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*Corresponding author: Paliwal G. N Centre for Bee Development, Nalwadi, Wardha, MS, India 442 001

ISSN: 0976-3031

Research Article

COMPARATIVE PHYSICO-CHEMICAL ANALYSIS IN SOME HONEYS OF COMMERCIAL BRANDS WITH THAT OF APIS DORSATA BEE-SPECIES

Paliwal S. G., Paliwal G. N*., Dhore R. D and Tembhare, D. B

Centre for Bee Development, Nalwadi, Wardha, MS, India 442 001

DOI: http://dx.doi.org/10.24327/ijrsr.2021.1207.6113

ARTICLE INFO ABSTRACT

The commercially available honeys in local market (probably resourced from Apis mellifera bee-species) and honeys of Apis dorsata, bee-species that gathered from different floral seasons have been analyzed and compared in the present study. The physicochemical parameters like specific gravity, moisture, non-reducing sugars (NRS), total reducing sugars (TRS), true fructose, true glucose, fructose/glucose (F/G) ratio, dextrose/water (D/W) ratio, acidity, ash, total dissolved solids (TDS), hydroxyl methyl Furfural (HMF), Fiehe’s test and Aniline Chloride test have been studied in detail. Based on these observations, we can state that honeys generated from A. dorsata bee-species and that of commercially available brands show their unique physico-chemical characteristics. The later has been ultimately seemed to be influenced and attributed with the type of bee-species from which honeys have been resourced along with the factors like specific floral composition and local geo-specific conditions prevailed.

INTRODUCTION

Honey is the naturally sweet substance derived from the wild- and cultivated bee-nests. Bees collect nectar and pollen from varieties of the flowers to make honey (Codex Alimentarius Commission, 2001). It is the food of bees and their young ones (James, 2009). It is the complex composition of moisture, reducing sugars, non-reducing sugars, proteins, fats, enzymes, vitamins, amino acids, organic acids, minerals, other phyto-chemicals, etc. (Ouchemoukh, et al., 2007; Paliwal, 2008; Gomes, et al., 2010; and Lazarevic et al., 2010, Saxena et al., 2010, Puja et al., 2020). All these microelements make honey the unique nutritional or medicinal substance (James, 2009). The basic composition and properties of honey depends upon the factors like floral origin, seasons, environmental factors, etc. (Da Costa Leite, et al., 2000, Marchini et al., 2006; Kaskoniene et al., 2010, Iglesias, et al., 2012; and El-Metwally, 2015). Moisture content is one of the important parameter that decides primarily the shelf life in any honey (AL-Naji and Hujazy, 1982; Cantarelli et. al., 2008; Bogdanov, 2009; Buba et al., 2013; Akhtar, et al., 2014 and EL-Metwally, 2015). Lesser the moisture betters the quality of honey. It is the riches resource of energy (Rahman, et al., 2010). The carbohydrates are the major constituents of honey, corresponding to 95-99%

of the dry matter (Olaitan, et. al., 2007). The higher sugars may be constituted of maltose, isomaltose, trisaccharides and tetrasacchatides (Anklam, 1998) while there are easily digestible carbohydrates like fructose and glucose and its ratio (fructose to glucose) defines granulation in honey (While and Doner, 1980). Honey has natural therapeutic applications like antimicrobial- and antioxidant agent (National Honey Board, 2002; Basualdo, et al., 2007, and Alvarez- Suarez, et al., 2010).

The Vidarbha region of Masharashtra State in Centre India has many forest pockets with full of Apis dorsata bee-population yielding ‘forest honey’ during the summer season. Hundreds of local traditional honey collectors gather and sale the raw-honey on adoption of scientific technology imparted to them by the local NGO namely Centre for Bee Development (CBeeD), Wardha, in the province of Maharashtra, India. This NGO is working for the conservation of Apis dorsata bee-population since past 20 years. The forest honey is yielded mostly in the summer season (March to June) from the wild flowering plants like Mangifera indica (mango), Butea monosperma (palas), Azadiracuta indica (neem), Ceiba pentandra (white katsawar), Bombax ceiba (red katsawar), Pongamia pinnata (karanj), Calycopteris floribunda (kukurangi, Madhuca indica (mahua), Hardwickia binata (anjan),Terminalia arjuna (arjun), T. bellirica (bahera),T. chebula (harda), Terminalia alata (ain);

Available Online at http://www.recentscientific.com International Journal of

Recent Scientific

Research International Journal of Recent Scientific Research Vol. 12, Issue, 07 (F), pp. 42538-42548, July, 2021

Copyright © Paliwal S. G et al, 2021, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.24327/IJRSR

