1. Introduction
Royal jelly (RJ), a substance secreted by the hypopharyngeal and mandibular glands of nurse honey bees (workers), serves as a crucial nutritional source for young larvae destined to become queen honey bees [
1]. The process through which RJ induces larvae to become queens involves DNA methylation and altered gene expression, as demonstrated by an earlier study [
2]. As nutrition plays a key role in the caste differentiation of honey bees, the chemical composition of RJ has attracted attention. Given the pivotal role of nutrition in honey bee caste differentiation, there is a growing interest in the chemical composition of RJ.
The nutrient composition of RJ is of significant interest, particularly its protein content, mineral composition, and high fructose content. RJ also contains the principal fatty acid, 10-hydroxy-2-decenoic acid (10-HDA), known for its diverse biological activities such as antimicrobial properties and immune modulation [
3,
4,
5]. These notable bio-functional properties, coupled with the growing awareness of natural products, have led to an increased demand for RJ and other hive products. China leads in RJ production, with other Asian countries notably increasing production over the past decade.
The exceptional biological properties of RJ have attracted considerable attention and commercial interest from various industries, including pharmaceuticals, functional foods, cosmetics, and manufacturing [
6].
Studies suggest RJ has potential bio-functional benefits, including antioxidant properties, immune support, anti-inflammatory effects, and potential contributions to skin health, cognitive function, energy levels, and hormonal balance [
7,
8,
9,
10]. The primary functional role of royal jelly is frequently associated with the major royal jelly proteins (MRJPs). MRJPs exhibit diverse biological functions beyond their role in honey bee larval development. They, alongside royalactin, contribute significantly to royal jelly’s antibacterial activity, particularly against Gram-positive bacteria [
11]. Notably, MRJP1 has been observed to promote cell proliferation and albumin production in rat hepatocytes, potentially serving as substitutes for growth factors in cell culture media [
12]. Additionally, it exhibits antioxidant, anti-tumor, and immune-modulating properties [
13]. MRJP2 and MRJP3 demonstrate immunomodulatory effects, with MRJP3 showing promise as an anti-allergic and anti-inflammatory agent [
14,
15]. While MRJP4 contributes nutritive components to royal jelly, its expression in the hypopharyngeal gland is relatively low compared to other MRJPs [
16,
17]. MRJP6, MRJP7, and MRJP8 appear to lack nutritional functions in
Apis cerana, with MRJP8 and MRJP9 being considered ancestral members of the MRJP family [
12].
This concern is reflected in the efforts of several countries, such as China, Switzerland, Bulgaria, Brazil, and Uruguay, to establish quality standards for RJ [
6,
18]. Leading producers of RJ include China, Japan, the USA, Canada, Australia, and certain European countries. While Korea’s RJ production is increasing, official statistics are not yet available.
Technological advancements, including the development of high-yielding honey bee genetic strains, modifications in feeding practices to increase RJ production, and improvements in harvesting techniques, have enhanced production [
19,
20,
21]. However, it is equally important to maintain the quality and standards of RJ [
22]. Nonetheless, there is currently no legislative framework for RJ standards in Korea. Consequently, it is essential to evaluate the chemical composition of RJ produced by local apiaries to assess its quality according to international standards and to investigate the factors influencing royal jelly quality.
Furthermore, the nutritional composition of RJ, a protein-rich substance typically comprising 9–18% protein for fresh RJ [
6], may be influenced by the honey bees’ dietary intake, particularly in terms of the protein content of their feed. Stimulating royal jelly production often involves providing selected colonies with supplementary feed, typically consisting of sugar syrup or honey, in addition to pollen patties. This practice ensures that the nurse bees receive adequate nourishment to produce high-quality royal jelly. Prior research by Ghosh and Jung [
23] has demonstrated that the composition of pollen patties, derived from various bee pollen sources, impacts the peptide levels within RJ. Nevertheless, the impact on additional nutritional factors such as proximate composition, amino acid profile, 10-HDA, and minerals was not discernible, possibly because of the relatively consistent protein content between oak and rapeseed bee pollen, and consequently, the resulting pollen patties [
23]. However, the impact of using honey and sugar syrup as feeding supplements on royal jelly quality remained to be determined. Honey, being nutrient-dense with compounds beyond carbohydrates compared to sugar syrup, could potentially enhance the quality and nutritional value of royal jelly. Therefore, to investigate this further, we collected RJ samples from 11 different commercial apiaries across diverse regions of South Korea to evaluate their nutritional characteristics. Stable carbon isotope analysis allowed us to categorize the samples into two groups, corroborated by information obtained from the respective apiaries regarding the diet of the honey bees. We identified samples produced by honey bees that were fed either honey or a sugar solution. Subsequently, we conducted a comparative analysis of the nutritional profiles of RJ from both feeding groups to ascertain whether the diet of honey bees influences the nutrient composition of RJ.
