Factors influencing the quality of Royal jelly and its components: a review

Abstract

Royal jelly (RJ) has various applications in cosmetics, health food, and apitherapy to treat chronic inflammation disorders. It is a milky white substance rich in proteins, carbohydrates, lipids, vitamins, and minerals. RJ acts as an antioxidant, anti–inflammatory, and antimicrobial agent, which helps in reducing inflammation and boosting immunity. This review provides an insight into the sensory and chemical analytical aspects of RJ, with a focus on water, 10–hydroxy–2–decenoic acid (10–HDA), protein, and sugar contents, as well as the national and international quality standards of major honey–producing countries. The chemical composition of RJ varies across locations due to external conditions, nectar sources, harvesting conditions, and honeybee species, which affect its quantity and quality. Despite having a consistent composition, the production of RJ per honeybee colony is influenced by environmental conditions, nectar source attributes, and mineral, vitamin, and amino acid content. Advanced analytical methods are required to accurately quantify the diverse ingredients of RJ, which are responsible for its biological and pharmacological effects. Understanding the intricacies of RJ is essential to unlock its therapeutic potential. Notably, apiculture research at the national level acts as sensitive bioindicators, emphasizing the global need for honeybee standards. This exploration contributes to understanding how external factors affect honeybee health and bee product quality.

Keywords:

• Beekeeping
• environmental condition
• floral source
• honeybee species
• major royal jelly proteins
• sensory analysis

Reviewing Editor:

• M. Luisa Escudero–Gilete, Universidad de Sevilla, Spain

Subjects:

• Food Analysis
• Food Laws & Regulations
• Food Safety Management

Introduction

Royal Jelly (RJ) is a milky–white, bee–derived secretion produced by nurse bees’ hypopharyngeal gland and mandibular glands of (Apis mellifera) to feed young worker larvae during the first three days and the entire life of queen bees. The chemical composition of RJ was first analyzed in 1888. Subsequent studies have shown that RJ comprises water, proteins, free amino acids, lipids, vitamins, and other trace components (Oršolić, 2013).

Extensive research has been done on RJ and other honeybee products, discussing their use in cosmetics, health food, and medication and in treating chronic inflammatory disorders, also called apitherapy (Collazo et al., 2021). Recent research has focused on the pharmacological activities of RJ and its components, including anti–inflammatory, antibacterial, immunomodulatory, neuromodulatory, and anti–aging effects in animal models and humans (Kunugi & Mohammed Ali, 2019). Bee scientists and beekeepers have undertaken considerable efforts to boost RJ output with excellent quality, beginning with basic apiculture instruments and continuing with the genetic selection of high RJ–producing species of bees (Altaye et al., 2019).

In recent years, extensive studies have been carried out to standardize RJ quality through assessments based on identifying the RJ’s components to ensure that industry standards and consumer needs are satisfied (Sabatini et al., 2009). RJ has a distinct composition, unlike honey, pollen, or other bee products. Multiple factors influence the water and sugar, 10–HDA, and protein contents of RJ, which is considered a criterion for its authenticity. These include seasonal and regional variability, botanical sources, bee species, and harvesting conditions.

RJ’s increasing demand and commercialization have led some countries to create their own national standards. However, there is a lack of consistency between these standards. The International Honey Commission (IHC) and International Organization for Standardization (ISO) published research for fresh and lyophilized RJ and established standards for sanitary and manufacturing RJ. Gulf countries follow the values recommended by the Gulf Cooperation Council (GCC) under the Gulf Standardization Organization. All aim to define standard criteria for precise RJ compositions for better assessment using robust analytical techniques (Arfa et al., 2021). Therefore, this review aimed to investigate the factors influencing the quality and activity of RJ and/or its biological compositions by evaluating the most relevant studies. The review helps gain insight into apiculture and honeybee products by understanding standard RJ specifications for national and international purposes.

Royal jelly comprehensive profiling

Sensory analysis

Sensory analysis, also known as organoleptic analysis, has great significance in ISO standards. RJ must be liquid at normal temperature and free of contaminants (Lercker, 2003). Furthermore, it must be spicy, acidic, and pasty or gelatinous in appearance. According to the literature, RJ has a milky white, pale yellow (Figure 1) with a pH between 3.4 and 4.5.

Figure 1. Royal jelly in royal cells inside a hive.