CODEN: IJRSFP (USA)

Article History:

Received 6th April, 2021 Received in revised form 15th May, 2021 Accepted 12th June, 2021 Published online 28th July, 2021 Key Words:

Physicochemical analysis, honey, Apis dorsata, Apis mellifera, Vidarbha

International Journal of Recent Scientific Research Vol. 12, Issue, 07 (F), pp. 42538-42548, July, 2021

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Wrightia tinctoria (dudhapuda), Adina corifolia (haldu), Syzygium cumini (Jamun); etc. Such honeys are multi-floral and available for formal sale at Centre for Bee Development, Wardha, MS besides some local outlets. Little quantity of honey is also seen produced during the winter season especially from the flowers like crops of Gossypium spp. (Cotton) and Cajanus cajan (Tur).

Besides CBeeD’s Apis dorsata honey, there are so many other brands of honey commonly available in the market which are known to be generated from the Apis mellifera bee-species. Lots of work has been undertaken in the past on physicochemical analysis in some honeys derived from different bee-species (Apis mellifera, melipona, etc.) and different geographical origins (Almeida-Muradian, et. al., 2013; EI-Sohaimy, et. al. 2015; Tesfye, et. al., 2016; Roshan, Abdul-Rahman, et. al, 2017; and Boussaid, et. al., 2018; ) but very few studies have been conducted on that of honey of Apis dorsata (Paliwal, 2008; Basavarajappa, et. al., 2011; Puja, 2020).

Therefore, in the present study, the comparative physicochemical analysis of some honeys of commercial brands with that of Apis dorsata, bee-species, has been evaluated.

MATERIALS AND METHODS

Materials

Total 6 different honey samples were collected for analysis. Out of these 6 samples, sample A, sample B, sample C, sample D were the honeys of popular commercial brands available in the local market at Wardha (MS, India) while sample E-S (E-Summer) and sample F-W (F-Winter) were gathered from Centre for Bee Development, Wardha (MS, India) of different floral origins as detailed in table. 1.

Table 1 Honey samples from different botanical origin exclusively from A. dorsata species

Samples of honeys

Seasons

Botanical names of dominant flora resourced by bees in making honey

Local names of the flora

Sample E-S

Summer Season

Butea monosperma, Azadiracuta indica, Bombax ceiba, Calycopteris floribunda Madhuka indica, Terminalia tomentosa, Syzygius cumini, etc.

Palas, Kadu Neem, Katesawar, Kukurangi, Mahua, Ain, Jamun, etc

Sample F-W

Winter Season

Gossypium spp. and Cajanus cajan

Cotton and Tur

Methods

For the detection of specific gravity, moisture, total reducing sugars (TRS), non-reducing sugars (NRS), true glucose, true fructose, fructose/glucose (F/G), dextrose/water (D/W), acidity, ash, total dissolved solids (TDS), hydroxyl methyl Furfural (HMF), Fiehe test and Aniline Chloride test, we used the standard quality control manuals and protocols viz. India Standards IS- 4941 and IS- 4941 B-2, as approved by the competent authorities.

The methods adopted are detailed as in below.

Determination of Specific Gravity of Honey (IS-4941)

Procedure

Clean and thoroughly dry the specific gravity bottle and weigh. Fill it uo to mark with freshly boiled and cooled distilled water which had been maintained at 27 ±10 C. and weigh. Remove the water, dry the bottle and fill it with the honey sample maintained at the same temperature. Weigh the bottle again.

Calculation

Specific gravity at 270C = C – A ---------- B – A Where,

C = mass in g of the specific gravity bottle with the honey sample, A = mass in g of the empty specific gravity bottle, and B= mass in g of the specific gravity bottle with water

Determination of Moisture in Honey (IS-4941)

Moisture was determined using the indirect refract metric method. All measurements were taken using an Abbe refractometer, and the percentage of moisture obtained from the refractive index of the honey sample by consulting a standard table for the purpose (Chataway, 1932).

The table is derived from a formula developed by Wedmore (2015).

1.73190 – log [RI - 1] W = -------------------------- 0.002243 W is the water content in g per 100 g honey and R.I. is the refractive index

Determination of Non Reducing Sugars (NRS) (IS-4941)

Reagents

Stock solution of honey, concentrated hydrochloric acid and sodium carbonate

Procedure

To 100 ml of the stock honey solution add one mililitre of concentrated hydrochloric acid, heat the solution to near boiling and keep aside overnight. Neutralize this inverted honey solution with sodium carbonate and determine the total reducing sugar as described in above.

Calculation

Sucrose, percent by mass = (reducing sugars after inversion, percent by mass) – (reducing sugars before inversion percent by mass) x 0.95.

