Bee Bread as a Promising Source of Bioactive Molecules and Functional Properties: An Up-To-Date Review

Bee bread is a natural product obtained from the fermentation of bee pollen mixed with bee saliva and flower nectar inside the honeycomb cells of a hive. Bee bread is considered a functional product, having several nutritional virtues and various bioactive molecules with curative or preventive effects. This paper aims to review current knowledge regarding the chemical composition and medicinal properties of bee bread, evaluated in vitro and in vivo, and to highlight the benefits of the diet supplementation of bee bread for human health. Bee bread extracts (distilled water, ethanol, methanol, diethyl ether, and ethyl acetate) have been proven to have antioxidant, antifungal, antibacterial, and antitumoral activities, and they can also inhibit α-amylase and angiotensin I-converting enzyme in vitro. More than 300 compounds have been identified in bee bread from different countries around the world, such as free amino acids, sugars, fatty acids, minerals, organic acids, polyphenols, and vitamins. In vivo studies have revealed the efficiency of bee bread in relieving several pathological cases, such as hyperglycemia, hyperlipidemia, inflammation, and oxidative stress.


Introduction
Bee products are natural products that are either secreted by the body of bees through glands, i.e., venom, wax, and royal jelly, or collected and processed by the bees, i.e., nectar, pollen from flowers, and resin [1]. In recent years, research trends for bioactive molecules of natural origin have led to a proliferation of studies on bee products, including honey, propolis, royal jelly, bee bread, and bee pollen [2]. Bee bread is a unique bee product that is poorly known because, until a few years ago, beekeepers did not know the appropriate method for collecting this product without partially destroying the hive [3].
For honey bees, the nutrients necessary for the survival and maintenance of the health of colony populations come from two main sources, namely nectar/honeydew and pollen from flowers. Nectar and honeydew provide carbohydrates and pollen provides other dietary needs, such as lipids, proteins, vitamins, and minerals [4]. However, bees do not directly consume nectar/honeydew or pollen; in both cases, they induce biochemical changes, such that the nectar/honeydew turns into honey and the pollen turns into bee bread [5]. The process of making bee bread begins with the collection of pollen from flowers, which is then mixed by bees with the nectar or honey and saliva containing digestive enzymes [6]. At this stage, the pollen from flowers is transformed into bee pollen, stored in the pollen basket in the bee's hind legs, and carried to the hive, where the non-flying bees fill the cells of the hive with a mixture of bee pollen and honey. Then, a thin layer of wax is added to protect the mixture from oxygen. An anaerobic lactic fermentation process takes stored in the pollen basket in the bee's hind legs, and carried to the hive, where the nonflying bees fill the cells of the hive with a mixture of bee pollen and honey. Then, a thin layer of wax is added to protect the mixture from oxygen. An anaerobic lactic fermentation process takes place, and the bee bread is produced (Figure 1). This type of lactic fermentation makes the bee bread more digestible and enriched with new nutrients [7]. Studies on the chemical composition of bee bread have shown that it is generally made up of water, protein, free amino acids, carbohydrates, fatty acids, and other bioactive molecules. This composition varies from one region to another depending on the honey plants' climatic conditions and seasonal variations. All these conditions make bee bread a potential functional food with distinct bioactive molecules [8,9]. In the life of bees, there are times when they are highly active, such as when colonies are reproducing or during periods of high wax production. At these times, the demand for pollen rises, and a decrease in pollen inflow (fall and early spring) or the existence of pollen of poor nutritional quality will have serious consequences for the colony. This directs bees to rely on their bee bread store for their needs [10]. In this review, we will underline the importance of bee bread, not only for bee food and the good health of the hive, but also its nutritional importance for humans and its richness in bioactive molecules with protective or curative effects.

Methodology
A comprehensive literature search was performed from October 2021 to January 2022. The search engines "Google-Scholar, Web of Science, Scopus, Science-Direct and Pub-Med" were used to collect information on bee bread, its chemical composition, and its biological activities in vitro and in vivo using keywords such as "composition of bee bread", "bee bread and patients", the antimicrobial effect of bee bread", "antiviral effect of bee bread", "bee bread and rats", and"effects of bee bread". The papers were collected, examined for relevance, and then their general ideas were summarized.