The sensory analysis of RJ can provide valuable insights into its sensory characteristics, which can help producers optimize production processes (Apostol et al., 2019). Sensory analysis of RJ involves evaluating its physical attributes, such as taste, aroma, texture, and overall appearance. It also plays a vital role in determining RJ quality and acceptability by consumers (Hassoun et al., 2022).

Representative samples should be collected from different sources or batches and stored properly to maintain their sensory properties. Before testing, samples should be brought to room temperature and evaluated by trained panelists. For panelists to develop a universal sensory vocabulary, training sessions are necessary to familiarize them with RJ’s characteristics. Light odors and ventilation should be controlled appropriately to prevent external influences on the sensory evaluation environment. The sensory evaluation of the RJ typically involves attributes such as taste (e.g., sweetness, bitterness, acidity, and overall flavor profile) and scent (e.g., floral, fruity, fermented). Other sensory attributes include the evaluation of texture (e.g., creamy, sticky, gel–like) and appearance (e.g., color, clarity, and presence of visible impurities). Sensory attributes can be evaluated using a hedonic scale (liking/disliking), a descriptive analysis, or a ranking. RJ sensory quality and acceptability are determined after analyzing the data statistically and applying quality control measures (Marx et al., 2021).

Chemical profiling

Numerous articles have demonstrated RJ’s protein, carbohydrate, and lipid compositions throughout ­history (Lercker, 2003; Garcia-Amoedo & Almeida-Muradian, 2007; Pourtallier et al., 1990). By developing and applying advanced analytical techniques to assess the quality of RJ, its chemical content has been well–defined to fit the standard criteria of production and marketing. However, chemical contents accessible in the literature are influenced by a wide range of factors, partially due to the different analytical methods, and the natural variations in RJ samples from different countries are most likely the result of varied sample counts made at various production stages and locations and due to the inhomogeneity nature of RJ by itself.

The chemical analysis tests applied to the RJ include determining preliminary contents of water, sugars (fructose, glucose, sucrose, total sugars), protein content, and amount of 10–HDA (Garcia-Amoedo & Almeida-Muradian, 2007; Lercker, 2003; Pourtallier et al., 1990). Table 1 displays the national standards, ISO (72), and IHC proposal drafts (Arfa et al., 2021; Sabatini et al., 2009) that have been adopted. The ISO organization has established precise standards to differentiate between two types of RJ: type 1 RJ, which is produced from beehives that are fed on natural nectar sources and where the bees feed naturally, and type 2 RJ, where bees are fed artificially on sugar syrups, carbohydrates, or proteins. Several factors contribute to the chemical profiling of RJ, which are mentioned below.

Table 1. Comparison between national and international standards regarding RJ chemical composition.

Characteristics (%)	ISO	IHC	China	Malawi	Poland	Brazil	Bulgaria	Turkey	Egypt	UAE
Moisture	62.5–68.5	60–70.0	62.5–67.5	62.0–68.5	Max: 67.5	60–70.0	62.5–67.0	62.5–68.5	Max: 66	62.5–68.5
10–HDA	Min:1.4	Min: 1.4	Min: 1.4	Min: 1.4		Min: 2.0		Min: 1.4	Min: 2.0	Min: 1.4
Protein	11.0–18.0	9.0–18.0	Min: 11	11.0–18.0	13.5–20.0	Min: 10	12.0–17.0	11.0–14.5	Min: 12	11.0–18.0
Fructose	2.0–9.0	3.0–13.0		2.0–9.0						2.0–9.0
Glucose	2.0–9.0	4.0–8.0				Max: 10.0				2.0–9.0
Sucrose	Max:3.0 (type1) Max: Not applicable (type2)	0.5–2				Max: 5.0	Max: 4.5			Max:3.0 (type1) Max: Not applicable (type2)
Total sugars	7.0–18.0	7.0–18.0		7.0–18.0	6.5–18.5		9.0–13.0		Min: 12	7.0–18.0

Water content

Water is a significant component of the raw RJ (Kausar & More, 2019). The RJ’s processability, shelf life, usability, and quality are impacted by its moisture content. Therefore, accurate moisture content estimation is essential for determining the RJ quality. The moisture content of the fresh RJ is within the range of 60% to 70%. It was reduced by 3.8% following drying steps of sublimation of lyophilized RJ (Lercker et al., 1993; Piana, 1996; Pourtallier et al., 1990). Inside the hive, the consistency of the moisture content is essentially guaranteed by a constant provision of new supplies by nurse bees, RJ’s inherent hygroscopicity, and the colony’s efforts to maintain a level of ambient moisture. In addition, the non–solubility of some compounds can explain the differences in water content (Sabatini et al., 2009).

