Assessment of essential minerals and physico-chemical analysis of floral origins fresh honey produced by Apis mellifera

Honey is one of the best nutritious substances in the world, having different services in the body functions regulation. Ten elements (K, Na, Ca, Co, Cr, Mn, Mo, Ni, Pb, Se) from honey samples were analyzed from 80 different locations of Punjab and ten floras. The aim of the present study was to determine the quality and quantity of minerals and Physico-chemical analysis in honey. A flame photometer was used to measure the concentration of major minerals (K, Ca and Na). The concentration of micro minerals (Co, Cr, Mn, Mo, Ni, Pb and Se) was analyzed using Atomic Absorption Spectrometer. The concentration of macro-elements obtained was as follow (in ppm): K (166-1732), Na (107-418) and Ca (07-99), while the concentration of microelements (in ppm) Co (1-2), Cr (>1), Mn (<1), Mo (1.818), Ni (1.911), Pb (<1) and Se (1.968). The most abundant minerals were potassium, calcium and sodium, ranging between 396-810.5, 17.5-640.63 and 169.88-238.62 ppm, respectively. However, the trace mineral elements of honey were obtained in the order of decreasing Se > Co > Ni > Pb > Cr > Mo > Mn. The findings showed that all the heavy metals like Co, Cr, Ni and Pb were present in trace amounts and close to International Honey Quality Standard. The result of given honey samples represented highest value of moisture (31.23%), color (80 mm pfund), pH (8.23), acidity (72.02 meq/kg), electrical conductivity (0.85 ms/cm) and ash contents (0.83%).

Honey is a divine food and is prescribed as the best treatment for allergies (Purbafrani et al., 2014) and gastric ulceration (Al-Waili et al., 2005;Mandal and Mandal, 2011). The ancient Egyptians and Greeks utilized honey as medicine to cure various diseases like gastrointestinal disorders such as burns, ulcers and healing wounds (Pećanac et al., 2013). It plays an important role in producing blood cells, purifying blood, controlling and promoting blood flow, and providing useful protection against arteriosclerosis. It also has essential antioxidant compounds such as organic acids, aromatic acids, ascorbic acid, catalases, flavonoids, glucose oxidase and compounds of carotenoids (Münstedt et al., 2008;Khalil et al., 2010).
Honey comprises essential nutrients and major minerals that perform vital functions (Bogdanov, 2012;Solayman et al., 2016). Macrominerals have been described as being extracted through flowering plants and land. Still, they have also been analyzed to be derived from human-induced resources because different compounds prevail in the atmosphere and pollute the environment. It has also been recognized that trace minerals obtained from flowering plant products play an important role in human welfare (Pohl, 2009). Honey's role as nutrition gives a better body production and a good accumulation of minerals for bone strength (Chepulis and Starkey, 2008;Solayman et al., 2016). The total number of minerals discovered in honey is about 54 till now. The minerals of honey can be categorized as major and trace minerals and elements that are needed in small amounts for organism metabolism are minor minerals (Pohl et al., , 2012Solayman et al., 2016). Keeping in view the importance of honey, present work had been planned to determine the quality and quantity of minerals in honey of different floral sources and areas of Pakistan and compare mineral levels of fresh Pakistani Honey with International Honey Standards.

Collection of samples
Different samples of honey were collected from the different locations of Punjab, Pakistan. Airtight plastic bottles were used to preserve these samples at room temperature. In order to determine the quality of minerals in fresh honey, 80 fresh honey samples were collected directly from beekeepers at various sites or areas or districts throughout Punjab. The concentration of macro minerals (K, Na, Ca) and micro minerals (Co, Cr, Mn, Mo, Ni, Pb, Se) was analyzed by Flame Photometer and Atomic Absorption Spectrophotometer and was compared with International Standards shown in (Table 1) (Bogdanov, 2012).