Free Amino Acids
Several studies have investigated the free amino acids content of bee bread. For instance, Malaysian bee bread was analyzed in two studies published by Mohammad et al. [11] and by Othman et al. [12]. In the first study, four multifloral bee breads were analyzed using chromatographic separation; the botanical origins of the samples were Mimosa pudica, Sphagneticola trilobata, Bidens pilosa, Cassia sp, Areca catechu, Peltophorum pterocarpum, Phaleria capitata, Cassia siamea, Citrus aurantifolia, and Ageratum conyzoides. In the second In the life of bees, there are times when they are highly active, such as when colonies are reproducing or during periods of high wax production. At these times, the demand for pollen rises, and a decrease in pollen inflow (fall and early spring) or the existence of pollen of poor nutritional quality will have serious consequences for the colony. This directs bees to rely on their bee bread store for their needs [10]. In this review, we will underline the importance of bee bread, not only for bee food and the good health of the hive, but also its nutritional importance for humans and its richness in bioactive molecules with protective or curative effects.
On the contrary, in a study published by Mohammad et al. [11], a sugar profile revealed that glucose was the major free sugar contained in four Malaysian multifloral bee breads analyzed by HPLC coupled with an evaporative light scattering detector (ELSD); the values ranged between 10.270 ± 0.140 g/100 g and 12.397 ± 0.980 g/100 g.The second highest sugar content was sucrose, ranging from 0.595 ± 0.000 g/100 g to 2.094 ± 0.141 g/100 g; then fructose, ranging from 0.396 ± 0.000 to 1.488 ± 0.140 g/100 g; and maltose, ranging from 0.694 ± 0.140 to 1.994 ± 0.000 g/100 g) ( Table 1). This high variability could be explained by numerous factors, such as the harvesting time, the botanical origin, and the decomposition of sugars by lactic acid bacteria [23].

Fatty Acids
Fatty acids are among the most important compounds in bee bread. Bakour et al. [20] analyzed Moroccan bee bread using gas chromatography coupled with flame ionization detection (GC-FID), and they identified and quantified fourteen saturated and eleven unsaturated fatty acids, in which the highest levels were represented by α-linolenic and arachidonic acid, 25 ± 1% and 23.2 ± 0.5%, respectively. On the other hand, Turkish bee bread was analyzed by Kaplan et al. [24,25] in two studies using GC-MS analysis and GC-FID analysis, and the results indicated that seventeen saturated fatty acids and twenty unsaturated fatty acids were detected in eight monofloral samples in the first study, and five monofloral samples in the second study. In a recent study published by Drancaet al. [21], the GC-MS analysis of Romanian bee bread showed a content of thirty-seven fatty acids, with a percentage of 23.13% for saturated fatty acids and 76.87% unsaturated fatty acids (Table 1). Keeping in view the previous investigations, it is well recognized that individual fatty acids are the same in different bee breads; however, their concentrations vary depending on the geo-climatic conditions, the floral origin of pollen flowers, or even within the same species of the same growth site [26,27].
It has been documented that fatty acids play a crucial role in the nutrition, reproduction, and development of bees [28]. Owing to their antibiotic functions, such as antibacterial activity and antifungal activity, fatty acids, especially lauric, myristic, linoleic, and linolenic acids, contribute greatly to bee colony hygiene [29].

Minerals
The minerals from soil are carried into plants by their roots, and into bee bread through the pollen of flowers, nectar, or honeydew. Bee bread is one of the richest hive products in terms of macro and microelements. Moroccan bee bread was analyzed by Bakour et al. [20],who showed that potassium (338 ± 8 mg/100 g) was the predominant mineral followed by phosphorus (251 ± 4 mg/100 g), calcium (198 ± 4 mg/100 g), magnesium (61 ± 2 mg/100 g), iron (27.3 ± 0.3 mg/100 g), sodium (14.2 ± 0.1 mg/100 g), zinc (3.31 ± 0.04 mg/100 g), and manganese (2.6 ± 0.1 mg/100 g). The mineral content of bee bread may vary depending upon the agro-climatic conditions, specific melliferous plants, growth site, and harvesting season. Moreover, the methods used for sample collection and conditioning storage could affect the concentration and the mineral composition of bee bread [11].
In Malaysian bee bread, the most abundant mineral was potassium (6524.9 ± 610.6 mg/kg), followed by phosphorus (6402.28 ± 163.29 mg/kg), and magnesium (1635.4 ± 215.4 mg/kg) [11]. Similarly, Eleazu et al. [30] reported that calcium, phosphorus, potassium, magnesium, iron, zinc, and manganese were detected at high concentrations in Malaysian bee bread. In addition, the ICP-MS analysis of Serbian bee bread samples showed their richness in sodium, magnesium, potassium, calcium, manganese, selenium, zinc, and copper. The most abundant minerals were potassium, calcium, and magnesium [31] (Table 1). Mineral elements are involved in the functioning of many biochemical and physiological processes in humans. For instance, a diet rich in potassium, an oligoelement that presents highly in bee bread samples of different floral and geographical origins, improves blood pressure and prevents cardiovascular diseases in both human and animal models [32,33]. Phosphorus, the sixth most abundant metal in the human body, is closely related to nucleic acid synthesis, enzyme activity, skeletal and non-skeletal muscle function, bone mineralization, energy, and lipid metabolism [34].
Micro-elements, including antioxidant minerals, such as zinc, iron, manganese, selenium, and copper, act as cofactors of many antioxidant and non-antioxidant enzymes. Therefore, they are effective components of metabolism and human body integrity [35].