Various accredited techniques have been established to quantify the amount of moisture in RJ, such as drying, Karl Fisher, and lyophilization (Teresa et al., 2009). In this context, there are many ways to dry the raw RJ samples, including freeze–drying oven, vacuum oven, and infrared drying (Garcia-Amoedo & Almeida-Muradian, 2007; Lercker et al., 1993; Pourtallier et al., 1990). Among all methods, the classic Karl Fischer titration is the most popular one; it is more accurate because it is based on a chemical reaction that requires the presence of water, extreme specificity for water determination without detecting other volatile substances, and extensive determination range from 0.001% to 100%. Moreover, it’s a rapid approach with minimal sample preparation, whereas other techniques are time–consuming or require equipment. Several studies reported a correlation between the moisture content of fresh RJ and the harvesting period. The moisture content increases rapidly between 24 and 48 hours after grafting, then gradually until 72 hours, and is extremely low on the fourth day (Kanelis et al., 2015; Lercker et al., 1984).

10–hydroxy–2–decenoic acid (10–HDA) content

10–HDA is the major lipid component of the RJ (Honda et al., 2015), also known as queen bee acid (QBA). It is considered a unique bioactive compound in RJ that hasn’t been discovered in other natural products. This compound corresponds to about 70% of its total fatty acids. Accordingly, the 10–HDA determines the pharmacological activities of the RJ and, therefore, serves as a marker component and international standard for the freshness and quality of the RJ (Sabatini et al., 2009; Takikawa et al., 2013). Several studies have shown that 10–HDA has significant antimicrobial activity against approximately 30 Gram–positive and Gram–negative bacteria (Uthaibutra et al., 2023). 10–HDA’s antibacterial activity results from its ability to disrupt bacterial cell membranes. Besides its antiviral properties, 10–HDA has also been found to be antifungal and anti–inflammatory (Ratajczak et al., 2021). Similarly, it inhibited the production of TNF–α, IL–6, and IL–1, pro–inflammatory cytokines, as evident from the minimum inhibitory concentrations (MIC) and minimal bactericide concentrations (MBC) of the 10–HDA on the growth of a pathogenic bacterium (Šedivá et al., 2018). Besides, 10–HDA promotes fibroblast tissue of the collagenous matrix (Kotronoulas et al., 2021).

The minimum concentration of 10–HDA in pure RJ was estimated to be 1.4% (Table 1) following the Chinese Ministry of Agriculture (MOA) regulations and the ISO RJ international standard (ISO 12824:2106, ISO/DIS 12824:2106, 12824:2106, 2016). Its concentration is one of the parameters used to determine RJ quality (Sabatini et al., 2009). The high concentration of 10–HDA in RJ makes it a valuable source of nutrition, offering a range of health benefits to those who consume it. Over time, various methods have been developed to determine the amount of 10–HDA in RJ, such as high–performance liquid chromatography (HPLC), which is considered the most reliable method related to its simplicity and sensitivity (Genç & Aslan, 1999). Other methods include gas chromatography (GC), gas–chromatography–mass spectrometry (GC–MS), and ultra–performance liquid chromatography (Zhou et al., 2007). Capillary zone electrophoresis (CZE) has been used to identify 10–HDA. The results were compared with HPLC and demonstrated a high correlation (p < 0.01). Further, CZE has been suggested as a fast routine analysis test for authenticity assessment (Ferioli et al., 2007). CZE is an effective technique for separating and identifying components in complex samples. Besides, CZE can be used as an alternative to HPLC, as it is faster and requires less sample preparation (Wang et al., 2022).

Recent research has reported the impact of pollen source on the content of 10–HDA by comparing three different monofloral pollen samples of tea (Camellia sinensis), coffee (Coffea arabica), and bitter bush (Eupatorium odoratum L) with the commercial RJ in Thailand. Results showed the lowest quantity of 10–HDA was found in the commercial RJ at 1.14%. In comparison, the highest quantities (1.66–1.67%) were detected in bee colonies fed by bitter bush and coffee (Pattamayutanon et al., 2018).

