed by IHC and one of the methods proposed by the AOAC. It is the most used method due to its simplicity and reproducibility. Temperature control is utmost for refractive index determination. Sugar crystals of honey have to be dissolved previously in a heating bath at 50 ˚C. Refractive index of the honey is measured at 20 ˚C or 40 ˚C with an Abbe or digital refractometer, evaluating moisture percentage by using an empirical formula or a relative conversion table.

ii.                  Direct drying

This is another method proposed by the AOAC (method). This method is a gravimetric determination after oven drying at <70 ˚C under pressure ≤50 mm Hg. The main disadvantage of direct drying is that the procedure is time consuming.

iii.                Other methods

 Infrared spectrometric methods have been used for moisture determination, such as NIR and Fourier transform midinfrared spectrometry with attenuated total reflectance (FT-MIR-ATR), in conjunction with multivariate calibration. The major drawback of infrared techniques is related to the compulsory previous calibration

with a large number of samples, in which moisture has been analyzed by other method(s).

Elements analysis

 Five grams of honey were accurately weighed in a 120 ml glass beaker.  25 ml of nitric, 10 ml of perchloric acid were added and heated with a hot plate to mineralize organic material. The mixtures were heated to almost dry. The acid clear solutions were transferred to 50 ml volumetric flasks and diluted with deionized water. Three replicates were analyzed per sample. Elements P, Al, Fe, Mn, Zn, Cu, Ca, Mg, Na and K were analyzed by emission measurements obtained by direct nebulization in an inductively coupled plasma optical emission spectromete

Sugar analysis

 Honey is a supersaturated sugar solution. Sugars are the main constituents of honey accounting for about 95 g/ 100 g dry matter. The methods used for the determination of the sugar composition validated by the International Honey Commission are apparent reducing sugars, apparent sucrose, HPLC-IR, HPLC-PAD and GC-FID; and the methods adopted by AOAC are apparent reducing sugars, apparent sucrose , HPLC-IR, column chromatography and thin layer chromatography for glucose determination. Other methods such as spectroscopy, enzymatic and capillary electrophoresis methods have been also developed.

i.                    Determination of total sugars

Apparent reducing sugars and apparent sucrose The method used for quantification of reducing sugars (mainly fructose and glucose) and apparent sucrose in honey is by reducing Soxhlet’s modification of Fehling’s method (a modification of the Lane and Eynon procedure) (AOAC, 2012; Bogdanov, 2009). Currently, volumetric procedures for honey reducing sugars’ and apparent sucrose determination are hardly used, since European regulation (OJEC, 2002) considers percentages of glucose + fructose, as well as percentages of sucrose, instead.

Techniques:

HPLC:

HPLC with refractive index detector (IR). This is the most popular method used for sugar identification. IR detector has several disadvantages such as the lack of sensitivity and selectivity signal dependence with temperature and with mobile phase flow rate, and the incompatibility with gradient elution. It requires a simple sample preparation. Silica-based columns of polar aminopropylsilane (–NH2) are the most used, being the main eluent a solution acetonitrile/water (proportion about 80:20 v/v) Identification is carried out by comparing the retention times of the peaks obtained from authentic commercial standards and quantification is normally achieved according to the external calibration method.

HPLC analysis of honey

Gas chromatography (GC):

 GC provides better resolution and sensitivity than HPLC for many important minor carbohydrates in relative short retention times, but it involves a previous derivatization reaction to make sugars volatile compounds, which is time-consuming and implies possible sample losses although I.N.A. method has been also used. After derivatization process, sugars are determined as their trimethylsilyl (TMS) derivatives . Gas chromatography with flame ionization detector (GC-FID) is the most used GC method. Identification is carried out by comparing their retention times with those of standard compounds and quantification is made with the internal standard method

Spectroscopy methods:

Spectroscopy methods such as NIR  FT-NIR-ATR (Fourier transform near infrared spectroscopy method with attenuated total reflectance), FT-MIR-ATR ,1H and 13C NMR (nuclear magnetic resonance spectroscopy) and Raman Spectroscopy in combination with chemometrics, have been applied for sugars analysis in honey. They are quick methods that need minimal sample amount and handling.

 

 

 

Experimental Materials and methods:

Sampling:

Five samples of natural  bee honey were collected from four  geographically different areas.one sample from Darfour states, and the second from Kurdofan states, the third from South Sudan state, and two samples from Blue Nile state (amber and white honey).

 

Physicochemical analysis:

Six physical parameters, PH, moisture content, refractive index, viscosity, free acidity, and ash content were determined for each sample. Results are shown in table 1.

 PH:        

  PH value was determined according to 350 PH meter -JENWAY. 10g of honey sample were weighed and diluted in 75 cm3 of distilled water. The PH electrode was immersed in the honey solution and the PH value was recorded.  Refractive index: (R. I)       

 

 Refractive index of the five honey samples were determined by KRUSSOptrnic Germany refractometer at constant temperature (26°C).

Moisture content:

The moisture content was determined by calculation and expressed as percentage.

Viscosity:

 The viscosity of the five honey Samples were measured at 26oC using capillary viscometer-schott GERATE. 

 

Free acidity: 

10.0 g of honey sample were weighed and diluted in 75 cm3 of distilled water. Sample was titrated against (0.2M) sodium hydroxide solution using phenolphthalein indicator, the end point was determind by pink color that persisted for seconds. The results expressed as mg/kg honey.

Ash Content:

 The ash content was determined using muffle furnace. 5.0 g of honey sample weighed in dry crucible and ignited at 550oC for 2 hours. The ash was determined by calculation and expressed as: Wa = (m1- m2) x 100                  m0 Wa = ash content (g/100g),  m0 = weight of honey taken m1 = weight of dish + ash  m2 = weight of dish

Sugar content:

The sugar content of honey samples was determined according to the external standard procedure. The result are shown in table (2). 

 

Instrument:

HPLC:

shimadzo- japan Column: normal- phase (shim-pack)CLC-NH2  equipped with a guard column of the same material.

Mobile phase:

Acetonitrile (75%):Deionized water (25%) (V/ V)

Sample preparation:

 2.5 g of honey sample were weighed into a beaker and dissolved in 50 cm3 water then shaked. The solution was filtered through membrane filter 0.45 µm.20 µl of extract was injected in HPLC-RI detector.

 Flow rate:

 1.00 ml /min.

Mineral content:

 5.0 g of honey sample were weighed in dry crucible; and placed in muffle furnace at 550°C for two hours. The contents were cooled and transferred to 250 cm3 beaker and, 10 cm3 of 5M HCl and 1 cm3 of concentrated HNO3 were added. The beaker was placed in water bath to boil for 15 mins.  50 cm3 of distilled water was added and the content was filtered through filter paper and the volume was made to 100 cm3 with distilled water. The Cu, Fe, Mg, Zn, were determined by F-AAS.  The  Na+, K+, Ca+2, were determined by flame photometer 410. The results are shown in table (3).

Ascorbic Acid content:

Instruments and Apparatus:

Measurements were made with a double beam  uv 1800 ultraviolet-visible spectrophotometer provided with matched 1.0cm quartz cell (SHIMADZU JAPAN). Jenway instrument HI 350 pH meter. Electronic balance type AY220. Preparation of sample: 1.0 g of five different samples were transferred into 100 cm3 volumetric flask and diluted to the mark with sodium oxalate solution. The absorbencies of standard solutions and sample were measured at 226nm against the sodium oxalate solution as blank. 

 

 

 

 

 

 

 

Results and discussion

 Table (1): pH-values of different honey samples 

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