Organic Acids
Organic acids are responsible for the microbial and digestive properties of bee products, and they are also used as food preservatives [36]. The information available in the literature on the organic acid content of bee bread remains limited. In a recent study conducted by Dranca et al. [21], the HPLC-DAD analysis of Romanian bee bread revealed the presence of the following organic acids: gluconic acid (79.2 g/kg), formic acid (6.75 g/kg), acetic acid (10.7 g/kg), propionic acid (1.3 g/kg), and butyric acid (0.33 g/kg). While in the Moroccan bee bread studied by Bakour et al. [20], the only organic acid found was oxalic acid. Organic acid content in bee bread varies depending upon the botanical origin (age, species, and vegetable tissue) ( Table 1). Since bee bread is a bee pollen-derived product, its organic acid content can be compared to that of bee pollen. Kalaycioglu and coworkers [36] analyzed six multifloral and four monofloral bee pollen samples collected from different regions of Turkey and reported that gluconic acid (5.9-32 g/kg), lactic acid (0.72-1.2 g/kg), tartaric acid (0.17-0.30 g/kg), succinic acid (0.092-0.40 g/kg), and citric acid (0.19-0.31 g/kg) were the most quantified organic acids. Gluconic acid, a main organic acid in bee bread, exhibited potent antibacterial activities against anaerobic (Porphyromonasgingivalis), Gram-negative (Escherichia coli), and Gram-positive bacteria (Staphylococcus aureus), as well as achieved effective biofilm penetration. Importantly, polymyxin B associated with gluconic acid exhibited good biofilm penetration and high antibacterial action [37]. In addition, acetic acid, another valuable component found in bee products, has been documented to have anti-fungal effects against toxigenic Aspergillus flavus [38]. Therefore, bee bread extracts could be used as promising antibiotic and natural food preservative agents.

Vitamins
Vitamins are organic compounds tha thave several biochemical roles in growth, the regulation of mineral metabolism, and cell differentiation; some also possess antioxidant activity and others play the role of precursors of enzyme cofactors [39]. According to the literature, vitamin content is highly dependent on the plant origin of bee bread. Loper et al. [40] conducted a comparative study of the vitamin content in bee pollen and bee bread samples after seven, twenty-one, and forty-two days from the same plant source (Prunus dulcis), and they found that the vitamin content decreased by passing pollen from the flowers throughout the forty-two days of storage. Until present, few studies (Table 1) have been conducted on the vitamin composition of bee bread. Bakour and coworkers [20] reported that Moroccan bee bread contained α-tocopherol and δ-tocopherol with concentrations of 10.5 ± 0.8 and 0.4 ± 0.04 mg/100 g of bee bread (BB), respectively. Vitamin quantification was also the subject of other investigations. In fact, Salma Malihah et al. and Chinedum et al. [11,20,31] studied Malaysian bee bread and revealed the presence of vitamin C (11.52 mg/100 g), α-tocopherol, δ-tocopherol, vitamin A (146.8 mg/100 g), vitamin E (46.27 mg/100 g), thiamine (6.20 mg/100 g), and riboflavin (1.50 mg/100 g) ( Table 1).
Since there is no specification for the vitamin content in bee bread, a bee pollenderived product, the specifications for the vitamin composition of bee pollen suggest contents between 0.6 and 2 mg/100 g for riboflavin and between 0.6 and 1.3 mg/100 g for thiamine [41], thus allowing us to highlight bee bread as a dietary supplement and natural source of valuable vitamins.

Polyphenolic Composition
Owing to the development of advanced techniques for the separation and purification of molecules, such as gas chromatography (GC) and high-performance liquid chromatography (HPLC), as well as other identification techniques, such as mass spectroscopy (MS), thin-layer chromatography (TLC), and other combined techniques, several bioactive components have been identified in bee bread from different geographical origins (Table 1).
Polyphenol compounds are secondary metabolites produced by plants in response to different biotic and abiotic stress conditions [42]. These chemical compounds are divided into two groups: hydrophilic antioxidants, such as vitamin C and phenolic acids, and lipophilic antioxidants, such as carotenoids, tocopherols, and flavonoids [43]. The high content of antioxidants in bee bread is responsible for its bioactivities. For instance, Oltica and coworkers [44] found that there was a good correlation between the content of antioxidants (polyphenols, flavonols, flavones, and flavanones) and the antioxidant activity of scavenging free radicals carried out by 2,2-diphenyl-1-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) tests, which explains their contribution to the antioxidant effects of bee bread extracts. It is well known that the soil nature, geographical conditions, and plant species highly influence the phytochemical composition of functional foods, including bee product extracts [7]. Furthermore, the solvent and extraction method used influence the quantity and the selectivity of the extract components [45]. Chemical variability has been observed in the composition of bee bread extracts of different geographical and floral origins.
The phenolic screening of multifloral Moroccan bee bread by HPLC showed the presence of thirteen phenolic compounds, where isorhamnetin-O-hexosyl-O-rutinoside was the most representative [20]. Similarly, Sobral et al. [46] analyzed six bee bread samples harvested from different apiaries located in the north of Portugal. High-performance liquid chromatography-diode array detection coupled to tandem mass spectrometry (HPLC-DAD-ESI/MS) analysis revealed the presence of thirty-two phenolic compounds, of which flavonol derivatives, mainly quercetin, kaempferol, myricetin, isorhamnetin, and herbacetin glycoside derivatives, were the main quantified antioxidant molecules.
Flavonoids, phenolic acids, and other antioxidant components are recognized as effective functional molecules, displaying numerous bio-pharmacological potentialities. In fact, several researchers suggest the use of bee bread as a common food preservative and bio-functional dietary supplement, or as a therapeutic strategy in the prevention of oxidative stress and associated ailments, such as cardio-metabolic, neurodegenerative, and cancerous diseases [46,51,52].