Amino acid contents

Raw RJ is made mostly of proteins, which make up around 12% to 15% of its content. The family of Major Royal Jelly Proteins (MRJPs) accounts for 80% of these proteins. The gene that encodes Major Royal Jelly Protein1 (MRJP1) is located on chromosome 11 of the honeybee genome (Drapeau et al., 2006). This protein contains essential amino acids and was first identified in 1992 using ion exchange chromatography and Sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The genome of Apis mellifera likely generated ten major royal jelly protein genes (MRJP1–10) via gene duplication of a single–copy protein. These genes are highly expressed in nurse bees’ hypopharyngeal glands (HPG) (Dai et al., 2021). MRJPs are named either according to their molecular weight or to the order in which they were discovered. Some MRJPs can be present in different forms, such as monomers (single structure), oligomers (combined structure), and water–soluble forms (Simuth, 2001).

MRJP–1, also known as royalactin, constitutes over 45% of water–soluble proteins in RJ and exists either as a monomer or as an oligomer called apisin, formed by non–covalent bonding with apisimin, a 5 KDa joining protein. MRJP1 is crucial in accelerating larval development and inducing queen differentiation in honeybees by enhancing body size, promoting ovary development, and reducing developmental time (Kamakura, 2011). MRJP–2 and MRJP–3 exhibit notable variations in molecular weights and isoelectric points across different honeybee species. MRJP–4, first detected in the hypopharyngeal glands (HPG) in 1998, along with MRJP–5, is consistently present in all developmental stages of Apis mellifera. However, MRJP–4’s presence in the HPG is relatively minor compared to other MRJPs. MRJP–5 is distinguished by an extensive repeat region between amino acid residues 367 and 540, spanning 174 amino acids. Proteomic analyses have identified MRJPs 6–9, with MRJP–6, MRJP–7, and MRJP–8 showing no apparent nutritional function in Apis cerana honeybees. MRJP8 and MRJP9 are considered the most ancestral members of the MRJP family. Both MRJP–6 and MRJP–7 are found in the HPG of worker and forager bees, and MRJP7 has also been identified in nurse bees’ brains. Additionally, certain MRJPs, including MRJP–8 and MRJP–9, have been detected in honeybee venom. In 2017, Helbing et al proposed the addition of MRJP–10, discovered in the phylogenetically oldest honeybee species, Apis florea, to the MRJP family.

Most of the RJ’s therapeutic advantages are believed to come from MRJPs (Hossen et al., 2019). MRJPs are effective in curing many diseases. They also have anti–tumor activity and can help reduce stress levels. MRJPs also have antioxidant and neuroprotective properties. Furthermore, MRJPs are known to boost the immune system and can help improve physical and mental performance (Wang et al., 2023). Additionally, they have the potential to reduce cholesterol levels and improve digestion. In addition, MRJPs have been shown to improve liver health, strengthen bone and joint health, lower cancer risk, and reduce diabetes risk (Guo et al., 2021). Furthermore, research has demonstrated that MRJPs can reduce inflammation, reduce heart disease risk, and protect against oxidative stress (Kunugi & Mohammed Ali, 2019). Studies have also shown that they can improve mental clarity, decrease anxiety, and improve overall well–being (Bălan et al., 2020).

MRJP1 is considered the freshness marker for evaluating the quality and authenticity of RJ (Shen et al., 2015). Due to its high specificity, sensitivity, and simple procedure, the enzyme–linked immunosorbent test (ELISA) has become a popular laboratory technique for estimating MRJPs. Various studies have proposed employing a combination of quantitative real–time PCR and mass spectrometry to determine the exact amount of MRJP1 concerning its function in honeybee age polytheism (Dobritzsch et al., 2019).

The protein and amino acid compositions could be modified during RJ storage, affecting its quality during shelf life. Browning reaction is one of these modifications that involve free amino acids (FAAs). By quantifying the FAAs compositions, the highest percentages belonged to proline, lysine, glutamate, β–alanine, phenylalanine, aspartate, and serine. The FAAs level can be determined using gas chromatography with mass spectrometric detection (GC–MS). In addition, storing RJ samples at 4 °C for 10 months preserved the amount of free amino acids, while storing them at room temperature showed a slightly lower level after three months (Boselli et al., 2003).