Determination of Total Reducing Sugar (TRS) (IS-4941)

Reagents

Soxhlet Modification of Fehling’s Solution- Prepared by mixing equal volumes of Solution A and Solution B immediately before using.

Paliwal S. G et al., Comparative Physico-Chemical Analysis In Some Honeys of Commercial Brands With That of Apis Dorsata Bee-Species

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Copper Sulphate Solution (Solution A)

Dissolve 34.639 g of copper sulphate crystals (CuSO4.5H2O) in water, dilute to 500 ml and filter through glass wool or filter paper.

Standardization of copper sulphate solution:- Using separate pipettes, pipette out accurately 5 ml of Solution A and 5 ml of Solution B in to a conical flask of 250 ml capacity. Heat this mixture to boiling on an asbestos gauze and add standard invert sugar solution from a burette, about one milliliter less than the expected volume which will reduce the Fehling’s solution completely (about 48 ml).Add one milliliter of Methylene blue indicator while keeping the solution boiling. Complete the titration within three minutes, the end point being indicated by change of colour from blue to red. From the volume of invert sugar solution used, calculated the strength (S) of the copper sulphate solution by multiplying the titre value by 0.001 (mg/ml of the standard invert sugar solution). This would give the quantity of invert sugar required to reduce the copper in 5 ml of copper sulphate solution.

Potassium Sodium Tartrate (Rochelle salt) Solution (Solution B):-

Dissolve 173 g of potassium sodium tartarate and 50 g of sodium hydroxide in water, dilute to 500 ml. Let the solution stand for a day and filter.

Hydrochloric Acid –Sp gr 1.18 at 200 C. (approximately 1/2 N).

Standard Invert Sugar Solution

Weigh accurately 0.95 g sucrose and dissolve it in 500 ml of water. Add 2 ml of concentrated hydrochloric acid, boil gently for 30 minutes and keep aside for 24 hours. Neutralize with sodium carbonate and make the final volume to 1000 ml, 50 ml of this solution contains 0.05 invert sugar.

Methylene Blue Indicator – 0.2 percent in water.

Procedure

Place accurately weighed about one gram (W) of the prepared sample of honey into a 250 ml volumetric flask and dilute with about 150 ml of water. Mix thoroughly the contents of the flask and make the volume to 250 ml with water. Using separate pipettes, take accurately 5 ml each of Solution A and Solution B in a porcelain dishes. Add about 12 ml of honey solution from a burette and heat to boiling over asbestos gauze. Add one milliliter of methylene blue indicator and while keeping the solution boiling complete the titration, within three minutes, the end point being indicated by change of colour from blue to red. Note the volume (H) in ml of honey solution required for the titration.

Calculation

Total reducing sugars, percent by mass = 250 x 100 x S H x M Where, S = strength of copper sulphate solution, H = volume in ml of honey solution required for titration, and M = mass in g of honey.

Determination of True Fructose (F), True Glucose (G) and F/G) Ratio

Reagents

Iodine Solution – 0.05 N Sodium Hydroxide Solution – 0.1 N. Sulphuric Acid – concentrated. Standard Sodium Thiosulphate Solution – 0.05 N.

Procedure

Pipette out 50 ml of honey solution in a 250 ml stopper flask. Add 20 ml of iodine solution and 25 ml of sodium hydroxide solution. Stopper the flask and keep in dark for 20 minutes. Acidify with 5 ml of sulphuric acid and titrate quickly the excess of iodine against standard sodium thiosulphate solution. Conduct a blank using 50 ml of water instead of honey solution.

Calculations

where,

B = volume of sodium thiiosulphate solution required for the blank,

S = volume of sodium thiosulphate solution required for the sample, and

A = mass of honey taken for test.

Determination of Acidity

Reagents

Standard Sodium Hydroxide solution – 0.05N Phenophthalein Indicator Solution – Dissolve 0.5 g of phenolphthalein in 100 ml of 50 percent ethyl alcohol (v/v ).

Procedure

Take 10 g of the sample in a suitable titration flask and dissolve it in 75 ml of carbon dioxide free water .Mix thoroughly. Titrate against standard sodium hydroxide solution using 4 to 6 drops of carefully neutralized phenolphthalein solution (pink colour of indicator should persist for at least 10 seconds). Determine blank on water and indicator and correct the volume of standard sodium hydroxide solution used.

Calculation

Acidity (as formic acid), percent by mass = 0.23 x V M Where,

V = corrected volume of 0.05 N sodium hydroxide solution required for titration, and M = mass in g of the sample taken for the test.

Determination of ASH

Reagents

Honey sample and pure olive oil.