Sample preparation
To examine major and trace minerals, 2-3 g of honey was collected in a beaker and weighted on electrical balance. Then added 10 mL of Nitric acid (HNO 3 ) in it and heated at 160 0 C temperature to evaporate HNO 3 . Subsequently, 5 mL of Perchloric acid (HClO 4 ) was added and heated until complete evaporation or colorless fumes

Introduction
Honeybees are biological engineers who convert the raw material (flower nectars, plant excretions of plants licking insects) by modifying them via increasing their specific compounds (Andrade et al., 2018). Honeybees belong to order Hymenoptera, family Apidae and genus Apis. There are 4 species, including Apis mellifera, Apis cerana, Apis dorsata and Apis florea exhibited in Pakistan (Qamer et al., 2013;Sajid et al., 2020). Approximately 25,000 bee species participate in food crop manufacturing and enhancing biodiversity (Gould, 2015). A. mellifera enhances crop amounts and fertilization in plants and shows an important function in the honey farming food supply chain. Honey has become one of the most horticulture commodities available commercially in the world and Pakistan at current times (Munawar et al., 2009;Waghchoure-Camphor and Martin, 2009;Adnan et al., 2014;Anjum et al., 2015). Honey is described as "the delicious substance from the nectar of flowers formed by collection, processing and deposition in honeycombs by the bees". Honey is the most important natural product and graded because of its large dietary contents (330 kcal/100 g) and rapid carbohydrate digestion (Abdulkhaliq and Swaileh, 2017;Sajid et al., 2020).
Honey's chemical constitution and characteristics depend on the kinds of plants visited by honeybees and the environmental factors under which the crops are cultivated. Substances such as toxic metals or other chemicals in honey are created by human actions in that particular area from which they are extracted (Pohl et al., , 2012. Honey has a higher nutrient value based on the high percentage of carbohydrates, including maltose, sucrose, fructose, glucose, oligosaccharides, polysaccharides, enzymes, pigments, aroma compounds, pollen grains, minerals, waxes, vitamins and acids (Haouam et al., 2016;Conti et al., 2018). Honey's botanical and regional source is largely decided by its constitution as the raw material and plant's capital portion strongly affected the honey production (Pohl, 2009). The chemical composition of honey also depends on several ingredients, including nectar sources afflicted by environmental conditions, botanical origin of floral sources, soil conditions and geographical origin (Solayman et al., 2016). Honey composition depends on the various plant types, ecological and climatic factors and effective methods of apiculture. Mostly honey comprises water, sugar, proteins, enzymes, nutrients, flavoring agents, phenolic constituents and more than 200 other compounds in trace amounts (Silva et al., 2009).
A wide range of parameters such as sugar, hydroxylmethyl furfural (HMF), color, pH, humidity, minerals and pollen contents are assumed to differentiate the quality of honey from various regions (Meda et al., 2005;Njokuocha et al., 2019). During honey production, the nature and performance of the honey depend on many ecological factors, including temperature, humidity, and honeydew concentrations in honeycomb. Honey quality depends on different parameters such as humidity, vitamins, amino acids, enzyme activity, electrical conductivity and solid corpuscles (Tuzen et al., 2007;Kiliç Altun et al., 2017;Njokuocha et al., 2019). came out. The remaining solution was just 2 ml in the beaker then placed in the water bath for one hour at 100 0 C for complete digestion. Eventually, the samples were filtered and transferred to 50 mL Eppendorf tube filled with distilled water up to mark. This procedure was carried out in triplicate (Grembecka and Szefer, 2013;Magna et al., 2018).

Standards for solutions
A stock solution of K, Ca, Na, Co, Cr, Mn, Mo, Ni, Pb and Se were 1000 ppm concentration. The working solutions of different concentrations were prepared from this stock solution using the following Equation 1.
where: C 1 = Standard stock solution concentration; V 1 = Standard stock solution volume; C 2 = Working standard stock solution concentration; V 2 = Standard stock solution of working volume.

Standard solution for Sodium (Na)
Working standard solutions of concentration 50 ppm, 75 ppm and 100 ppm were used. These solutions were made following the Formula 2.