Microorganisms
Bee bread is a complex product that contains different microorganisms, such as bacteria and fungi, involved in the production process of bee bread to enrich it with newly produced nutrients. The micro-organisms found in bee bread originate from the bee's saliva, added to pollen as a raw matter of bee bread. This microbiome plays an important role in the sustenance of larva and in adult bee's health, including in making carbohydrates digestible and providing different nutrients with added value. Furthermore, the richness of bee bread in microorganisms can give it the properties of a probiotic product [53]. A study conducted by Dimov et al. [54] showed the following fungi composition in bee bread: Cladosporium, Penicillium, Alternaria, Monilinia, Sclerotinia, Ascosphaera, Toxicocladosporium, Botrytis, Pseudopithomyces, Camerosporium, Paraconiothyrium, Podosphaera, Golovinomyces, Paraphaeosphaeria, Periconia, and Septoriella. In addition, it was found that bee bread was rich in lactic acid bacteria, such as Apilactobacilluskunkeei, Lactiplantibacillus plantarum, Fructobacillusfructosus, Levilactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus musae, Lactobacillus crustorum, and Lactobacillus delbrueckii [54][55][56].

Enzymes
Bee bread contains high amounts of enzymes, of which amylase, invertase, phosphatases, transferases, and glucose oxidase are the most important. Invertase and glucose oxidase are mainly produced in the hypopharyngeal glands, and are added by the bees to complete the honey maturation process. However, some enzymes, such as catalase and phosphatase, originate from nectar, honeydew, or pollen [57]. Enzyme cofactors, such as biotin, glutathione, and NAD, have also been found in bee bread. These enzymes can transform high molecular weight compounds into other low molecular weight molecules, such as polysaccharides and proteins. This makes bee bread more digestible than bee pollen [58,59].  Aspartate, Glutamate, Asparagine, Serine, Glutamine, Histidine, Glycine, Threonine, Arginine, Alanine, γ-aminobutyric acid, Tyrosine, Cysteine, Valine, Methionine, Tryptophan, Phenylalanine, Isoleucine, Leucine, Lysine, and Proline

Antioxidant Capacity of Bee Bread
As already mentioned, antioxidant molecules are one of the most important constituents of bee bread. Several studies have evaluated the antioxidant capacity of these molecules using spectrophotometric techniques. Akhir et al. [61] and Othman et al. [12] tested the antioxidant capacities (DPPH, ABTS, and FRAP tests) of different Malaysian bee bread extracts (hexanoic, ethanolic, and distilled water extracts). The authors showed that the solvents used influenced the bioactivity of the bee bread. The most potent extract was the ethanolic extract, followed by the hexanoic extract and then the aqueous extract. Similarly, 15 Colombian bee bread samples were analyzed by Zuluaga et al. [62] for their antioxidant activity. The authors revealed that all the extracts had good antioxidant activity with FRAP and TEAC methods; their total flavonoid content ranged between 1.9 and 4.5 mg equivalent of quercetin/g, while their total phenolic content ranged between 2.5 and 13.7 mg equivalent of gallic acid/g.
The antioxidant activity of Moroccan bee bread was analyzed in three studies conducted by Bakour et al. [20,63,64] using three different solvents for extraction (ethanol, ethyl acetate, and methanol). The results showed that the antioxidant activity evaluated by three tests-DPPH, ABTS, and reducing power-had the lowest recorded values of IC 50 /EC 5050 forthe ethanolic extract, followed by the methanolic extract and then by the ethyl acetate extract.

The Antitumoral Effect of Bee Bread
The antitumor activity of bee bread has been evaluated in vitro in two studies, the first performed by Markiewicz-Żukowska et al. [50], in which different ethanolic extracts of bee bread samples collected from Poland were tested for the viability of the glioblastoma cell line (U87MG) after 24 h, 48 h, and 72 h. The results showed that bee bread ethanolic extract reduced the viability of cancer cells with percentages ranging from 49% to 66%. This inhibitory effect appeared mainly after the passage of 72 h of contact.
The second study is that of Sobral et al. [46],who evaluated the antitumor activity of bee bread from northeastern Portugal against different human tumor cell lines-NCI-H460 (non-cellular lung cancer), HepG2 (carcinoma hepatocellular), HeLa (cervical cancer), MCF-7 (breast adenocarcinoma)-and also against non-tumor liver cells (porcine liver cells, PLP2). The bee bread samples showed low to moderate cytotoxicity ranging from >400 to 68 µg/mL; however, none of the bee bread samples showed toxicity against normal cells (Table 2).