Sugar content

Determination of the sugar level in RJ samples is imperative for assessing their quality. Sugars have a significant role in promoting and regulating lipids and carbohydrates metabolic pathways. Moreover, sugars could potentially be used as a criterion to check for probable honey and RJ adulteration (Serra Bonvehi, 1991). Consequently, sugars are considered an important part of assessing RJ quality. Research has proven that RJ sugars are mainly fructose, glucose, and sucrose and comprise about 7–18% of fresh samples. In lyophilized RJ, carbohydrates are destroyed by freeze–drying to reach 1.28% (Kausar & More, 2019).

Multiple factors should be considered when assessing the level of major and minor sugars in RJ samples, such as the geographical origin diversity, harvesting season, and the availability of forage sources in the environment compatible with the bees’ natural diets, including nectar and pollen. Various methods were used to quantify sugar, including gas chromatography, which required purification and derivatization phases. Additionally, an enzymatic method developed by Tourn et al. (1980) determines each kind of sugar separately (Lercker et al., 1986; Pourtallier et al., 1990; Serra Bonvehi, 1991). An alternative technique to HPLC has been developed to analyze three types of sugars (fructose, glucose, and sucrose) in RJ. It’s proposed for sugar detection due to its simplicity and ability to detect the presence of maltose in RJ. Maltose content is supposed to be an adulteration sign in RJ. One drawback of using HPLC to analyze RJ is the need for sample preparation to remove lipids and proteins (Bogdanov et al., 1997a; Serra Bonvehi, 1991).

Sugar content tends to fluctuate on a mean basis with dietary changes. Samples collected after some bees were fed sugar syrup and others were left to forage for their food revealed differences in the sugar composition. Hence, research on sugar content can help distinguish between RJs made using different techniques, determine the mode of manufacture, and serve as a supplementary means of detecting unspecified commercial RJs (Daniele & Casabianca, 2012).

Major external factors influencing the quality of the RJ

Environmental conditions

Due to changing feeding patterns, shifting seasons, and climatic extremes, the external environment plays a role in determining bee products’ protein content and amino acid compositions. The key to understanding protein composition diversity in bee products is to consider geographical and temporal (i.e., spatiotemporal) variations in nectar supplies. Temperature–dependent changes in the microstructure and rheological characteristics of RJs indicated that the Brix value, a measurement of the sugar particles in the samples, increased as the temperature climbed. This boost is attributed to the fact that particle swelling causes yield stress (Saricaoglu et al., 2019).

In addition, various anthropogenic activities threaten honeybees’ ability to locate food sources, forage for nectar, and reproduce. Many studies have confirmed pesticides’ harmful effects on honeybees, including neonicotinoids, even if treated correctly. This coincides with the widespread use of pesticides in planted fields. It demonstrates that neonicotinoids affect honeybees’ motor, sensory, and cognitive processes, thereby reducing their foraging activity and possibly lowering their food intake, which could adversely affect the health of the entire colony (Ohlinger et al., 2022).

However, as urbanization spreads, bees’ natural habitats and nectar–rich pastures are diminished, leading to a reduction in the overall population of pollinators worldwide, particularly honeybees. This decrease in pollinator numbers is alarming, as bees are an integral part of the global ecosystem, providing pollination services to many plant species, as well as acting as keystone species in many habitats (Jarvis, 2023). As such, it is imperative to protect bees and their habitats. It has been reported that the risk of disease transfer between wild and managed honeybee colonies has increased (Youngsteadt et al., 2015).

Source of nectar

It has been reported that different floral nectar contains between 2.90% to 33.51% of proteins with more than 16 amino acids (Nicolson & Human, 2013). The properties of bee products, such as honey and RJ, are defined exclusively based on the content of crude proteins in the nectar and pollen grains. The distinctive origin of bee pollens reveals a significant concentration of essential amino acids and proteins. Consequently, the bees feeding on different pollen floral origins at different blooming seasons express distinct protein ratios in their RJ. For example, a study in Saudi Arabia tested the relations between pollen sources, such as alfalfa trees (Medicago sativa L.), summer squash (Cucurbita pepo Thunb), and date palms (Phoenix dactylifera L.) and their protein compositions determined by the chemical measure of protein quality and Essential Amino Acid Index (EAAI) (Taha et al., 2019). The results indicated that the sunflower pollens have the lowest compositions of essential amino acids. At the same time, the highest concentration of crude proteins was detected in alfalfa and date palm pollens. Similarly, a variation in major RJ protein 5 (MRJP5) and minor sugars (raffinose, erlose, and sucrose) was detected in distinct floral periods when the metabolic and proteomic profiles of high RJ–producing strains were observed during migratory beekeeping in China at floral periods of Rape, Cherry, Acacia, Chaste and Tea. Moreover, a considerable difference in the antioxidant activity of RJ was also caused by changes in antioxidant compounds, indicating the importance of the chemical composition data in evaluating RJ quality (Ma et al., 2022).