4. Discussion
RJ samples derived from colonies fed with sugar syrup showed significantly elevated levels of sucrose and stable carbon radioisotope values and lower levels of fructose compared to samples from colonies fed with honey as a feed supplement. On the other hand, royal jelly samples produced by honey bees fed with honey, characterized by higher fructose, lower sucrose, and elevated levels of certain minerals, indicate that nutrient-dense honey might positively impact the quality and nutritional value of royal jelly.
Moisture content was found to be in agreement with previously published scientific reports on RJ [
29,
30]. However, the value obtained for RJ samples in the present study was less than the moisture content reported in some other studies and standards [
22,
31,
32]. Overall, the moisture content of the fresh RJ is within the range of 60 to 70% [
6]. The slightly lower moisture content of our royal jelly can be attributed to the fact that it was harvested from temperate regions with relatively low relative humidity (RH). This likely explains why the moisture content of royal jelly samples in this study falls on the lower end of the range reported in the scientific literature. For instance, honey harvested from tropical regions with high RH and temperatures requires honey bees to expend more energy to reduce moisture during ripening compared to honey from temperate regions with lower RH and temperatures. This indicates that indigenous honey bees in tropical areas might ultimately cap honey at higher moisture levels than those traditionally shown for
A. mellifera honey [
33]. Moisture content is important to maintain the freshness and consistency of RJ. Higher moisture content may lead to faster spoilage. The moisture content of the RJ samples in this study was in the lower margin, which implies that it could potentially retain beneficial properties for human consumption or other applications, and at the same time, it helps increase preservation.
Sabatini et al. [
6] reported that total fructose, glucose, and sucrose content of RJ accounted for 7 to 18%. Based on the available literature, Sabatini et al. [
6] also provided a range for fructose (3–13%), glucose (4–8%), and sucrose (0.5–2%) content of RJ. Wytrychowski et al. [
34] reported the carbohydrate content in commercial RJ as follows: fructose ranged from 2.41% to 7.53%, averaging 5.42%; glucose ranged from 3.22% to 7.62%, averaging 5.70%; and sucrose ranged from not detected (n.d.) to 3.85%, averaging 1.42%. For Italian RJ, fructose ranged from 2.73% to 5.10%, averaging 4.04%; glucose ranged from 2.00% to 5.85%, averaging 4.55%; and sucrose ranged from 0.07% to 3.18%, averaging 0.93%. In French RJ, fructose ranged from 2.80% to 6.04%, averaging 4.55%; glucose ranged from 4.17% to 7.40%, averaging 5.79%; and sucrose ranged from n.d. to 1.29%, averaging 0.16% [
34]. In this study, however, a contrasting scenario with respect to sucrose content was revealed in case of six samples where significantly higher sucrose content was found in RJ. The higher sucrose content presumably depends on the sugar solution feeding of honey bee. The consumption of sugar syrup as a feeding supplement for honey bees results in royal jelly with significantly higher sucrose and lower fructose content compared to the honey-fed group. This suggests that the high sucrose content in the feed is likely incorporated into the synthesis of royal jelly. For instance, sugar-fed honey bees produce honey with notably high sucrose levels and low fructose levels [
35], which aligns with the findings of this study on royal jelly. However, the specific metabolic pathways involved in this process have yet to be investigated. The observation of higher sucrose levels in RJ from sugar-syrup-fed bees aligns with the findings of Daniele and Casabianca [
36], who demonstrated elevated sucrose content in RJ from the hive fed with cane sugar. Typically, RJ is considered to contain a little higher amounts of fructose than glucose [
30], a trend consistently observed in our routine laboratory analyses (CJ, unpublished data), but this study revealed a higher glucose content relative to fructose, a pattern also noted in several earlier studies despite variations in the fructose-to-glucose ratio [
34,
36,
37,
38,
39,
40]. Based on the carbohydrate composition obtained in the present study, we assume that it is a common practice to feed honey bees with sugar solution, which in turn is being reflected in the sugar composition of RJ.
