**4. Discussion**

In this study, UPLC-Q-Exactive Orbitrap/MS analysis was conducted to explore the effects of different types of bee pollen consumption by honeybees on the lipid composition of royal jelly. PC and PE belong to the phospholipids, Cer and SM belong to the sphingolipids. PC is known as the "third nutrient" and has a variety of biological activities. It can regulate lipid metabolism, prevent vascular diseases, counteract inflammation and oxidation, improve brain and nerve function, and prevent senile dementia [21,22]. PEs maintain the structure and function of cells, repair nerve cell membranes, and promote normal metabolism in brain neurons. The PC/PE ratio is an important factor affecting metabolic dysfunction and insulin sensitivity [23]. SMs are phospholipids containing sphingosine or dihydrosphingosine; these are primarily metabolized in the small intestine and colon and have various physiological functions. For example, SM can reduce intestinal absorption of cholesterol, down-regulate proteins related to cholesterol absorption, and reduce blood cholesterol levels [24]. SMs can also promote colon cancer cell apoptosis, inhibit colon cancer cell proliferation, improve skin barrier function, and maintain physiological functions of skin [25,26]. SMs are important initial substrates of the sphingomyelin signaling pathway and can be hydrolyzed by sphingomyelinase to produce Cer. In the reverse reaction, sphingomyelinase synthesizes SMs from Cers [27]. Cer is an important signaling molecule in many basic cellular physiological and biochemical processes, such as inflammation, immune cell transport, stress responses, apoptosis, and autophagy [27]. Notably, PCs can be hydrolyzed into linoleic acid (FA 18:2) and α-linolenic acid (FA 18:3) which can participate in linoleic acid and α-linoleic acid metabolism. Stearic acid (FA 18:0) and oleic acid (FA 18:1) are also associated with glycerophospholipid metabolism through the FA biosynthesis pathway (Figure 5). Therefore, the nutritional and functional properties of royal jelly may be affected by changes in the lipid composition. Our findings suggested that the optimum time point for collecting royal jelly from artificial feeding bee pollen is 18~24 days, because the content of functional lipids in royal jelly is the most abundant during this period.

**Figure 5.** Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway annotations for the detected lipids. SM, sphingomyelin; Cer, ceramide; So, sphingosine; S1P, sphingosine-1-phosphate; PE, phosphatidylethanolamine; DG, 1,2-diacyl-sn-glycerol; PC, phosphatidylcholine.

Studies have shown that honeybee foraging preferences may be related to the protein/lipid ratio in pollen. Honeybees can selectively balance their intake of amino acids and FAs during natural foraging [28]. Moreover, royal jelly is secreted by the hypopharyngeal and maxillary glands, development of which is positively correlated with the type and quantity of pollen ingested. A previous study found that consumption of mixed bee pollen is better for hypopharyngeal gland development than single pollen [29]. In addition, the increase in lipids before day 24 may therefore be attributed to a gradual adaptation to the single-pollen diet. However, the forced change from a natural mixed bee pollen diet to the single bee pollen diet may destroy the nutritional balance and therefore affect the development of honeybee hypopharyngeal glands over time, resulting in the observed decrease in lipids after 24 days of single bee pollen consumption.

Moreover, FAs can be used as fuel, bioactive lipid media precursors, and cell membrane components (in the form of phospholipids and glycolipids). SFAs do not contain unsaturated double bonds, which are generally considered the main trigger of high blood cholesterol, obesity, and coronary heart disease in humans [30,31]. Unsaturated FAs are classified as MUFAs or PUFAs, with classification depending on the number and positions of double bonds. Dietary unsaturated FAs have various physiological functions, such as inhibiting inflammatory mediators and cytokines, regulating lipid metabolism through transcription factors, and preventing cardiovascular diseases [32,33]. Ahad unique MUFA with antibacterial, antioxidative and anti-inflammatory activities, 10-HDA, accounts for over 50% of free FAs in royal jelly [12]. Honeybees that consume bee pollen from different plant sources secrete royal jelly with different levels of 10-HDA compared to those that consume single pollen [34]. In honeybee maxillary glands, 10-HDA is converted from an 18-carbon FA (stearic acid) through hydroxylation and β-oxidation to shorten the carbon chain [35]. Consumption of oleic acid can also affect 10-HDA levels in royal jelly [36]. Therefore, our results indicated that feeding bees with BP-Am was beneficial to increase the content of 10-HDA in royal jelly than feeding with BP-Pa, due to the significantly higher content of stearic acid and oleic acid in BP-Am than in BP-Pa.