Lipid composition and content also control the RJ quality. Most fatty acids contain phospholipids (4.34–6.62 mg/100 g). The distinct phospholipids composition of RJ provides special health advantages. In addition, 10–HDA, a monounsaturated fatty acid, comprises more than half of the total fatty acids in RJ, while 96.38 to 125.47 mg/100g returns to polyunsaturated fatty acids. It has been reported that the source of forage plants had a marked effect on the composition of lipids, especially phospholipids and monounsaturated fatty acids (MUFAs) contents (Martin et al., 2019; Yan et al., 2022). Therefore, the botanical origins affect the RJ lipid profiles, affecting RJs targeted bioactivities and quality. A recent study used ultra–high pressure liquid chromatography/ion mobility–quadrupole–time–of–flight–mass spectrometry (UHPLC–IM–Q–TOF–MS) combined with GC–MS quantified nine classes of lipids in RJ samples of Apis mellifera fed on various pollen sources: Camellia sinensis L., Brassica campestris L., maize and pine pollen grains (Yan et al., 2022). The results indicated that RJs of honeybees fed on Brassica campestris L. pollen had more phospholipids than the other RJs. Using stable isotopes with machine learning analyses recently assessed the relationship between environmental conditions and RJ compositions. Specifically, the artificial neural network (ANN) model concluded that isotopes were affected by environmental factors such as heat (Liu et al., 2022).

Harvesting conditions

Harvesting RJ doesn’t require any special techniques like honey. The main challenge is to ensure the hive remains undisturbed while harvesting. RJ is a highly valued product, so it is important to harvest it in a way that does not disrupt the bees or their environment. On average, a colony produces 900 grams of royal jelly throughout the 5–6 month blooming season, making it far less abundant than honey. Most RJ is produced while feeding larvae in the queen cell when the larvae are five days old. At that time, a worker bee’s RJ production exceeds its consumption. Three main steps for extracting the RJ. First, the cell’s thin end is sliced off; second, the RJ is carefully extracted using a micro spatula so as not to harm the larva. Finally, the RJ is stored correctly (Gemeda et al., 2020). The RJ can be collected, strained, and frozen from the cells for long–term storage. It can also be processed further to create a powder or other product (Krell, 1996).

It has been reported that harvest time affects RJ yield and chemical composition. For example, RJ harvested 72 hours after larva grafting has the highest yield, maximum concentrations of crude protein, ash, fructose, and glucose and lowest water content compared with RJ harvested after 24 and 48 hours of grafting. However, the highest lipid contents have been reported to be obtained in harvest after 24 hours of grafting (Al–Kahtani & Taha, 2021). Conversely, another study reported high levels of proteins, polyphenolic chemicals, and many other beneficial components in RJ harvested 24 hours after grafting (Liu et al., 2008). The difference in the composition of RJ in both studies may have arisen from variations in experimental conditions, beekeeping practices, or regional factors influencing RJ production. The post–harvest storage condition is crucial in protecting RJ’s quality by preventing the loss of valuable nutrients, including vitamins and proteins. A study was conducted to monitor protein changes in RJ samples stored at different temperatures for 12 months. A protein identification tool was built on a protein engine and applied to the honeybee genome. Other methods included two–dimensional polyacrylamide gel electrophoresis (2D–PAGE), matrix–assisted laser desorption ionization–time–of–flight mass spectrometry (MALDI–TOF/MS), gel filtration chromatography, nano–liquid chromatography–mass spectrometry, and mass spectrometry (Li et al., 2008). RJ’s proteins can be affected by temperature, MRJP1 was considered sensitive to storage temperature. Studies also showed that the highest concentrations of minerals, including Mg, Ca, K, Na, Fe, Cu, and Mn, were high after two days (48 h) and three days (72 h) of RJ grafting (Al–Kahtani & Taha, 2020).