Hypotensive Effect
The hypotensive effect of bee bread was evaluated by Nagai et al. [69] using enzymatic hydrolysates from bee bread prepared by three proteases: pepsin, trypsin, and papain. The obtained hydrolysates showed inhibitory activities for the angiotensin I converting enzyme of 1.48 mg protein/mL obtained by the hydrolyzate of pepsin, 2.16 mg protein/mL obtained by the hydrolyzate of trypsin, and 5.41 mg protein/mL obtained by papain hydrolysate (Table 2).

The Inhibition Effect of Carbohydrate-Hydrolyzing Enzymes
The good management of diabetes necessitates the control of the approach to the first line of treatment, such as discovering new agents with a high inhibition potential for carbohydrate-hydrolyzing enzymes. In vitro studies have demonstrated the ability of bee bread methanolic extract to inhibit alpha-amylase with an IC 50 % of 3.57 mg/mL [67]. The techniques of molecular docking, ultraviolet absorption, and fluorescence quenching tests have demonstrated that the bee bread contains functional fatty acids thatinteract with the amino acid residues of hydrolyzing enzymes through hydrogens bonds and vander Waals interactions [70]. Bee bread is a complex product with a high flavonoid content; flavonoids are known for their capacity to embed in the active site of the enzyme by bond and alkyl interactions, which are facilitated through the diverse methyl and hydroxyl groups that characterize the flavonoid's structure [71]. The ability of bee bread to control metabolism disorders through the inhibition of carbohydrate-hydrolyzing enzymes has been confirmed by in vivo studies detailing the pharmacological effects of bee bread, as shown in the in vivo investigations section (Table 2).

Probiotic Properties of Bee Bread
Probiotics are defined by the Food and Agriculture Organization and the World Health Organization as "live microorganisms that when administered in adequate amounts confer a health benefit on the host" [74]. Human probiotic strains frequently belong to the Lactobacilli, Bifidobacteria lactococcus, Streptococcus, and Enterococcusgenera, and some yeast strains belonging to the genus Saccharomyces [75]. Recently, the field of study on probiotics has gained great scientific and public interest due to their beneficial effects in the treatment of several human diseases, notably inflammatory bowel diseases, gut infections, allergy, asthma pulmonary infections, and even psychiatric illnesses [76,77]. A recent study suggested that probiotics could be beneficial in the framework of COVID-19 infection through their immunomodulatory effect [78]. Indeed, the relationship between probiotic bacteria and human well-being has been well established and, as a result, consumers are becoming more concerned and require more functional food in their diet, especially fermented food and beverages [79].
In a similar context, bee bread is a natural probiotic-rich product that contains a different and complex spectrum of microorganisms, such as bacteria and fungi, involved in the bee bread production process through bee pollen lactic fermentation [80]. Indeed, Toutiaee et al. [81] reported the isolation of a Bacillus species with probiotic properties.
The microbial composition with probiotic properties of bee bread confers interesting therapeutic effects to this product. Khalifa et al. [82] in their review study suggested that bee bread exhibits a remarkable hypolipidemic effect through the cholesterol-lowering factors produced by Lactobacillus bacteria. Moreover, the presence of Lactobacillus bacteria and fructophilic lactic acid bacteria in bee bread provides a promising source of compounds with high techno-functional properties that are used in the food industry as a food preservative or in functional culture for the production of fermented food at an industrial scale.

Antimicrobial Activity of Bee Bread
Bee bread from different regions of Morocco was evaluated by Abouda et al. [83] for antibacterial activity against the following bacteria: Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Escherichia coli. The results showed that all bee bread samples exhibited strong antimicrobial activities against the bacterial strains, with greater sensitivity to Gram-positive bacteria than Gram-negative bacteria.
Baltrušaitytė et al. [65] tested four samples of bee bread against Staphylococcus aureus and Staphylococcus epidermidis. The results obtained after the neutralization of the product and its treatment with catalase revealed that the samples of bee bread had non-peroxide antibacterial activity. Similarly, Ivanišov et al. [66] showed that samples of bee bread from five regions of Ukraine possessed antimicrobial activities against four bacterial strains, two Gram-positive-Bacillus thuringiensis and Staphylococcus aureus-and two Gram-negative-Escherichia coli and Salmonella enterica. The findings revealed that the minimum inhibitory concentrations varied between 6.40 µg mL −1 and 25.58 µg mL −1 .
In most studies, it seems that Gram-positive bacteria are, in general, more susceptible to bee bread extract than Gram-negative bacteria. This may be due to the cytoplasmic membrane of Gram-negative bacteria, which contains less anionic phospholipids than Gram-positive bacteria; this particular composition is the cause of the resistance of certain Gram-negative bacteria to antibiotics [89].
Until now, the antiviral property of bee bread was revealed only in a study published by Didaras et al. [90] against the EV-D68 virus. The results showed promising IC 50 values, ranging between 0.048 and 5.45 mg/mL, and CC 50 values ranging between 0.17 and 8.60 mg/mL. Numerous reports have suggested that the antiviral effect of bee products is attributed to their content of polyphenols, such as caffeic acid, chrysin, galangin, and rutin [91,92]. Recently, in silico studies have shown that flavonoids may inhibit SARS-CoV-2 [93,94].