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Procedure

Weigh accurately 5 to 10 g of the sample in a silica or platinum dish; add a few drops of pure olive oil to prevent spattering, heat carefully over a low flame until swelling ceases. Ignite in a muffle furnace at 600 + 20c till white ash is obtained. Cool the dish in a desiccator and weigh. Incinerate to constant weight.

Calculation

Ash, percent by mass = 100 (M2 –M) M1 - M Where, M2 = mass in g of the dish with the ash M = mass in g of the empty dish, and M1= mass in g of the dish with the material taken for the test.

Determination of Total Dissolved Solids

Procedure- Put one drop of honey sample on the glass surface of portable sugar refractometer made by Erma Japan (60-100 Brix) make refractometer and focus against the light by eye and note down reading of calibrated scale. The reading at partition between blue and white will give the result ie. 100 – X (reading) = total dissolve solids in %.

Determination of HMF Content

Reagents

1. Barbituric Acid Solution

Weigh out 500 mg barbituric acid and transfer to a 100 ml graduated flask using 70 ml water. Place in a hot water bath until it dissolve, cool and make up to volume.

2. P – Tolidine Solution Weight out 10.0 g toluidine, analytical grade and dissolve in about 50 ml isopropanol by gentle warming on a water bath. Transfer to a 100 ml graduated flask with isopropanol and add 10 ml glacial acetic acid. Cool and make up to volume with isopropanol. Keep the solution in the dark. Do not use the solution for at least 24 hours.

3. Distilled Water (Oxygen Free) 4. Nitrogen gas is passed through boiling distilled water

and cooled.

Procedure

Preparation of Test Sample

10g of honey sample is weighed and dissolved without heating in 20 ml oxygen free distilled water. This is transferred to a 50 ml graduated flask and made up to volume (honey solution). The sample should be tested after preparation without delay.

Photometric Determination

2.0 ml of honey solution is pipetted into each of two test tubes and 5.0 ml p toluidine solution is added to each. Into one test tube 1 ml water is pippeted and into the other 1 ml barbituric acid solution and both mixture are shaken. The one with added water serves as the water blank. The addition of the reagents should be done without pause and should be finished in about 1 to 2 minutes.

The extinction of the sample is read against the blank at 550 nm using a 1 cm cell, immediately after the maximum value is reached.

Calculation

The method may be calibrated by using a standard solution of hydroxymethyl furfuraldehyde (HMF) standardized by dissolving commercial or laboratory prepared.

HMF and assaying spectro-photometrically where e = 16.830 at 284 nm using 0.3000 ug standards. An equation by which result may be worked out is given below: Mg/100 g HMF = Absorbance Thickness of Layer Determination of Fiehe’s and Aniline Chloride Tests

Fiehe’s Test

Reagents

Resorcinol Crystal and Hydrochloric acid Ether – Sulphuric ether

Procedure

Transfer about 5 g of the honey sample into a mortar, mix the honey with 10 ml of ether. Decant the ether in to a porcelain dish. Repeat the extraction to evaporate and collect the extract in the same dish. Allow the extract to evaporate to dry at room temperature and add a large drop of freshly prepared resorcinol solution. The production of cherry red colour appearing instantly indicates a positive reaction. Faint pink colour disappearing after a short time or yellow to salmon pink colours indicate a negative reaction.

Aniline Chloride Test

Reagents

Aniline Chloride solution (Aniline and Hydrochloric Acid)

Procedure

Place 5 g of the sample in a porcelain dish and add, while stirring 2.5 ml of recently prepared aniline chloride solution. In the presence of commercial invert sugar within one minute, the reagent assumes orange red colour turning dark red. Yellow to salmon shades has no significance.

RESULTS AND DISCUSSION

The samples of total 6 different honeys have been analyzed to explore the comparative physicochemical characteristics. The parameters like specific gravity, moisture, non-reducing sugars (NRS), total reducing sugars (TRS), true fructose, true glucose, fructose/glucose (f/g) ratio, dextrose/water (d/w), acidity, ash, total dissolved solids (TDS), hydroxymethylfurfural (HMF), fiehe’s test and aniline chloride test have been studied thoroughly (table 2). These results were compared (Figs. 1 to 12) in between the honey samples of commercial brands generated from A. mellifera (Samples A to sample D to be) with that of A. dorsata honeys (samples E-S to F-W).

Specific Gravity at 270C

It is the very important physical characteristic used to determine honey quality (Rehman et. al., 2008). The specific gravity of honey depends upon moisture content and temperature (Wedmore, 2015). More the value of specific gravity thicker is the honey i.e. lesser the moisture.

