Bee Pollen as a Functional Product – Chemical Constituents and Nutritional Properties

Nutritional supplements play a role in promoting human protein synthesis, fitness recovery, and mental health protection. Pollen is considered a natural food with excellent nutritional value. The use of bee-gathered pollen as a nutritional supplement is now widespread around the world, and it is appreciated for its healing qualities. How - ever, depending on the floral species and the region of origin, pollen has a wide range of nutritional value. It was found that clover pollen had the highest protein content (31.4 g/100 g DM), while the lowest content was observed in maize pollen (21.3%). Eucalyptus pollen had the highest lipid amount (9.49 g/100 g DM), while clover pollen had the lowest content (7.46 g/100 g). Phytochemical analysis showed that eucalyptus pollen occupied the highest total antioxidant activity (67.02%), followed by clover (58.25%) then maize (52.18%), whereas clover pollen had the highest phenolic content (1165 mg GAE/100 gm), compared with the other pollen varieties (949.4 to 1073 mg - GAE/100 gm). The contents of branched-chain amino acids were 29, 33.3, and 38.4 mg/g for maize, eucalyptus, and clover respectively, representing 17.87, 18.44, and 16.53% of total amino acids. The contents of total essential amino acids (EAAs) were 61.8, 73.0, and 83.5 mg/g for maize, eucalyptus, and clover pollen, respectively. Only eucalyptus pollen ultimately met the minimum requirements of EAAs for adults. In contrast, the other two types of pollen contained at least 3–4 limiting amounts of the essential amino acids leucine, lysine, phenylalanine, and valine. In conclusion, pollen is a good, affordable source of nutrients that can be utilized as beneficial dietary supplements for human health.


INTRODUCTION
In the anthers of angiosperm stamens, pollen, a male floral gametophyte, is a significant source of chemicals with potential health advantages (Sadeq et al., 2021). It has a high protein content and a reliable supply of bioactive substances that are typically used to identify the nutritional value of pollen (Mayda et al., 2020). Hence, it is consumed by humans as a nutritional supplement and is recognized as a superior functional food ingredient (Kostić et al., 2020). As reported by Food and Agriculture Organization (FAO), pollen may contain more than 16 amino acids (Taha et al., 2019).
recently been discovered to modulate the gut microbiota to support gut health (Kalaycıoğlu et al., 2017;Wu et al., 2019).
Consumers who are concerned about their health these days prefer to eat value-added goods that allow them to replace traditional food ingredients with components that have a high nutritional value in order to enrich the already existing processed goods. The human diet should include energy and other essential nutrients in amounts that fulfil criteria for physical and mental development and health (Kieliszek et al., 2018). Pollen can be supplemented in a healthy diet, offering a significant daily intake of nutrients because of its superior nutrient profile. Even elderly persons can consume it as a stand-alone diet or supplement, because of its nutritionally balanced composition and low-calorie count (Campos et al., 2021).
Branched-chain amino acids (BCAA) leucine, isoleucine, and valine which make up 33% of human muscle protein, are also important for protecting mental health and promoting protein synthesis (Günalan et al., 2022). According to the situation, the effects of BCAA, which are necessary amino acids, on metabolic health, have been described as both negative and beneficial (Bishop et al., 2022). Only outside food can be used to obtain BCAA because the body cannot, or can only do so in very little amounts. Leucine is one of the branched-chain amino acids and makes up a significant fraction of the amino acids in proteins. It is also the principal by-product of protein catabolism. The Office of Dietary Supplements at the National Institutes of Health advises taking 10-20 grams of BCAA a day, either as supplements or in the form of protein that is eaten (National Institutes of Health Office of Dietary Supplements, 2017).
More attention is paid to the production of pollen in most countries, since it has beneficial effects on the economy, society, and environment. The top producing countries of pollen are Australia, Argentina, Brazil, China, Spain, and Vietnam (Lorini et al., 2020). The main composition of pollen has drawn the attention of researchers across a variety of disciplines, including plant physiology, biochemistry, nutrition, food biotechnology, and even material science. More than 200 different compounds have been identified in pollen, demonstrating the wide variety in its chemical composition (Komosinska-Vassev et al., 2015).
The main goal of this study was to see if certain types of pollen collected in Egypt might be used as nutritional supplements. For this reason, the chemical composition, which comprises carbohydrates, proteins, lipids, fiber, and ash, was determined. The total phenolic content and antioxidant potential of pollen from maize, clover, and eucalyptus was also checked. In addition, the nutritional value of pollen samples was demonstrated by using chemical scores for amino acids, specifically branched-chain amino acids "BCAA", following the Food and Agriculture Organization's recommendations for human nutritional needs of pollen samples from eucalyptus, clover, and maize. Finally, the ability of various pollen types to give vital nutrients for individuals' daily needs was examined.