The storage environment affects Natural honeybee products, including factors such as heat, light, and air. RJ’s shelf life varies depending on storage conditions. For example, storing RJ in a dark, translucent, or opaque ceramic, glass, or plastic container is the most appropriate for preserving its integrity and nutritional content. Storing RJ frozen prevents the degradation of the active proteins. Fresh RJ can be used for up to two years without deteriorating when stored in the freezer (< –18 °C). In contrast, the refrigerator freezer (3 to 5 °C) has a six–month holding period. It is best to avoid storing RJ in rooms with high temperatures or direct sunlight as it can cause a quick loss of nutrients. On the other hand, freezing RJ slows down the process of oxidation, which is the primary cause of RJ’s degradation. Oxidation happens when oxygen reacts with the nutrients in RJ, causing them to break down. This helps maintain the RJ’s nutritional quality for longer (Bogdanov, 2011).

Honeybee species

The queen bee gained many benefits from consuming RJ. Several factors, such as genetics, colony conditions, food flow, and egg–laying of the queen, can affect the production of RJ. In addition, beekeepers and honeybee scientists have improved many effective ways to enhance RJ production. Breeding and genetic selection of specific bee races and strains with high RJ production began in China in the 1980s when they introduced the Italian bee strain (Apis mellifera ligustica) for a high yield of RJ. This hybrid can now produce RJ at a rate 10 times higher than native Italian honeybees (Han et al., 2015; Li et al., 2010).

Several studies have confirmed that different honeybee races have unique activity and colony productivity characteristics. These differences are likely due to the different environmental conditions in which bees live (Khan et al., 2021). Besides, some bee races are more resistant to mites, while others are better pollinators (Le Conte & Navajas, 2008). Beekeepers often crossbred different bee races to create a bee population that exhibited the desired traits, resulting in increased productivity of the colonies. Hybridization is a very effective way to improve bee colonies, and beekeepers have long used this technique to increase productivity. Furthermore, this practice is also beneficial for the environment, as it helps to promote the health of wild bee populations (Ellis & Ellis, 2009).

Other characteristics that distinguish different bee races are the ways to collect and store pollens, the speed at which a colony expands, and the amount of honey and other bee products that can be produced under specific weather and light circumstances (Mouro & Toledo, 2004). Similarly, race factors affect the size of the worker and drone broods and the adult population (Taha et al., 2019). Much research has compared different bee races and monitored their adaptation to specific climate conditions. Each bee race has distinct physical measurements such as body size, weight, head structure, and abdominal characteristics. Therefore, those variations affect pollen production, eggs, larvae, and pupae. All these factors have a significant role in the rate of honeybee survival (Page et al., 2012).

A significant difference between honeybee races appeared in queen cell acceptance and RJ production. Khan et al. (2021) compared two honeybee races, the Italian and Carniolan bee races, and showed a significant difference (p < 0.001) in the quantity of produced RJ per colony, which was 13.10 ± 0.42 g in Italian honeybees, compared with 9.66 ± 0.43 g in Carniolan races. Therefore, studies are necessary to assist beekeepers in selecting bee races with higher potential for RJ production, considering the different characteristics among different honeybee species. Table 2 represents the relation between physicochemical parameters and the RJ’s compositions from the national bibliography of three countries.

Table 2. Physicochemical parameters affecting quality of RJ from various countries’ bibliography.

A. China.

Race	Forage plant	Period	Area	Climate	Elevation	Moisture %		Crude protein %	Total sugar %	Acidity %	Ash %	10–HDA %
Apis mellifera ligustica–hybrid of A. m. ligustica and A.m. arnica	–Brassica campestris L., Tilia amurensis, Vitex negundo L. variety heterophylla	March 2008–June 2009	Northeastern	Extremely cold T = 5.68 °c	+300m above the sea	65.15 ± 0.30		14.11 ± 0.23	12.73 ± 0.30	36.65 ± 0.48	1.08 ± 0.02	1.87 ± 0.05
			West	Drought and cold T= <10 °c	+250m above the sea	64.43 ± 0.25		13.93 ± 0.24	13.58 ± 0.24	36.41 ± 0.51	0.99 ± 0.02	2.01 ± 0.05
			East	Warm and humid T = 16 °c	–200m Lower than the sea	64.72 ± 0.21		14.25 ± 0.10	13.20 ± 0.13	36.04 ± 0.29	0.96 ± 0.02	1.75 ± 0.03
Techniques						Weight loss after drying in a vacuum–drying oven	Multiplying the content of nitrogen	Fehling’s method	Titration with NaOH	Preheating and adding concentrated sulfuric acid	HPLC

