Mechanical methods break down pollen walls using shear or friction forces. Equipment such as ball mills or high-speed shearing machines is commonly used. These methods are relatively simple and the equipment is cost-effective (58). Ball milling grinds materials using hard balls in a rotating container, applying compression and friction to reduce particle size, mix powders, and modify structures. In bee pollen research, it is used to break down tough pollen walls, thereby improving the accessibility of nutrients and bioactive compounds (7, 59). However, the studies have shown that ball milling alone is less effective in breaking cell walls than ultrasonication. In contrast, the combination of ultrasonication and ball milling achieves significantly better results, enhancing the release of nutritional and bioactive compounds and providing the highest antioxidant and antimicrobial activity. This combined method produced the largest inhibition zones in tests against E. coli, S. aureus, L. monocytogenes, C. albicans and Saccharomyces cerevisiae, and also showed activity against P. aeruginosa, which was resistant to other bee pollen treatments. Moreover, compared to prolonged ball milling alone, this combined approach caused minimal degradation of the main bioactive compounds in bee pollen (59).

Due to its high water content (20–30 %), nutritional value and poor aseptic conditions in hives, bee pollen is susceptible to contamination. To prevent this, bee pollen is typically dried after collection to reduce moisture below 10 %, usually to 5–8 % (60-62), ensuring stability and quality during storage. Effective drying requires careful control of temperature, consideration of product characteristics, and treatment scope to prevent thermal degradation of sensitive components and to enhance subsequent processing of the pollen wall (60). The drying method and plant species significantly influence the outcome, as different species respond differently. Studies have examined the effects of drying on monofloral bee pollen compounds and properties from Castanea sativa, Hedera helix, Salix spp. (63), Cistus ladanifer (64), Helianthus annuus (36), Rubus spp., Eucalyptus spp., Cistus spp., Cytisus spp., Echium spp. and Erica spp. (65).

Traditional drying methods include sun drying, hot-air drying and the freeze-thaw method (6, 61). However, to avoid many negative effects associated with traditional drying techniques, these have largely been replaced by industrial methods such as spray drying, freeze-drying, microwave drying and vacuum drying (60). Although high quality and yield are desired, drying methods are closely linked to cost. Sun drying is the cheapest but is rarely used due to environmental contamination, product loss, insect and bird interference, space requirements, process control difficulties, and odour issues (66). Hot-air and convection drying offer the best balance of cost, quality and speed (67). Higher temperatures shorten drying time but can reduce nutrient content, alter colour and negatively affect antioxidant, anti-inflammatory and antimicrobial activity (63, 68, 69). Drying at 40 °C has been shown to optimally preserve bee pollen quality (67).

Freeze-drying (lyophilization) is considered the most suitable method for preserving the colour, taste, bioactive compounds and biological properties of bee pollen (60, 61). The process involves freezing at very low temperatures, followed by sublimation under reduced pressure, which disrupts the bee pollen wall and increases nutrient availability (70). Compared to hot-air drying, freeze-drying better preserves nutrients and bioactive compounds (65, 68).

When studying the antimicrobial activity of bee pollen after drying, lyophilization consistently maintained stronger activity across most plant species. The lowest MICs were observed against S. aureus for methanolic bee pollen extracts from Erica species, followed by extracts from Castanea sativa, Echium and Cistus species (65). Table 2 (22, 36, 65, 71) summarizes the studies that investigated the effects of different drying methods on the antimicrobial activity of bee pollen. Both monofloral bee pollen and polyfloral bee pollen are included in the review, due to the very limited number of studies investigating monofloral bee pollen so far.

Lyophilization also proved superior to conventional drying in a study using polyfloral bee pollen, showing stronger activity against all studied bacteria and yeasts (22). For sunflower bee pollen extract, lyophilization and freezing were comparably effective when comparing dried, frozen and freeze-dried bee pollen against human and bee bacterial pathogens. Antifungal activity was highest in frozen pollen against Aspergillus ochraceus and in freeze-dried pollen against A. niger, with antibacterial activity stronger than antifungal activity (36). When comparing drying methods using a chiller (4 °C) versus oven drying, antimicrobial activity was higher in bee pollen processed with the 4 °C chiller method (71). Despite its effectiveness, freeze-drying has limitations. It is relatively expensive and time-consuming, making it less suitable for industrial use where mass production is required (60, 61).