X 19.2


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In the present study the values of Specific Gravity in the commercial honeys and Apis dorsata honeys were found in the permissible level (AGMARK, 1984; and Codex Alimentarius Commission, 2001; FSSAI, 2011) i.e. from 1.40±0.11 to 1.41±0.12 (table 2) supporting the earlier observations (Bogdanov, et. al.., 1999; Kamal et. al., 2002; Nanda et al.,2003; Ouchemoukh, et. al., 2007). The specific gravity of most commercial honeys was found higher than honeys of Apis dorsata bee-species derived in both seasons (fig.1). The Specific Gravity is the physical property directly depending upon the quantity of moisture in any honey. During processing of honey, the moisture reduction is undertaken to increase the Specific Gravity (Wakhale, 1996; Acquarone, et. al., 2007).

Moisture %

Honey naturally contains moisture as its one of the most important component and many methods have been derived for its quantitative analysis (Chataway, 1932 and Wedmore, 2015). It has been reported that higher the moisture content there are more chances of yeast fermentation in honey (Singh and Bath, 1997; Chirifie et al., 2006; Iglesias, et al., 2012). Thus, the moisture content decides the shelf life in any honey (Terrab et. al., 2003). However, moisture content in honey depends upon varies factors like harvesting seasons, flora in abundance, temperature, relative humidity and maturity of honey harvested (Crane, 1979; Acquarone, et al., 2007; Kahraman et al., 2010).

As during processing of raw honey, the practice of moisture reduction is undertaken so as to maintain the moisture level between 19 to 25 % (Wakhale, 1996; Acquarone, et. al., 2007, Paliwal, 2008; Puja et al, 2020).

In the present study, the moisture level in all 6 samples was found from 19±1.00 to 20±1.00 % (table 2). These results (fig. 2) are within the prescribed range as approved by certifying agencies (AGMARK, 1984; Codex Alimentarius Commission, 2001; Council Directives of the European Union, 2001 and FSSAI, 2011). These observations are well in support with earlier data on honey moisture carried in the case of honeys of different bee-verities- and geographical origins (Bogdanov, et. al.., 1999; Paliwal, 2008; Feas et. al.., 2010; Gomes, et. al., 2010; Vanhanen et. al.., 2011; El-Bialee and Sorour, 2011; and Boussaid, et. al., 2018; Puja, 2020).

Non-Reducing Sugars (NRS) %

In honey, Iurlina and Fritz (2005) reported the NRS constitute about 12% by mass. However, higher sucrose in honey indicates that honey harvest was premature wherein sucrose was not converted fully into glucose and fructose (Ozcan et. al, 2006). As per the National and International honey quality regulatory bodies the level of NRS should not be more than 5% by mass. (AGMARK, 1984; Codex Alimentarius Commission, 2001; Council Directives of the European Union, 2001; European Economic Committee, 2002; EOSC, 2005 and FSSAI, 2011). In the present study, the NRS values obtained in all 6 samples have been noticed from 2.36±0.15 to 3.72±0.25 (table 2) and thus, all the samples pass the mandatory maximal requirements by the law. It is noteworthy to report here that all

1.394

1.396

1.398

1.4

1.402

1.404

1.406

1.408

1.41

1.412

A B C D E-S F-W

Fig. 1. Specific Gravity at 270C

Specific Gravity

Honeys of 'commercial brands' Honeys of 'Apis dorsata bees'

18.4

18.6

18.8

19

19.2

19.4

19.6

19.8

20

20.2

A B C D E-S F-W

Fig. 2. Moisture %

Moisture


Table 2 Physicochemical characteristic of commercial and Apis dorsata honeys

Parameters↓

Honey samples of 'commercial brands' seems generated from A. mellifera bee-species

Honey samples of 'Apis dorsata’ bee-species

A B C D E-S

(E-Summer) F-W

(F-Winter) Specific Gravity 1.41±0.09 1.41±0.12 1.40±0.11 1.41±0.09 1.40±0.11 1.40±0.11

Moisture 19±1 19±1 20±1 19±1 20±1 20±1 Non-Reducing Sugars (NRS) 3.21±0.21 2.94±0.18 3.72±0.25 3.39±0.23 2.70±0.18 2.36±0.15 Total Reducing Sugars (TRS) 64.29±2.57 57.40±2.39 60.43±2.45 67.94±2.72 72.01±2.88 75.02±3.01

True Fructose 31.97±1.59 22.45±1.12 28.54±1.43 35.77±1.79 44.18±2.21 43.37±2.17 True Glucose 34.72±1.74 36.64±1.83 34.63±1.73 34.85±1.74 30.68±1.53 33.99±1.69