Sabatini et al. [
5] reported that for fresh RJ, 10-HDA content should be more than 1.4%. Wang et al. [
30] estimated 10-HDA for 2-day-old RJ on a fresh-weight basis 3.72 g per 100 g, but after that, it was within the range of 2.10 to 2.58%. The difference in 10-HAD content might be due to geographical location, honey bee subspecies, and flora, which significantly influence the 10-HAD content of royal jelly, as demonstrated by Wei et al. [
41]. 10-HDA also exhibits functional properties in regard to human health like bacteriocide and anti-inflammatory activity in human colon cancer cells [
2], anti-inflammatory effect [
42], melanogenesis inhibitor [
8], and antiproliferative effect on human neuroblastoma cells [
43].
Stable carbon isotope analysis detects honey adulteration by measuring the ratio of
13C to
12C isotopes. Natural honey from C3 plants has a distinct isotopic signature, while common adulterants like corn syrup or cane sugar (from C4 plants) have a different isotopic ratio. By comparing the isotopic ratios, deviations from the expected values can indicate the presence of adulterants [
44]. We hypothesize that if honey bees are fed with a sugar supplement, the royal jelly they produce will have a high
13C content (low absolute value). Conversely, if they are fed with a honey supplement, the royal jelly will have a low
13C content (high absolute value), similar to what is observed in honey [
44]. In our current investigation, we found a significant difference in
13C stable isotope levels between the two types of royal jelly.
Not many reports are available on the amino acid composition of RJ. Sabatini et al. [
5] reported that the total protein content of fresh RJ was within the range of 9–18%. Given the potential for overestimation when using different methods to measure total protein content, since nitrogen may be present in non-protein compounds also [
45], assessing the total amino acid content, particularly essential and non-essential proteinogenic amino acids, offers a more accurate alternative for understanding nutritional value. The total amino acid content for the RJ samples was within the range of 10.7 to 13 g per 100 g of RJ on an as-is basis, which is in agreement to the protein content (11.6–12.2%) of harvested RJ reported by Howe et al. [
29]. The results of this study align with the recent findings of Wang et al. [
46], which demonstrated no significant difference in the majority of amino acids in RJ between the sucrose-fed and honey-fed treatment groups. Overall, essential amino acids accounted for 40.7 to 43.3% of the total amino acids. Aspartic acid was found to be predominant, followed by glutamic acid. Except tryptophan, all the essential amino acids were present in the RJ samples. Among the essential amino acids, leucine was the most abundant. A similar distribution pattern of amino acids was found in previous studies [
32,
47]. Essential amino acids are indispensable for human health, serving as the fundamental building blocks of proteins and participating in numerous physiological functions. Lysine, for instance, is crucial for collagen synthesis, tissue repair, and calcium absorption, impacting bone health and wound healing, supporting immune function, among others [
48,
49]. Branched-chain amino acids (BCAAs) like leucine, valine, and isoleucine are vital for muscle protein synthesis, energy production, and exercise performance [
50]. Additionally, amino acids contribute to neurotransmitter production, immune function, and hormone regulation [
51]. Since the body cannot synthesize essential amino acids endogenously, their intake through dietary sources is imperative for maintaining overall health and wellbeing, supporting growth, development, and optimal bodily function. Therefore, consumption of RJ could be beneficial for human health.
The findings of this study indicated that there was no impact of feeding on 10-HDA and amino acid levels, suggesting that the metabolic pathways related to lipid and protein synthesis remain largely unaffected by the type of feed provided. However, a comprehensive investigation into the specific metabolic pathways influenced by different feeding practices has yet to be conducted. This future research could provide deeper insights into how various feeds affect the overall metabolic processes in honey bees.
The mineral contents, which are nutritionally significant, of the royal jelly samples examined in this study were consistent with those reported in previous research [
23]. The differences in mineral content, particularly potassium, iron, manganese, and magnesium, between the two groups may be attributed to the higher nutrient density, especially in minerals in honey compared to sugar syrup. RJ holds a pivotal role in human health owing to its rich mineral content. Essential minerals such as calcium, phosphorus, and magnesium support bone health by aiding in bone formation and density. Potassium is essential for maintaining fluid balance, nerve function, and muscle contraction, including heart rhythm. It also helps regulate blood pressure and supports bone health by neutralizing acids that can deplete calcium. RJ’s potassium-to-sodium ratio could help maintain electrolyte balance, crucial for nerve function and blood pressure regulation. Additionally, minerals like zinc, iron, and copper facilitate various metabolic processes, bolstering immunity and energy production. Moreover, RJ’s provision of antioxidants, including copper, manganese, and zinc, combats oxidative stress, promoting healthy aging [
7,
23]. These minerals also contribute to hormonal balance and overall wellbeing. In essence, integrating RJ into the diet offers a comprehensive array of minerals vital for optimal physiological functioning and resilience against illness.