Standard for Potassium (K)
Potassium was determined in Apis mellifera fresh honey by using the working standard concentration of 50 ppm, 75 ppm and 100 ppm, respectively.

Standard for Calcium (Ca)
For calculating calcium, the working solution of 50 ppm, 75 ppm and 100 ppm were prepared.

Standard for Cobalt (Co)
Similarly, cobalt (Co) was measured using the 1 ppm, 2 ppm, 4 ppm, and 5 ppm working solution of the same element.

Standard for Chromium (Cr)
The working solution of chromium (Cr) had the concentration of 0.1 ppm, 0.5 ppm, 3 ppm and 5 ppm to measure this element in honey.

Standard for Manganese (Mn)
Manganese (Mn) in Apis mellifera fresh honey was estimated by a working solution of 1 ppm, 2 ppm, 3 ppm and 4 ppm concentration.

Standard for Molybdenum (Mo)
The standard working solution of 0.1 ppm, 0.2 ppm, 0.3 ppm, and 0.5 ppm strength was used to determine honey's molybdenum (Mo) concentration.

Standard for Nickel (Ni)
Nickel (Ni) was determined in Apis mellifera fresh honey using the working standard with the concentration of 0.5 ppm, 1 ppm, 1.5 ppm and 2 ppm, respectively.

Standard for Lead (Pb)
The working standard of 0.1 ppm, 0.4 ppm, 2 ppm, 3 ppm and 5 ppm were used to calculate lead (Pb) in fresh honey samples.

Standard for Selenium (Se)
The honey samples were subjected to analyze selenium (Se) in honey using the working standard of 0.5 ppm, 1.5 ppm, 2.5 ppm and 3 ppm concentration.

Flame photometer
An instrument used in inorganic chemical analysis to determine the concentration of certain metal ions of different macro minerals. Samples were subjected to the Flame Photometer to compare some minerals Potassium, Sodium and Calcium.

Atomic absorption spectrometer
Atomic absorption spectrometry is an analytical technique that measures the concentration of elements. It is so sensitive that it can measure down to parts per billion of a gram in a sample. The technique uses the wavelengths of light specifically absorbed by an element. For analysis of micro minerals, samples were subjected to the Atomic Absorption Spectrometer. Operational conditions are given in Table S1 (Supplementary Material).

Physicochemical analysis
Moisture contents, color, pH values, total acidity, electrical conductivity and ash contents were analyzed according to the method of (Bogdanov et al., 2002).

Statistical analysis
The data of all honey samples were analyzed to the statistically ANOVA one-way using Tukey's test. The data was analyzed at the significance level 0.05.

Calcium (Ca)
Calcium (Ca) concentration recorded the highest 90 ppm in honey sample 52 collected from Narowal, whereas, lowest 7 ppm in Murree honey sample 10. The results revealed that Ca, being a macro element, was not more than 100 ppm in all the samples, far less than International Standard limits of 200-2300 ppm (Table 2).