F/G ratio 0.92±0.05 0.61±0.03 0.84±0.04 1.02±0.05 1.45±0.07 1.27±0.06 D/W 1.83±0.09 1.93±0.10 1.7±0.08 1.83±.0.09 1.58±0.08 1.59±0.07

Acidity 0.06±0.01 0.03±0.003 0.14±0.014 0.05±0.01 0.15±0.02 0.13±0.013 Ash 0.21±0.014 0.19±0.013 0.22±0.015 0.24±0.016 0.26±0.017 0.29±0.019 TDS 79 ±1 79±1 80±1 80±4.02 79±1 79±1 HMF 23.01±1.15 39.07±1.95 57.83±2.89 43.04±2.15 46.5±2.33 44.06±2.20

Fiehe’s Test -ve -ve -ve -ve -ve -ve Aniline Chloride test -ve -ve -ve -ve -ve -ve


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the samples of commercial honeys in the present study have more NRS (higher sugars) contents than the honeys of Apis dorsata derived in the summer and the winter seasons (fig. 3). It also means that honeys of Apis dorsata bees contain NRS below 3% by mass which certainly proves its uniqueness and species specific characteristics.

Total Reducing Sugars (TRSs) %

The both reducing sugars i.e. fructose and glucose are the main constituents of any natural honey (White and Doner, 1980). Although, no limit have been fixed for their individual values but their sum (fructose + glucose) should be over 60 or 65%. As per the quality assuring parameters, the TRS should not be less than 65% by mass (AGMARK, 1984; Adgaba, 1999; FSSAI, 2011) while that limit is 60% as per Codex Alimentarius Commission, 2001. However, in the present study, sample no. D (67.94±2.72%) from commercial brands and both samples i.e. ES (72.01±2.88%) and FW (75.02±3.01%) of A. dorsata honey clearly passes the required level while sample no. A (64.29±2.57%) was found on the edge and that of sample nos. B (57.40±2.39%) and C (60.43±2.45%) do not at all meet the minimal requirement (table 2 and fig. 4). In the present study TRS in Apis dorsata honeys have been detected beyond 70%. It clearly indicates the bee-specific, geo-specific and flora-specific character and supporting the earlier observations (Bogdanov, 2002; Paliwal, 2008; Basavarajappa, 2011; EI-Sohaimy, et al., 2015; Tesfaye et. al., 2016; Boussaid, 2018).

Fructose %

The fructose is main reducing sugar found in most honeys. It eventually maintains the honey in liquid form (Al., et al., 2009). In the present study, concentration of fructose in the commercial honeys and A. dorsata honeys was found to be from 22.45±1.12 to 35.77±1.79% and 43.37±2.17 to 44.18±2.21%, respectively (table 2). We also demonstrated that commercial honeys contend comparatively less fructose than the A. dorsata honey (fig. 5). Lots of work have been carried out on quantitative analysis of fructose in honeys derived from various bee-species of different geo-specific locations having varied floral spectrum (Almeida-Muradian, et. al., 2013; EI-Sohaimy, et al., 2015; Tesfaye et. al., 2016; Boussaid, et. al., 2018). All these studies revealed the quantity of fructose in varied samples of honeys in the range of 35 to 51% depending upon the bee-species and flora. However, Basavarajappa, et. al., (2011) reported presence of fructose from 58.03 to 59.69% in A. dorsata honey that seems unique characteristics. In the present study, we could record just 43.37±2.17 to 44.18±2.21% fructose in our honey samples.

Glucose %

Glucose is the other important reducing sugar available in honey. Its quantity profoundly determines the granulation in honey (Paliwal, 2008; Ouchemoukh, et. al., 2010; Venir, et. al., 2010). However, in most honeys, the percentage of glucose is relatively less than that of the fructose (Almeida-Muradian, et. al., 2013; Buba, et. al., 2013; Manzoor et. al., 2013; EL- metwally, 2015; EI-Sohaimy, et al., 2015; Tesfaye et. al., 2016; Boussaid, et. al., 2018). It is noteworthy to mention here that monofloral honeys derived from the yellow colored flowers (i.e. sunflower, brassica and pigeon pea) contain more glucose than fructose and ‘such’ honeys granulates even itself in the combs or during the storage (Paliwal, 2008). The present study evaluated quantitative analysis of glucose in the commercial and A. dorsata honey. The percentage of glucose was found in the range of 34.63±1.73 to 36.64±1.83 and 30.68±1.53 to 33.99±1.69 in the case of commercial and A. dorsata honeys, respectively (table-2). These results also suggesting that the quantity of glucose in A. dorsata honey was less than that in the commercial honeys (fig. 6). However, overall observations are in the support of earlier works carried out on glucose estimation in the honeys of varieties of bees of different geographical locations (Almeida-Muradian, et. al., 2013; EI-Sohaimy, et al., 2015; Tesfaye et. al., 2016; Boussaid, et. al., 2018).