Microwave drying uses electromagnetic waves for efficient heating and dehydration, preserving bioactive compounds while minimizing thermal stress (60, 61). Controlled power and moisture levels are essential to ensure product quality. At lower power, microwave drying produces nutritionally rich bee pollen faster than other methods (61). To the best of our knowledge, there are no studies on the antimicrobial activity of bee pollen before and after microwave drying. Despite this gap in the literature, existing studies indicate that the antimicrobial activity of bee pollen is generally retained following various drying techniques, with lyophilization being the most effective. The choice of drying method, in combination with the plant species, also has a significant influence, as not all species respond equally to the same drying technique (36, 65). However, only a limited number of studies have examined the effect of drying on the antimicrobial activity of bee pollen – particularly monofloral bee pollen. This gap should be addressed in future research focused on developing quality parameters for monofloral bee pollen.

Ultrasound is a cost-effective, simple and energy-efficient method for disrupting the walls of bee pollen. It increases membrane permeability and induces cavitation, which fragments cellular structures (72). Low-power ultrasound is mainly used for monitoring, while high-power ultrasound causes structural changes. As a non-thermal process, it preserves heat-sensitive compounds (73). However, prolonged use can reduce enzyme activity and efficiency (74).

Ultrasonication significantly enhances the bioactivity of bee pollen. After 5 h of treatment, increases were observed in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), total phenolic and total flavonoid content. Similar enhancements were observed with supercritical fluid extraction (75). Combining ultrasonication with complementary methods, such as high shear or enzymatic treatment, further disrupts pollen walls, enhances the release of bioactive compounds, improves protein yield and solubility, and boosts functional properties like digestibility, emulsifying capacity and gelling ability (57, 76).

The antimicrobial activity of treated bee pollen showed high variability. Some studies reported no antimicrobial activity against several enteric pathogens, with either conventional or sonication-based extraction (77), while others observed enhanced antioxidant activity (75, 78, 79), and both antioxidant and antimicrobial activity – particularly against S. aureus (59, 80). S. aureus appears especially sensitive to ethanolic bee pollen extract when processed with ball milling, ultrasonication, or their combination, unlike P. aeruginosa, which shows little to no inhibition. When comparing ball milling and ultrasonication, the latter demonstrated a stronger cell wall-breaking effect. However, the combination of both methods proved most effective, resulting in the highest antioxidant and antimicrobial activities (59).

Recent advances in sustainable biotechnology have introduced innovative processing methods for bee pollen. Ultrasound and green extraction methods, such as deep eutectic solvents (DES) and supercritical fluid extraction, enhance the antioxidant and antimicrobial activity, and the functional properties of bee pollen. These eco-friendly techniques improve the yield of bioactive compounds and produce high-value ingredients for food supplements (59, 75, 80, 81). DES-treated bee pollen extracts exhibited strong antimicrobial activity, especially against Gram-positive bacteria like S. aureus. Antifungal activity was limited, indicating lower effectiveness against yeast-like fungi. Broad-spectrum antibacterial activity was observed across all tested strains, with extracts at molar ratios of 1:1.5 and 1:2 showing slightly higher inhibition than 1:1, suggesting that the hydrogen bond donor (HBD) to hydrogen bond acceptor (HBA) ratio influences antibacterial potency (80). To date, no published research has directly compared the antimicrobial activity before and after ultrasound treatment of monofloral bee pollen.