Sodium (Na)
The amount of Na as macro-mineral was analyzed in all Apis mellifera fresh honey samples collected from various Punjab districts. The concentration of Na recorded highest 418 ppm in honey sample 49 collected from Hafizabad. Whereas the lowest 107 ppm in Sheikhupura honey sample 2. Overall, most honey samples had Na less than International Standard (300-1000 ppm) ( Table 2).
In the present study, ten minerals, including three macro minerals (K, Ca and Na) and seven micro minerals (Co, Cr, Mn, Mo, Ni, Pb and Se) were analyzed. The most abundant elements identified in the present research were K, Ca and Na with concentrations ranging from 166-1732, 7-99 and 107-418 ppm, respectively. And the presence of trace elements (Co, Cr, Mn, Mo, Ni, Pb and Se) in honey samples indicates a nutritional value and potential component for table sweeteners. These three minerals are also discovered in the highest concentrations in honey from the Canary Islands, Spain (Fernández-Torres et al., 2005). (Cantarelli et al., 2008;Pisani et al., 2008;Kiliç Altun et al., 2017;Altunatmaz et al., 2019) found similar results with honey samples from Turkey, Argentina and Italy. According to the results, K was the most prevalent mineral in all local and exotic honey varieties, followed by Na and Ca. (Mesallam and El-Shaarawy, 1987) determined that K and Na were the most abundant elements in the honey. The Dietary Guidelines for Americans 2010 Advisory Committee recognized K as the first significant insufficient nutrient. Its advantages for organic anions are connected and occur in foods such as honey. The values of K vary between 1.18 and 268 ppm. According to our results, greater levels of K in honey samples have already been recorded in Eastern Slovakia (Kováčik et al., 2016). Na is required for appropriate cell activity, plasma volume maintenance, acid-base balance, and nerve impulse transmission (Kiliç Altun et al., 2017). Ca is a necessary vitamin for bone health because it promotes bone calcification (Tuyen et al., 2016).
The trace minerals are also common in honey, and they are present in smaller quantities than major minerals. The trace mineral elements of honey were obtained in order of decreasing Se > Co > Ni > Pb > Cr > Mo > Mn. According to European Union (2002) and Sobhanardakani and Kianpour (2016), the quantities of trace elements Co, Cr, Mn, Mo, Ni, Pb, and Se found in several honey samples in this study are relatively low and fall below acceptable international limits. Although necessary in minute amounts in the body, these trace elements are essential to the normal biological activities of the human body but may be harmful when present in excessive concentrations.
Normally, the most abundant elements in the given honey samples were potassium, calcium, and sodium, ranging between 396.00-810.50, 17.50-640.63 and 169.88-238.62 ppm honey, respectively. The findings of this study revealed that the honey sample was highly rich in macro minerals. (Boussaid et al., 2015) determined the sodium range from 497.54 to 362.55 ppm and 251.34 to 521.22 ppm, almost equal to the current research. All of the fresh honey samples had Ca + levels lower than the international range (200-2300 ppm). (Abdulkhaliq and Swaileh, 2017) investigated potassium and sodium concentrations ranging from 183.86 ppm to 104.66 ppm, which are lower than the previous study. The findings agreed with those of (Agbagwa et al., 2011) and(Joel, 2014), who found potassium dominance in the honey. On the other hand, microelements such as Cobalt, Chromium, Manganese, Molybdenum, Nickel, Lead and Selenium were found in the normal range. Bari samples had the greatest levels 56,57,59,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79 and 80 had Pb as a micro element within the International limit (˂1 ppm) ( Table 2).
The composition of minerals in fresh 80 honey samples were analyzed as shown in Table 3. In macro elements, the maximum range of potassium, calcium and sodium was found in Berseem (Trifolium alexandrinum), Sarson (Brassica campestris) and Sheesham (Dalbergia sissoo) samples i.e, 810.50, 640.63 and 238.62 ppm, respectively. Whereas lowest concentration of these three elements was present in Orange (Citrus xsinensis) ranged 396, 17.50 and 169.88 ppm, respectively.
In micro elements, the optimum concentration of Cobalt is 201 ppm in Berseem while lowest in Clover sample 1.29 ppm. Maximum range of Cr was 1.27 in Beri, its minimum range was present in Sheesham which was 0.22 ppm. Manganese highest value (0.71 ppm) in Sheesham and lowest value (0.37 ppm) in orange. Molybdenum and nikal containing maximal range in Beri and Sarson samples i.e., 0.92 and 0.88 respectively, however its minimal range present in Berseem (0.34 and 0.35 ppm). Samples of orange containing the highest concentration of Lead and Selenium (1.52 and 0.85, respectively) whereas least value (0.39 ppm) of lead was observed in Black locust and minimum selenium concentration (0.38) was found in sarson (Table 3).
The physicochemical parameters of the 80 honey samples were analyzed. The lowest moisture content was present in both Phulai and Sheesham samples which were 15.00%, whereas 31.23% highest moisture contents were measured in Clover. In the present study, variation of color was observed in different honey samples collected from various flora. The value of color variation was observed 19.5, 25.89 and 32.01 mmpfund (white) in Clover, Bhaiker and Black locust, respectively, 34.67 to 50.00 mm pfund (extra light amber) was observed in granda, serson, orange, sheesham, barseem, and phulai samples while bari samples contained 80.00 mm pfund (light amber). pH value of Black locust and Sheesham were neutral i.e., 7.01 and 7.2, respectively, serson samples have basic pH (8.23) while rest of the samples contained acidic pH values. The acidity range vary between 32.29 to 72.02 meq/kg in the observed honey samples in the similar way EC values of given samples was observed between 0.21 to 0.83 ms/ cm. The bari samples have highest (0.83%) and sheesham (0.20%) lowest ash contents (Table 4).