0

0.5

1

1.5

2

2.5

3

3.5

4

A B C D E-S F-W

Fig. 3. Non-Reducing Sugars (NRS) %

Non-Reducing Sugars (NRS)


0

10

20

30

40

50

60

70

80

A B C D E-S F-W

Fig. 4. Total Reducing Sugars (TRS) %

Total Reducing Sugars (TRS)


0

5

10

15

20

25

30

35

40

45

50

A B C D E-S F-W

Fig.5. Fructose %

True Fructose



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Fructose/Glucose (F/G) ratio

In order to maintain the liquidity in honey, the concentration of fructose must be higher than the glucose (AGMARK, 1984; Ouchemoukh et. al., 2010; Venir et. al., 2010; FSSAI, 2011). EI-Sohaimy, et al., (2015) claimed that if the glucose content is lower than the fructose content, it means that there must be the natural ‘floral’ feeding by the bees which ultimately confirms the quality of honey. In the present study, the F/G in commercial honey noticed ranges from 0.61±0.03 to 1.02±0.05 while that on case of A. dorsata honey from 1.27±0.06 to 1.45±0.07 (table 2). It also mean that only samples A and D of commercial brands and E-S and F-W samples of A. dorsata bees passes the quality criteria as decided by the national and international regulations (AGMARK, 1984; Codex Alimentarius Commission, 2001; FSSAI, 2011). The honeys of A. dorsata derived from both the summer and winter seasons may not granulate easily because of the better F/G ratio while that of sample B and C of commercial honey may show granulation on the longer storage (Fig. 7), thus, supporting the earlier observations (Amir et. al., 2010; Draiaia et. al., 2015).

Dextrose/Water (D/W)

The dextrose/water ratio in honey is an indicator for honey quality (White, et al., 1962; Nour, 1988; Oddo and Piro, 2004; Soria et. al., 2004; Buba, et al., 2013; EI-Sohaimy, et al., 2015). The D/W is considered an appropriate indicator than F/G ratio for the prediction of honey crystallization (EI-Sohaimy, et al., 2015). The least ability of honey crystallization is obtained when the D/W ratio is less than 1.58 while it is faster or completely crystallizes when that ratio is more than

2.0 (White, et. al., 1962; Manikis and Thrasivoulou, 2001; Amir et. al., 2010).

In the present study D/W ratio in commercial honeys has been found in the range of 1.7±0.08 to 1.93±0.10 thus, noticed no significance difference, while that in the case of A. dorsata honeys is from 1.58±0.08 to 1.59±0.07 (table 2). These results clearly indicates that all the commercial honey investigated may crystallize to some extent on the longer storage as compared to the honeys of A. dorsata (fig.8), thus, confirming the earlier observations (EI-Sohaimy, et al., 2015).

Acidity %

The acidity in honey is the result of fermentation of sugars by yeasts. It is well known that during fermentation, glucose and fructose are converted to alcohol and carbon dioxide. Alcohol is further hydrolyzed into acetic acid (Ajlouni and Sujirapinyokul, 2010). As per the honey certifying agencies (AGMARK, 1984; Codex Alimentarius Commission, 2001; FSSAI, 2011) acidity in honey should be within 0.2% by mass. In the present study, all the samples have been reported the acidity within the prescribed limit (table 2 and Fig. 9) and thus, supporting the earlier observation in the case of varieties of honeys derived from the different bee-species and varied floral recourses (Perez-Arquillue, et. al., 1994; Bogandov et al., 1999; Boussaid, et. al., 2018). It also proves that none of the honeys evaluated in this study have been found fermented.

Ash Content %

The ash contents in honey express its richness in minerals and constitute quality parameter too. (Andrade et. al., 1999; Marchini, et al., 2007). In honey, the minerals like potassium, sodium, calcium found in abundant while sulfer, copper, iron,

27

28

29

30

31

32

33

34

35

36

37

38

A B C D E-S F-W

Fig. 6. Glucose %

True Glucose


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

A B C D E-S F-W

Fig. 7. Fructose/Glucose (F/G) ratio

F/G ration


0

0.5

1

1.5

2

2.5

A B C D E-S F-W

Fig. 8. Dextrose/Water ratio

D/W


0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

A B C D E-S F-W

Fig.9. Acidity %

Acidity



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zinc in the meager quantity (Vanhanen et. al., 2011; Boussaid, et. al., 2018). As per the honey certifying agencies the ash contents in honey should be within 0.5% by mass (AGMARK, 1984; FSSAI, 2011). In this study, in all the samples tested, the ash content was found to be below 0.3% by mass (table 2), thus, meets the upper prescribed limit and thus, supports the earlier studies (Cano, et. al., 2001; Estevinho, et. al., 2012). Moreover, A. dorsata honey contains more minerals as compared with the honeys of commercial brands (fig. 10). It also means that ash contents has been associated in a qualitative way with different botanical and geographical origins of the honeys supporting the earlier observations (Felsner et al., 2004).