Microbial fermentation is an efficient method for transforming basic food ingredients, enhancing unique flavours and other sensory properties, improving nutritional profiles by degrading ant-nutrients and increasing beneficial nutrients, and extending shelf-life (82). In vitro fermentation of bee pollen, modelled after its natural transformation into bee bread in the hive, can enhance its antimicrobial activity and functional properties (15, 83). In the absence of oxygen, yeasts, lactic acid bacteria or a combination of both break down the multilayered pollen wall and degrade macromolecules into smaller, more bioavailable compounds. This process increases nutrient content and improves the accessibility of bioactive compounds (7, 20, 84, 85). Additionally, lactic acid bacteria produce bacteriocins, substances that disrupt bacterial membranes, which are effective against pathogens such as L. monocytogenes, Listeria innocua, S. aureus, E. coli, M. luteus, P. aeruginosa, B. subtilis, S. Enteritidis and S. Typhimurium (86).

Across multiple studies, fermentation consistently enhanced the antimicrobial activity of bee pollen extract. Both spontaneous and induced bacterial fermentation have been shown to increase antimicrobial effects, with Gram-positive bacteria generally being more sensitive than Gram-negative strains (86-88). Kaškoniene et al. (87) confirmed improved antimicrobial activity, showing an almost twofold increase in inhibition zones for M. luteus after spontaneous fermentation. Urcan et al. (88) reported that ethanolic bee pollen extracts showed a substantial reduction in MIC values after fermentation, nearly a twofold decrease for S. aureus, E. faecalis, E. coli and P. aeruginosa, indicating strongly improved antimicrobial potency against both Gram-positive and Gram-negative bacteria. Similar trends were observed in methanolic bee pollen extracts, with inhibition zones moderately increased after spontaneous and bacterial-induced fermentation of bee pollen (86).

Enzymatic hydrolysis enhances nutrient release and bioactivity in bee pollen by breaking down polysaccharides, proteins and other macromolecules (60, 89). This improves digestibility, permeability and bioavailability, resulting in increased antioxidant and antimicrobial activity (90). The efficiency of enzymatic hydrolysis depends on several factors, including enzyme type, concentration, pH, temperature, and duration of hydrolysis (91). Controlled hydrolysis can produce bioactive peptides with strong angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activity (92), as well as improved protein solubility and functionality compared to physical methods (76). Proteases (e.g. Protamex) enhance the release of proteins, phenolic compounds and flavonoids, while cellulases, pectinases and carbohydrases facilitate the release of bioactive compounds and improve functional properties (7, 89, 93). Combined methods, such as enzymatic hydrolysis with ultrasound or freeze-thaw cycles, can further enhance yield and functionality (58, 76).

Only a few studies have investigated the impact of enzymatic hydrolysis on the antimicrobial activity of bee pollen. Available research shows that enzymatically hydrolyzed bee pollen exhibits increased effectiveness against both Gram-positive bacteria (S. aureus, L. monocytogenes) and Gram-negative bacteria (S. Enteritidis, S. Typhimurium) (86, 89).

Gram-positive bacterial strains showed greater sensitivity to bee pollen extracts of both natural and enzymatically hydrolyzed bee pollen, likely due to differences in bacterial cell wall structure. In the study by Damuliene et al. (89), the first published investigation optimizing enzymatic hydrolysis parameters such as duration, enzyme concentration and substrate pH, the antimicrobial activity before and after hydrolysis was strongly correlated with total phenolic content, total flavonoids and antioxidant activity. This confirms that both the composition and quantity of bioactive compounds significantly influence the antimicrobial properties of bee pollen. In this study, cellulase and Viscozyme® L achieved the best results (89).

Further research on combined pollen wall treatment methods, including fermentation and enzymatic hydrolysis, showed that both spontaneous and induced bacterial fermentation significantly enhanced the antimicrobial activity of bee pollen. Induced bacterial fermentation with L. rhamnosus produced the highest increase. Enzymatic hydrolysis further boosted antimicrobial activity more consistently than fermentation, with Clara-diastase, Viscozyme® L, and cellulase providing the greatest improvements (86).

To date, no studies have investigated the effects of pollen processing methods such as ball milling, ultrasonication, fermentation and enzymatic hydrolysis on the antimicrobial activity of monofloral bee pollen. However, given the growing number of studies on the antimicrobial properties of monofloral bee pollen, such research is expected in the near future. Table 3 (59, 80, 86-89) summarizes the current information about the influence of processing on the antimicrobial activity of polyfloral bee pollen, showing the highest and lowest observed activity.