Discussion
The concentration of macro and microelements in bees' bodies varied widely and based on various circumstances, including soil and nectariferous plant varieties, beekeeping procedures, and the physiological and health statuses of The presence of trace amounts of heavy metals such as Pb, Cr, and Mo in all fresh honey samples indicated a clean environment, while the presence of other critical metals indicated the high nutritional value of Pakistani honey. The values of physicochemical parameters of given honey samples were very close to the suggested limit of international standards. of chromium and lead. Cadmium was not found in any honey samples tested. (Mena et al., 1996) determined that cadmium and lead are non-essential components in plant nutrition and one of the most harmful chemicals that accumulate in biological systems. According to mineral analyses, fresh honey is high in nutritional components and low in harmful metals.
The range of moisture contents varied in the given honey sample between 15.00 to 31.23% (Table 3). It depends on various factors like the floral origin of honey, way of storage, and manufacturing techniques. Kumar et al. (2018) was determined 18.37 to 25% moisture contents, which is comparable to the present findings. The moisture contents in honey samples are based on various circumstances like environmental factors, degree of maturity and producing season (Acquarone et al., 2007). For classifying different flora of honey, color determination is an effective technique. The color of honey in the present samples varies 19.5 to 80 mm pfund of clover and bari samples, respectively. (Boussaid et al., 2015;Khalafi et al., 2016;Aazza et al., 2018) determined the range of color 71.72 mm pfund, 36.46-50.73 mm pfund and 19.01-46.7 mm pfund, which is very similar to the present research. pH is an essential parameter while extracting and storing honey. Terrab et al. (2002) determined that pH impacts the stability, texture and shelf life of honey. In the present study, all samples showed acidic pH except black locust, sheesham and serson samples, which showed neutral and slightly basic pH values. Ouchemoukh et al. (2007) found the pH value was comparable to the earlier reported studies in Spanish, Algerian and Portugal honeys, which ranged between pH 3.50 to 6.58.
The range of acidity varied between 32.29-72.02 meq/kg in given honey samples. (Ajlouni and Sujirapinyokul, 2010) found that a high level of acidity causes yeast fermentation of honey sugar. High free acidity levels have been observed to indicate yeast fermentation of honey sugar. Glucose and fructose are generally converted into carbon dioxide and alcohol during fermentation. In the presence of oxygen, alcohol is more hydrolyzed and transformed into acetic acid. De La Fuente et al. (2011) observed that the EC value of honey is directly linked to the mineral's concentration or total ash, organic acid, salts, and protein. This parameter differs widely and depends upon the floral origin of the honey samples. After observing 80 samples of honey collected from different floral species, the EC values ranged between 0.21 to 0.83 ms/cm. Ash content is a parameter used to find out the floral origin (Boussaid et al., 2015). Parviz et al. (2015) and Sousa et al. (2016) found ash contents showed values 0.03 to 0.53 and 0.5-0.8, respectively, which is very close to the present study.

Conclusion
The present study showed the findings concerned honey and their level of micro and macro elements. Some of the variation in the honey sample was seen deviated from the international standard. It has been noted from the honey freshness and purity that the flora affects the micro and macro elements level in the honey composition.