Total Dissolved Solids (TDS) %

The TDS means water soluble matters and water contents in honey. More is the TDS lesser the water contents. All the samples in the present study, TDS was found to be from 79 to 80% (table 2 and fig. 11). It also means that there would be around 21 to 20% moisture, respectively. On knowing the TDS, we get immediate idea of moisture percent in any raw honey before preceding it for processing for moisture reduction.

Hydroxymethylfurfural (HMF) in ppm

In honey, the HMF is a compound increases during the storage period. It also means that honey gets darker and darker due to action of acids on simple sugars, fructose/glucose ratio; temperature, processing time, storage conditions, etc. (Doner, 1979; Anklam, 1998; Terrab et. al., 2002; Fallico., et. al., 2006; Meda, et. al., 2005; Gomes, et. al., 2010). Besides these factors, the HMF in honey is again based on the bee-species

from which honey has been sourced (Souza et. al., 2007; Almeida-Muradian, et. al., 2013). The HMF estimation in any honey is the parameter that suggests honeys freshness and overheating (Codex Alimentarius Commission, 2001). As per European Economic Community (2002), the HMF limit in honey is 40ppm. All the honey samples analyzed in this study passes the upper limit (80ppm) set as per the quality control authorities in India (AGMARK, 1984; FSSAI, 2011). The range of HMF observed from 23.01±1.15 to 57.83±2.89 in commercial brands while 44.06±2.20 to 46.5±2.33 in the honeys of A. dorsata bee-species (table 2). In this study, some significant variations recorded in the values of HMF in honeys of commercial brand- and A. dorsata bee-species (fig. 12) supporting the earlier studies undertaken on honeys of other bee-species (Anacleto, et al, 2009; Boussaid, et. al., 2018).

Fiehe Test

Fiehe’s test in honey is based on detection of hydroxymethylefurfural which is the result of hydration of fructose. This hydroxymethylefurfural reacts with resorcinol to form the red colour. The test is considered positive and indication that honey is old or over heated (Almeida- Muradian & Matsuda, 2007). In the present study, the Fiehe’s test was negative in the entire honey samples tested (table 2). This confirms that honey samples did not develop hydroxymethylefurfural beyond the permissible limits (AGMARK, 1984; FSSAI, 2011).

Aniline Chloride test

The aniline chloride test is performed to identify the adulteration in honey (AGMARK, 1984; FSSAI, 2011). In the present study, the Fiehe’s test was negative in all the honey samples tested, thus, passes the requirements by the certifying agencies (table 2). All the honey samples were found genuine and of high quality.

CONCLUSION

The comparative physicochemical analysis in some honeys of commercial brands- and that of Apis dorsata bee-species in Vidarbha region of Maharashtra State in India was critically undertaken in the present study. The analytical results clearly suggest that ‘each’ honey has uniqueness that probably seems influenced and attributed with the bee-species-, floral- and geo-specific specifications besides processing technology adopted for honey processing and storage. Most honeys passes though

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

A B C D E-S F-W

Fig.10. Ash Contents %

Ash


78.4

78.6

78.8

79

79.2

79.4

79.6

79.8

80

80.2

A B C D E-S F-W

Fig. 11. Total Dissolved Solids %

TDS


0

10

20

30

40

50

60

70

A B C D E-S F-W

Fig. 12. Hydroxy Methyl Furfural in ppm

HMF



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the specifications set by national and international quality regulatory laws.

Acknowledgements

We are thankful to Mission Samriddhi, Chennai for the financial help. We are also grateful to Centre for Bee Development, Nalwadi, Wardha (MS-India) for availing honey samples and offering the laboratory facilities during the present studies.

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How to cite this article:

Paliwal S. G et al.2021, Comparative Physico-Chemical Analysis In Some Honeys of Commercial Brands With That of Apis Dorsata Bee-Species. Int J Recent Sci Res. 12(07), pp. 42538-42548. DOI: http://dx.doi.org/10.24327/ijrsr.2021.1207.6113

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