Proposed Mechanisms of Antimicrobial Action
The investigation of new, safer, and more effective antibacterial molecules from food and food by-products has attracted the interest of many researchers worldwide. Owing to their hydroxyl groups (OH), phenolic compounds and some antioxidant molecules interact with the bacterial cell membrane by hydrogen bonding and achieve their antibacterial action by two possible combined mechanisms. The first mechanismis through the disruption of the bacterial membrane structure and thus the loss of the cellular contents [95]. The second mechanism depends on the delocalization of electrons, which results in the depolarization of the cell membrane and the reduction in the pH gradient across the membrane, and thus a reduction in ATP synthesis [96]. The antibacterial efficacy of phenolic compounds is mainly influenced by the relative position of the hydroxyl group on the phenolic nucleus of each antioxidant molecule [95].
Bee bread contains different valuable molecules (Table 1) with potent bactericidal properties. Several studies have shown that flavonoids induce their antibacterial actions by targeting different pathways and mechanisms, including the inhibition of nucleic acid synthesis, the disruption of cytoplasmic membrane function, the modification of cell membrane permeability, and the interaction with some vital bacterial enzymes. A recent study conducted by Wang and coworkers demonstrated that quercetin, a ubiquitous flavonol, exerts its bactericidal effect on Escherichia coli and Staphylococcusaureusbydamaging the bacterial wall and membrane structure as well as by inhibiting bacterial protein synthesis [97]. Furthermore, quercetin inhibits bacterial energy metabolism and DNA synthesis [98]. It was shown that kaempferol, a flavonol compound commonly found in bee bread, inhibits DNA PriA helicase and decreases ATPase activity in Staphylococcus aureus, which suggests its application as a natural component in the development of new active antibiotics against Staphylococcus aureus [99]. The antibacterial effect of other flavonoids, such as apigenin, has also been evaluated. Yu et al. [100] reported that apigenin inhibits RNA polymerase and DNA gyrase in Staphylococcus aureus.
In addition to phenolic compounds, organic acids (aromatic and aliphatic) have been reported to possess bacteriostatic and bactericidal effects [101]. Organics acids can permeate facilely through the bacterial membrane; once inside the cells, they release protons (H + ), decreasing intracellular pH, attacking macromolecules, and destabilizing bacterial walls [102]. The antibacterial properties of individual organic acids, such as gluconic, acetic, and formic acids, were widely investigated by several researchers. It was shown in one study that gluconic acid, a major organic acid present in bee bread, caused membrane cell depolarization and disrupted membrane integrity [103]. Moreover, a recent study showed that acetic acid is implicated in the sabotage of bacterial gene expression, including DNA replication enzymes, the elongation factors TU and GOS, polymerase alpha subunit, C-acetyltransferase 1OS, and chaperone proteins [104]. Besides its antibacterial effect, acetic acid exhibited a high inhibition of Conidia germination and aflatoxin production [105]. Finally, the antimicrobial efficacy of bee bread extracts is potentially due to the interaction between its active ingredients and microbial cells through one or more different mechanisms of action ( Figure 2). Current data suggest that bee bread could be used as a promising antibiotic and natural food preservative ingredient.  One gram of bee products was dissolved in 20 mL of aqueous ethanol (500 mL/L) for 6 h The inhibition diameters ranged between 9 and 22.8 mm [73] Greek (v/w).

MBC values ranging between 2.5 and 20%
Gram-negative bacteria: MIC values ranging between 5 and 20% (v/w) MBC values ranging between 10 and >20% (v/w) [85]   The inhibition diameters ranged between 4 and 8 mm Fungi species: The inhibition diameters ranged between 1 and 3.7 mm

