3.2. Sugars

Bees use sugars to produce the energy necessary for their survival. Several studies have been carried out on the sugar content of bee bread. For instance, bee workers need about 4 mg/day of sugars to cover their daily energy [3,19]. The free sugar contents in bee bread were determined using high-performance liquid chromatography−refractive index detection (HPLC-RID) in a Moroccan multifloral sample (Bupleurum spinosum, Anethum graveolens, Calendula officinalis, Anacyclus, Quercus ilex, Eucalyptus, Punica granatum, and Acacia). The results showed that fructose was the major representative sugar with a concentration of 11.8 ± 0.6 g/100 g, followed by glucose (5.7 ± 0.4 g/100 g), and a small amount of trehalose (0.92 ± 0.01 g/100 g [20]). Similarly, Dranca et al. [21] documented that Romanian bee bread contains 19.73 g/100 g of fructose and 8.82 g/100 g of glucose, and a low percentage of melezitose and raffinose (0.97 g/100 g and 0.96 g/100 g, respectively). Moreover, the findings of Urcan et al. [22] for five multifloral bee bread samples (Brassicaceae, Poaceae, Myrtaceae, Rutaceae, Asteraceae, Fabaceae, Tiliaceae, Fabaceae, Rosaceae, Plantaginaceae, Fabaceae, Asteraceae, Lamiaceae, Salicaceae, Rosaceae, and Fagaceae) from Romania and India revealed by high-performance liquid chromatography diode array detection (HPLC-DAD) analysis showed that fructose was the major individual sugar, with values ranging from 13.97 ± 0.05 g/100 g to 19.58 ± 0.03 g/100 g, followed by glucose (6.40 ± 0.010 g/100 g to 15.13 ± 0.02 g/100 g), and smaller quantities of maltose and turanose(0.82 ± 0.02 g/100 g to 1.00 ± 0.01 g/100 g and 0.56 ± 0.02 g/100 g to 0.87 ± 0.01 g/100 g, respectively).

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].

3.3. 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].

3.4. 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].

3.5. 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.

3.6. 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 pollen-derived 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.

3.7. 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.

A study conducted by Urcan et al. [22] revealed that five samples of bee bread from Romania and India contained kaempferol-3-O-glycoside, a hydroxycinnamic acid derivative, quercetin-3-O-sophoroside, a kaempferol-3-O-derivative, a hydroxycinnamic acid derivative, myrcetin, trycetin, luteolin, herbacetin-3-O-glycoside, quercetin-3-O-glycoside, and isoramnethin-3-O-glycoside. Similarly, Lithuanian, Romanian, Turkish, Portuguese, and Georgian bee bread samples showed the presence of several polyphenolic components belonging to different chemical groups, such as p-coumaric acid, kaempferol, chrysin, apigenin, caffeic acid, rosmarinic acid, myricetin, luteolin, naringin, rutin, and quercetin [15,21,47,48,49]. In addition, the GC-MS analysis of three bee bread samples from Poland showed the presence of kaempferol and apigenin [50] (Table 1).

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].

3.8. 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].

4.2. 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).

4.3. 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).

4.4. 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 IC₅₀ % 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).

4.5. 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.

4.6. 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.

The hydromethanolic extract of Moroccan multifloral bee bread (35% Anethum graveolens, 24% Quercus ilex, 16% Eucalyptus, and 25% other pollens) was evaluated against six bacterial strains (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, Salmonella typhimurium, and Listeria monocytogenes), and it was shown that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged between 0.04 mg/mL and 0.25 mg/mL and between 0.08 and 0.35 mg/mL, respectively. The bee bread sample was also effective against Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus niger, Penicillium funiculosum, Penicillium ochrochloron, and Penicillium verrucosum var. cyclopium. The obtained MIC and MBC values ranged between 0.35 and 1 mg/mL and between 0.70 and 1.40 mg/mL, respectively [20] (Table 3).

In a study conducted by Didaras et al. [84], it was reported that the aqueous extract of Greek bee bread was effective against Gram-positive bacteria (MIC values ranging between 3.9 mg/mL and 48 mg/mL) and against Gram-negative bacteria (MIC values ranging between 7.8 mg/mL and 90.4 mg/mL). It has also been shown that bee bread ethanolic extracts (70%, 95%, 80%, and 50%) have strong antibiotic activity against Gram-positive bacteria (Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Bacillus Subtilis, methicillin-susceptible Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogene), Gram-negative bacteria (Haemophilus influenza, Klebsiella pneumonia, Salmonella enteric, Escherichia coli, Shigella, Salmonella typhi, and Pseudomonas aeruginosa)and fungi (Candida albicans, Aspergillus niger, Candida glabrata, Candida tropicalis, Aspergillus clavatus, Aspergillus flavus, Aspergillus versicolor, Penicillium expansum, Penicillium chrysogenum, and Penicillium griseofulvum) [52,85,86,87,88].

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 IC₅₀ values, ranging between 0.048 and 5.45 mg/mL, and CC₅₀ 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].

6.2. 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%).

6.3. 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.

Special thanks to Badiaa lyoussi, recently awarded at Sidi Mohamed Ben Abdellah University, for her long career in the Apitherapy and Natural Products Research. Open access funding provided by University of Helsinki.