Chemicals
The chemicals and solvents were high purity. Folin-Ciocalteu solution, Gallic acid, and all chemicals were obtained from El-Nasr Pharmaceutical Chemicals Company, Cairo.

Pollen samples
As shown in Table 1, three pollen varieties were selected. Eucalyptus pollen was gathered from Nasr City, Cairo Governorate, Egypt, while maize and Egyptian clover pollen were collected from Bassioun Center, Gharbia Governorate, Egypt. For collecting pollen, Italian and Carniolan hybrids were used in the present study. During the collecting phase, a typical pollen trap was placed to the hive entrance. Fresh pollen loads were stored in paper bags and traps were regularly emptied. Following that, pellets underwent cleaning and color-based separation. The collected pollens were kept in polyethylene bags at -20 °C until further tests.

Proximate analysis
Proximate analysis (moisture, crude proteins, crude fats, crude fiber, and ash contents) of pollen was conducted using the standard methods of reference (Official methods of analysis of the association, 2016).

Energy values
The energy value was calculated as the following equation (cal/100g-1).

Preparation of pollen extraction
The extraction was performed following the steps specified in the reference (Keskin & Özkök, 2020). Using a mechanical shaker (150 rpm) for 12 hours at room temperature, three grams of each pollen sample were added to 20 mL of ethanol absolute. At 3000 rpm, the extract was centrifuged for 15 minutes. A final amount of 30 mL of ethanol absolute was then added after filtering. The extracts were utilized to determine the antioxidant capability and total phenol content.

Total phenolic content (TPC)
Folin-Ciocalteu colorimetric method was applied for evaluating the total phenolic content (TPC) of samples (Amarasinghe et al., 2021), where 2.5 mL (10 %) Folin-Ciocalteu reagent, was added to 0.5 mL extract. The mixture was settled for 8 min. in the absence of light, then 2 mL of Na 2 CO 3 (7.5%) was added and allowed to stand at room temperature for 60 min in the dark. Absorbance was read at 765 nm using a UV-VIS spectrophotometer (Cary 50 Bio Varian, Australia). The results were calculated as mg Gallic acid (GA)/100 g of dry matter (DM).

Antioxidant activity
The antioxidant compounds of pollen samples was evaluated by the DPPH radical-scavenging method according to reference (Sakooei-Vayghan et al., 2020), the DPPH radical-scavenging technique was used to assess the antioxidant components in pollen samples. To 2 mL of extract, 0.16 mM DPPH solution was first added. After being vortexed, the reaction mixture was incubated for 30 min. in the dark. To quantify the absorbance, a UV-visible spectrophotometer was used at 517 nm (Thermo Electron Corporation, Beverly, MA, USA).
This equation was used to compute the % inhibition of the radical scavenging activity: (3) where: AS -absorbance of the sample extract; AC -absorbance of control sample.

Amino acids
An automatic amino acid analyzer was used to determine the content of amino acids (AAA 400 INGOS Ltd. Czech Republic). About 100 mg of pollen samples were hydrolyzed in 10 mL of 6 M HCl in a sealed tube at 110 °C for 24 h. After hydrolysis, the acid was evaporated at 80 °C in a vacuum evaporator under reduced pressure. The HCl-free residue was dissolved in 2 mL of loading buffer (0.2 M, pH 2.2) to inject into the apparatus (Block et al., 2016).

Amino acid score
To calculate amino acid scores (%) the observed value of an essential amino acid (g/100g of protein) was compared to the reference pattern as provided by FAO, 2017 according to the following formula: (4) where: P -refers to amino acid in tested protein; R -refers to amino acid requirements for adult humans.