The Pharmacological Effect of Bee Bread: In Vivo Investigations
Moroccan bee bread was evaluated in type 1 diabetic Wistar rats; the sample was extracted using ethyl acetate and it was given orally at the dose of 100 mg/kg for 15 days. The results showed that the bee bread was effective at decreasing blood glucose levels, it protected against loss weight induced by diabetes, it possessed a hypolipidemic effect, and it protected diabetic rats against the increase in the coronary risk index, atherogenic index, and cardiovascular index [64]. In addition, the ethanolic extract of the same sample was evaluated by Bakour et al. [51] against the toxicity of titanium dioxide nanoparticles in Wistar rats. The results revealed that the bee bread reduced the elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), blood glucose, sodium, potassium, chloride, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL), and it exhibited a highly protective effect against the decrease in albumin and total protein. Additionally, bee bread, at a dose of 500 and 750 mg/kg, alleviated the biochemical changes induced by aluminum in rats by increasing hematocrit, hemoglobin, red blood cells, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), urine sodium, and creatinine clearance and decreasing platelets, monocytes, lymphocytes, leukocytes, ALT, AST, C-reactive protein (CRP), and blood urea [63] (Table 4).
Malaysian bee bread was tested in two studies in high-fat diet-induced obese rats; the dose used in both investigations was 0.5 g/kg via oral routes using distilled water. The first study conducted by Eleazu et al. [30] revealed that the administration of bee bread in obese rats decreased the percentage change in body weight, BMI index, kidney weight, MDA concentrations, inflammatory cells in kidney tissue, NFkB, TNF-α, interleukin-L-beta, and Bax, while it increased the levels of SOD, GPx, GST, and TAA and reduced the Bowman's capsule space in the urinary chambers of the kidneys. In the second study conducted by Othman et al. [106], the administration of bee bread in obese male Sprague-Dawley rats improved their lipid profile, aortic inflammatory markers, and their impaired vasorelaxation activity, and showed its ability to enhance nitric oxide release and promote endothelial nitric oxide synthase (eNOS) and cyclic guanosine monophosphate (cGMP) immunoexpression. The same protocol forhigh-fat diet-induced obese rats was used by Suleiman et al. [107] to examine the protective effect of bee bread at a dose of 0.5 g/kg once daily for 12 weeks on testicular oxidative stress, inflammation, apoptosis, and lactate transport in the testes of obese rats (Table 4).
Several studies have examined the ability of bee bread to lower blood pressure. These studies have evaluated the effects of the oral administration of bee bread extract on vascular inflammation and impaired vasorelaxation in vivo using an obesity-induced vascular damage rat model [30,108]. The authors reported that bee bread improved the impaired vasorelaxation activity by enhancing the release of nitric oxidase, endothelial nitric oxide (sNOS), and cyclic guanosine monophosphate immunoexpression, which provide vascular protection. In addition, it has been proven that bee bread boosts the aortic antioxidant activities impaired in high-fat diet-induced obese rats by ameliorating aortic proinflammatory markers (tumor necrosis factor-α and nuclear factor-κβ) and preventing aortic structural damage perturbation in the vasorelaxation response to acetylcholine [30]. The phytochemical analysis of bee bread reveals its diverse phytochemical compounds with high antioxidant potential, including ferulic acid, caffeic acid, kaempferol, apigenin, and isorhamnetin. These bioactive molecules have proven their ability to act as antiatherogenic agents against the perturbations induced by a high-fat diet [108].
In the same context, Martiniakova et al. [109] reported that the oral administration of monofloral bee bread extract (Brassica napus L.) significantly lowered blood glucose levels, prevented lipid abnormalities, and impaired the bone morphology of Zucker diabetic fatty rats. In addition, Haščík et al. [110] showed the effect of dietary bee bread powder on the chemical composition of Japanese quails meat. In the breast muscle of quails, the bee bread decreased the water content, increased the crude protein content, and decreased fat and cholesterol, while in the thigh muscle of quails, the dietary bee bread increased the water content, fat, and cholesterol.
In Turkey, the effect of bee bread on leptin and ghrelin expression in obese rats was studied by Doganyigit et al. [111] in Sprague-Dawley adult female rats using two doses via oral administration; the results showed that bee bread decreased ghrelin immunoreactivity, increased leptin immunoreactivity, decreased the apoptotic cell numbers in the hypothalamus, and decreased MDA levels. In addition, it was shown that bee bread from Slovakia reduced femoral bone structure and improved glucose and lipid metabolism in Zucker diabetic fatty rats [112]; bee bread from China has also been shown to have a capacity for the regulation of lipid metabolism [113].    ↓ % change in body weight, BMI index in rats that received HFD treated or protected with BB. ↓ Kidney weight in rats that received HFD treated or protected with BB. ↑ SOD, GPx, GST, TAA in rats that received HFD treated or protected with BB ↓ MDA concentrations in rats that received HFD treated or protected with BB ↓ Inflammatory cells in kidney tissues of HFD groups treated or protected with BB. ↓ Bowman's capsule space in the urinary chamber of kidneys of HFD groups treated or protected with BB. ↓ NFkB, TNF-α, interleukin-1-beta, and Bax in rats that received HFD treated or protected with BB [30]

Hepatoprotective Effect
Bee bread was tested byČeksterytė et al. [114] in patients with chronic hepatitis. The most important clinically relevant finding was a significant improvement in blood parameters, including erythrocyte count, hemoglobin, leukocytes, C-reactive protein (CRP), blood sugar, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin.

Anti-Atherogenic Dyslipidemic Effect
Kas'ianenko et al. [115] evaluated the effectiveness of treating patients with atherogenic dyslipidemia with a mixture of honey, pollen, and bee bread. The parameters of atherogenic dyslipidemia were examined in 157 patients (64 men and 93 women) aged 39 to 72 years. These patients were divided into four groups: (1) treated with a lipid-lowering diet only; (2) treated with a lipid-lowering diet and honey or pollen; (3) treated with bee bread only; and (4) treated with honey and pollen. The results obtained showed that a significant lipid-lowering effect was recorded in the patients taking honey in combination with pollen (total cholesterol decreased by 18.3% and LDL-C decreased by 23.9%) and bee bread (total cholesterol decreased by 15.7% and LDL-C decreased by 20.5%).