B. Saudi Arabia.

Race	Period	Area	Moisture %	Crude protein %	Fructose %	Glucose %	Sucrose %	Maltose %	Lactose %	Acidity [(1mol/1N NaOH) ml/100g}]	Electrical conductivity (µS/cm)	10–HDA%
Apis mellifera	2020	Al–Baha area of Saudi Arabia’s southwest	66.53 ± 4.19	9.88 ± 2.01	3.67 ± 0.81	3.25 ± 1.01	0.91 ± 0.82	0.45 ± 0.53	0.05 ± 0.12	42.4 ± 0.16	646.96 ± 62.91	3.83 ± 1.56
Techniques			refractive index	the total nitrogen method	HPLC	HPLC	HPLC	HPLC	HPLC	Titration with NaOH	Benchtop pH Meter	HPLC

C. Egypt.

Forage plant	Period	Area	Moisture %	Crude protein%	Lipids%	Carbohydrates%	Ash %	10–HDA%
Citrus	April of 2019	Northern of Egypt	66.5 ± 1.27	12.1 ± 0.75	5.34 ± 0.21	15.39 ± 1.00	0.67 ± 0.02	3.2 ± 0.12
Egyptian clover	June of 2019		64.4 ± 0.47	11.5 ± 0.78	5.52 ± 0.25	17.86 ± 0.83	0.72 ± 0.04	2.92 ± 0.17
cotton	September of 2019		64.2 ± 0.62	11.8 ± 0.22	5.25 ± 0.08	18.12 ± 0.59	0.63 ± 0.03	3.1 ± 0.10
Techniques			weight loss	kjeldahl method	Soxhelt extraction	formula: Carbohydrates = 100–(moisture%+protein%+lipids %+ash%)	removing the moisture and organic matter	HPLC

Conclusion

There is an increasing interest in RJ due to its potential benefits in medicine, nutrition, and cosmetics, which has led to significant investment in research and development. The biological and pharmacological properties of RJ are based on its composition, which includes water, 10–HDA, protein, and sugar content. This opens up opportunities for new products that can leverage its unique benefits. The standardization of RJ is crucial for consistency in its quality and effects, which requires advanced techniques in extraction, identification, and quantification. However, the quality of RJ can be influenced by various factors such as environmental conditions, bee nutrition, and the source of nectar, which necessitates careful management in harvesting and storage. The race of honeybees and beekeeping practices also play important roles. Adopting best beekeeping biosecurity practices based on research can enhance RJ production standards globally. It is essential to establish key physical and chemical benchmarks for RJ for international standardization, aiding consumer choice, ensuring safety, and supporting a stable market for RJ producers.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Notes on contributors

Fatima Khalfan Saeed Alwali Alkindi

Fatima Khalfan Saeed Alwali Alkindi is enrolled in master’s degrees in biotechnology from the University of Sharjah. Her thesis was inspired by her upbringing in a mountainous environment, where she developed an interest in beekeeping and its cultural significance. Now, as a livestock researcher in the Department of Agriculture and Livestock at Sharjah University, she emphasizes the importance of preserving this national heritage.

Ali El–Keblawy

Professor Ali Al-Keblawy holds a Ph.D. in Desert Plant Ecology.He is affiliated with The College of Sciences, Department of Applied Biology at the university of Sharjah and Department of Biology, Faculty of Science, Al–Arish University, Arish, Egypt. His research interests include biodiversity conservation, propagation of native plants, and sustainable uses of natural resources.

Fouad Lamghari Ridouane

Dr. Fouad Lamghari Ridouane, Director of Fujairah Research Centre, earned his Ph.D. in Chemistry. His interests include environmental studies and product development.

Shaher Bano Mirza

Dr. Shaher Bano is the Principal Investigator of Data Science and Head of Startup and IP at Fujairah Research Centre, Fujairah, UAE. Her research interests include dietary supplements, native plants, computer-aided drug discovery, and designing.