Analytical statistics
Every measurement was taken three times using the Duncan's Multiple Range test. Using the SPSS statistics package application, the data was statistically analysed (SPSS software version 21). The data were analyzed utilizing quantitative approaches using a one-way ANOVA to compare any significant differences between the means. Table 2 provides an overview of the chemical composition of the pollen samples under investigation. The results illustrated signifi cant diff erences between the chemical contents of the three tested pollen. The highest moisture content was observed in the pollen of clover (26.12%), followed by maize (23.21%), while the least value of moisture was detected in eucalyptus pollen (19.85%) with signifi cant diff erences (Figure 1).

Nutritional value of pollen samples
Pollen's protein concentration is seen as a clear indicator of how nutrient-dense it is. Proteins are the largest component in pollen after carbohydrates. It is highly (p < 0.05) varying in pollen collected from diverse plant sources ( Figure 2). The results showed that the protein contents of pollen from maize, eucalyptus, and clover were, respectively, 21.     Figure 3, presented the crude lipid content percentages of pollen. The highest lipid content (9.94%) was found in the pollen of eucalyptus, followed by maize (8.67%), while the least value was recorded in clover pollen at 7.46%. Moreover, from Figure 4, the average ash contents for the three tested pollen types were determined as 3.54, 5.33, and 5.55%, respectively for maize, clover, and eucalyptus. These values are within the range revealed by Kieliszek et al., 2018. All pollen samples of diff erent plant sources contain a moderate amount of ash. Our results were higher than that of Almeida-Muradian et al., 2005 who found that the ash content of pollens had an average of (2.2%).
Over the plant sources, pollen of clover contained the highest (p < 0.05) fi ber (6.78%) followed by eucalyptus (5.51%), while the lowest fi ber was found in maize pollen 5.14% ( Figure 5).
The majority of pollen's dry weight, or approximately two-thirds of it, is made up of    (Table 2).
Moreover, as reported in Table 2, FAO, 2011 recommends that adults (>18 years), for an individual of 75 kg consume, 50 g/day of protein, 80 g/day of fat, 30 g/day of fiber and 320 g/day of carbohydrates including fiber. For these individuals, a 25 g of eucalyptus pollen (the equivalent of one soupspoon) can provide up to 8.6, 2.49, 3.68 and 3.95% of the recommended daily intake of protein, fat, fiber, and carbohydrates, while it can provide up to 11.6, 1.72, 4.18, and 3.22%, respectively, for pollen of clover. Meanwhile, it is provided 8.18, 2.08, 3.29, and 3.99% of daily requirement intake for adult humans form the main components of maize pollen, respectively. Figure 7 shows that the fresh pollen samples had an energy value ranging from 307.31 to 342.29 kcal. The energy has been primarily attributed to its higher sugar content which is easily digestible (White, 1980). The enzymes break down polysaccharides into the necessary energy sources monosaccharaides and disaccharides.
Polyphenols have great importance, because they help the human body to fight against diseases. Determining the functions of foods or dietary supplements used as antioxidants has grown crucial, especially in recent years as the prevalence of several diseases such as cancer has increased  three types of pollen collected by honeybee colonies. The tested pollen contains considerable amounts of phenolic compounds. Total phenolic ranged from 949.4 to 1165 mg/100 g of the pollen samples. The largest amount was detected in the pollen of clover (1165 mg/100 g), followed by maize pollen (1073 mg/100 g), while the lowest value was recorded for the eucalyptus sample (949.4 mg/100 g), with statistically signifi cant diff erences (p < 0.05) between all pollen types as shown in Figure 8 The DPPH free radical scavenging activity is one of the most widely used assays for assessing pollen antioxidant properties. Figure 8 shows the ascorbic acid equivalents and depicts the antioxidant capabilities of the pollen under consideration. Eucalyptus pollen exhibited the highest DPPH scavenging activity (67.02%), whereas maize pollen had the lowest percentage (52.18%), with signifi cant variations. According to the fi ndings, the high phenolic content may be responsible for the free radical scavenging eff ect. Pascoal et al. (2014) showed an association between pollen's total phenolic content and antioxidant activity, which supported the conclusions of this study.