Strengthening Visual Acuity
Jarušaitienė et al. [116] studied refractive status, visual acuity, and ocular prophylaxis in 34 children (aged 6 to 17) with thyroid disease before and after taking bee bread. The results of this study showed an increase in visual acuity in the subjects who used bee bread.

Ergogenic Effect and Improvement of Athletic Performance
Chen et al. [117] examined the effects of bee bread supplementation during recovery on athletic performance. Twelve athletes were chosen for the study. During the experimental trials, the participants ran on a treadmill for 90 min and then rested for four hours. During this recovery period, the participants consumed 30 g/h of bee bread or a placebo. Heart rate and tympanic temperature were measured at 20-min intervals during this period. Blood samples were taken to determine plasma glucose, hemoglobin, and hematocrit. Participants then performed a 20-min trial on a treadmill. The distance traveled in the bee bread trial was significantly longer than the placebo trial (3.45 ± 0.4 km vs. 3.24 ± 0.4 km, respectively). The plasma glucose levels in the bee bread trial were significantly higher compared to those in the placebo trial during recovery. These results showed that the supplementation of bee bread during the recovery phase appears to provide improved athletic performance in athletes.
In a second study conducted by Fadzel et al. [118], bee bread supplementation was tested on the running performance of athletes. This time, the athletes were given 20 g of bee bread or a placebo every day for 8 weeks. After the first experimental run, there was a 4-week washout period. Then, they continued with the supplementation for 8 weeks before the second experimental trial. During the experimental trials, the participants ran at 60% VO 2 max for 90 min, immediately followed by a 20-min trial. Heart rate, oxygen uptake, ear temperature, perceived exertion rate, ambient temperature, and relative humidity were recorded during the tests. Blood tests were performed to determine the levels of plasma glucose and free fatty acids. The results of the study showed that there was no significant difference between the bee bread test and the placebo test for heart rate, oxygen uptake, tympanic temperature, the rate of perceived exertion, and plasma glucose levels. However, the distance traveled in the bee bread trial was significantly greater than that in the placebo trial (3.41 ± 0.2 km vs. 3.28 ± 0.2 km, respectively). In addition, the free fatty acid plasma levels in the bee bread trial were significantly higher than in the placebo trial. This allowed the researchers to conclude that supplementation with bee bread appeared to improve the running performance of athletes [118].

Bee Bread Adulteration
Hive products, namely honey and bee pollen, have long since been considered as a functional food for their broad-based composition and wide range of biological activities. Recently, the use of these products has increased all over the world because of public awareness of their nutritional and therapeutic properties, which could lead to a substantial economic-motivated adulteration.
Bee bread is a bee-derived product with an extended range of bioactive compounds; however, it is still not well-known and has a scarce production in the apiaries because of the difficult harvesting methods and the beekeepers' conviction that bee bread reserves in the hives should not be reduced to preserve the colonies' development [119]. The growing consumer attention regarding the health benefits of bee bread has increased its demand in the market compared with its production, which sets the stage for its adulteration and becomes an issue of concern for all bee product producers. However, the international scientific community has just started to define the regulation and quality criteria of bee products. Thus far, there are no acknowledged standards for bee bread, which makes it difficult to detect its falsification [120,121].
Based on the composition previously discussed in the present work, the detection of fraudulent bee bread manipulations requires the combination of multiple techniques mainly based on the composition of pollen in bee bread [122], including the identification of the floral origin of the bee pollen (starter matrices to the bee bread production) by a palynological survey [123] and the spectroscopic determination of the chemical composition of the bee bread, notably its vitamins (vitamin C, vitamin E, and β-carotene), amino acids, and fatty acids (taking into consideration the relationship between the botanical origin and chemical composition of bee pollen) [124,125]. Recently, advanced foodomics technologies have been used to characterize food products and have been applied to bee bread and other bee products to assess authenticity, safety, and quality issues and to determine the bioactive compounds present and their biological activities. In their review article, Kafantaris et al. [126] summarized the different methods used for the study of bee bread, including genomics as an alternative for the palynologic/microscopic determination of the botanical origin using the metabarcoding of DNA isolated from bee pollen and bee bread samples. The proteomics and metabolomics technologies can be performed using GC-MS, HPLC-DAD, SDS-PAGE, 2D-electrophoresis, and MALDI-MS methods for the identification of proteins, enzymes, fatty acids, the phenolic profile, and carotenoid composition. Additionally, metagenomics can beapplied to investigate the microbial community of bee bread, especially lactobacillus bacteria [126].
On this basis, we can conclude that the determination of the botanical origin of bee pollen, its chemical and enzymatic composition, and the characterization of its microbiome and biological properties are necessary for the authenticity, traceability, and safety of bee bread.

Conclusions
In conclusion, bee bread is a functional food produced by the fermentation of bee pollen and honey in the honeycombs of the hives. It contains a wide range of components, such as sugars, polyphenols, vitamins, and free amino acids. Bee bread is characterized by considerable pharmacological properties, proven in vitro and in vivo. Thus, it can be stated that bee bread offers a wide field for the exploitation of its benefits in the food and pharmaceutical industries.