Amino acid profi le
The content of individual amino acid in the eucalyptus, Egyptian clover, and maize pollen were investigated and are presented in Table 3. From the Table, at least 17 amino acids, including nine essential amino acids (EAAs) and eight nonessential amino acids (NEAAs) were determined. Tryptophan was not determined, presumably due to the limitation of the acid hydrolysis process.
Amongst essential amino acids, leucine shows the highest quantities, ranging from 11.7 to 16.9 mg/g DM and making up 7.21-8.19% of total amino acids. Valine (9.7-11.7 mg/g), lysine (9.2-11.7 mg/g). Moreover, moderate amounts of other essential amino acids were observed in pollen samples. Among non-essential amino acids, glutamic and aspartic acid were found predominant In clover pollen, proline followed by glutamic and aspartic acid present 16.7, 11.02, and 10.25 % of total amino acids (39.4, 25.6 to 23.8 mg/g DM). Additionally, in maize pollen, proline followed by aspartic then glutamic acid present 12.33, 11.16, and 10.36%, respectively, of total amino acids (20, 18.1, and 16.8 mg/g DM). Meanwhile, moderate amounts of other nonessential amino acids were observed in both pollen samples.
Leucine, isoleucine, and valine are branchedchain amino acids (BCAA) that play a role in stimulating protein synthesis and constitute 33% of muscle protein in humans, recovery processes from exercise, and protecting mental health. From Table 3, BCAA was 29, 33.3, and 38.4 mg/g of maize, eucalyptus, and clover pollen, respectively, it is present at 17.87, 18.44, and 16.53% of total amino acids.
For total amino acid contents, pollen showed variations as presented in Table 4. The average total amino acid amount was 191.6 g/100g DM. The highest values of total amino acids (232.2 mg/g DM) were obtained from clover pollen, followed by pollen of eucalyptus (180.5 mg/g DM). Meanwhile, the lowest values (162.1 mg/g DM) were recorded for maize pollen.
Based on the total measured amino acids, the amounts of total essential amino acids (EAAs) in  Note: *total amino acids, **TEAs % of total essential amino acids/total amino acids, ***TNEAs % of total nonessential amino acids/total amino acids.  9% (FAO, 1991). The nutritive value of proteins for any biological function is limited by the relative proportions comprising EAAs. Adult humans may benefit nutritionally from high quantities of EAAs. These findings support the notion that pollen is a "healthy" source of superior protein.
An indication for determining the effectiveness of proteins and amino acids needed for various demographic groups is amino acid scoring. It is based on the idea that body cells cannot synthesise proteins unless they are provided with the necessary amino acids through nutrition. In light of the amount of essential amino acids contained in food material, the amino acids score reflects the protein quality, and amino acids present in low concentrations are referred to as limiting amino acids (FAO, 2013). Therefore, concerning the minimal amino acid requirements of humans FAO/WHO/UNU (FAO, 2007) the chemical scores of the EAAs of pollen can be calculated, reflecting the nutritional value of pollen for humans Table 5. According to the chemical scores of essential amino acids, only the essential amino acid content of the pollen eucalyptus met the requirements of adult humans ( Table 5). The pollen of maize had three limiting amino acids (leucine, lysine, and phenylalanine). In addition, four limiting amino acids were found in the clover pollen (lysine, leucine, methionine, and valine). The lowest values for chemical scores indicate that there were fewer amino acids present than was necessary.

CONCLUSIONS
In this research, three pollen varieties, eucalyptus (Eucalyptus sp.), Egyptian clover (Trifolium alexandrinum), and maize (Zea mays) were evaluated, for the proximate chemical composition, energy value, amino acid, and antioxidants contents. The pollen samples' energy values ranged from 415.98 to 429.19 kcal. Egyptian clover has a greater total amino acid content (232.2 mg/g) than eucalyptus (180.5 mg/g) and maize (162.1 mg/g). Leucine, lysine, valine, threonine, and phenylalanine were the highest essential amino acid concentrations in the examined pollen samples. Compared to other non-essential amino acid types, proline, glutamic, and aspartic acids were found to have the highest concentrations in both pollen samples. The minimum necessary amount of essential amino acids for adult humans was entirely satisfied by the eucalyptus pollen.