1. Introduction
The nutritional value of honey is determined by sugars, proteins, enzymes, amino acids, phenols, vitamins, aroma compounds and other substances present in honey [
1]. The composition of the variety of compounds in honey determines its biological properties such as its antimicrobial, antioxidant, antiviral, antifungal and anti-inflammatory properties, among others [
2,
3]. It has been revealed that honey inhibits the activity of about 60 bacteria, which are aerobic, anaerobic, Gram-positive and Gram-negative [
4]. Honey is used to preserve food due to its antibacterial properties [
5]. The main compound responsible for honey’s antibacterial effect is believed to be hydrogen peroxide. Hydrogen peroxide in honey is mainly formed during the oxidation of glucose, which is catalyzed by the bee enzyme, glucose oxidase (GOD) [
6]. GOD was detected in honey of different origins, and the amount of hydrogen peroxide produced by GOD and antimicrobial activity varied among honey samples [
7]. Among other components attributable to the non-peroxide antimicrobial activity of honey are bee-secreted proteins, such as major royal jelly (MRJ) proteins (1–5) [
8], defensin-1 [
9,
10] and hymenoptaecin [
10]. The antibacterial properties of honey are employed in the food industry for prolonging the shelf life of the production; thus, they are often added in juice or dairy products, such as yogurt for activating bifidobacteria [
11,
12]. According to our recent studies, GOD was found in fresh bee pollen, dried bee bread, pollen–honey and bee bread mixtures with honey, along with vegetable oils that have been used as supplements in bee products. The activity of this enzyme was higher in the studied bee bread compared to pollen, as well as in all bee bread mixtures with honey and added vegetable oils. The data revealed that fresh pollen and all conserved mixtures with fresh pollen can be stored in a refrigerator at +4 °C room temperatures for one year [
13]. Other substances such as honey sugars and organic acids—i.e., gluconic, acetic, lactic, formic and propionic, among which gluconic acid is predominant—have an influence on honey preservation and the physicochemical and sensory properties of honey [
3,
14].
The content of lipids in fresh and lyophilized royal jelly (RJ) amounts to 4–8 and 15–30%, respectively [
15]. RJ is a product secreted by bees from their hypopharyngeal glands (HPGs) and fed to the bee queens and larvae [
16]. Researchers point out that fatty acids (FAs) present in RJ are mostly composed of medium chain fatty acids, containing 8–12 carbon atoms [
17]. Among FAs,
trans-10-hydroxy-2-decenoic acid (10-HDA) is the main monounsaturated fatty acid (MUFA), accounting for more than 70% of the total fatty acids in RJ [
18,
19]. According to the requirements of the International Organization for Standardization, 10-HDA content for RJ should be more than 1.4% of the total RJ composition [
20]. The 10-HDA is studied mostly via all components of a lipid fraction in RJ. For a long time, these FAs were considered to be a key factor in the antibacterial activity of RJ; however, recently, this role for the above FAs has been questioned [
21,
22], as more detailed RJ lipid profiles of different botanical origins have been determined [
15]. The authors identified 11 FAs in RJ samples which were collected from
Apis mellifera colonies fed by different plant pollen. Among them, decanedioic acid and 2-decenoic acid were the most abundant FAs in all RJ samples. According to the data of this study, it is determined that phospholipids and MUFAs have the greatest influence in differentiating RJs samples of different botanical origins. The findings showed that FAs content and the quality of RJ depends on the type of pollen on which bee colonies are fed [
23]. Pollen collected by bees is necessary for honeybee colonies to produce RJ [
15]. Pollen lipids make up to 13%, and their content and variety depend on the plant source [
24]. Studies show that the contents of 18-carbon-saturated and unsaturated FAs in RJ change when feeding bees with single bee pollen derived from
Acer mono Maxim. and
Phellodendron amurense Rupr. The data indicate that the best quality RJ was collected after 18 or 24 days of bee feeding with selected pollen. A diet with single pollen for bees may destroy their nutritional balance; therefore, pollen mixture is recommended as a better feed for bees [
23].
FAs in monofloral bee bread (BB) and bee pollen (BP) loads collected from
Trifolium pratense L. were identified and quantified via gas chromatography, indicating that the content of two FAs possessing n-3 and n-6 structures was found to be the highest [
25], where α-linolenic (C18:3n-3) in BB and BP varied in the range of 38.3–45.1% and 20.4–35.5%, respectively. The value of linoleic acid (C18:2-n-6) in BB and BP was 4.74 ± 0.13% and 7.57 ± 1.32%, respectively. Docosahexaenoic acid (DHA) was presented both in monofloral BB and BP from
T.
pratense. Bee products containing health-benefiting n-3 fatty acids are exceptional because these FAs cannot be synthesized in the human body and must be obtained through food intake. The enzymes enlongase and desaturase convert alpha-linolenic acid into EPA and DHA, though they produce a relatively small amount of these FAs [
26]. Most studies have confirmed that intake of food with higher n-3 fatty acid content is associated with better body fitness [
27,
28,
29].
Bees collect pollen from different plants, so the content of FAs in this product varies [
30,
31]. Among saturated FAs found in Romanian BP, the predominant ones are palmitic (C16:0) and stearic (C18:0) acids [
31]. The same FAs dominated in the BP from 11 different floral sources from Taiwan [
32]. Among the saturated FAs found in bee pollen samples collected in the Tuscany region of Italy, three dominated, including palmitic acid (C16:0), stearic acid (C18:0) and margaric acid (C17:0) [
33]. Fatty acids composition was determined in Brazilian honey BP. The unsaturated fatty acid (USFA) content in Brazilian pollen varied from 18.6% to 55.9% of the total FA content. Researchers believe that pollen is a good source of USFA [
34], suggesting that the ratio between USFA and SFA is of great importance. Pollen is considered to have high nutritional value when the USFA/SFA ratio is greater than 1. An USFA/SFA value less than 1 indicates reduction of USFA due to storage and dehydration processes [
35]. The USFA/SFA ratio ranged from 2.2 to 6.7 in the pollen collected in India and from 1.91 to 5.86 in the pollen collected in Portugal [
36,
37]. In addition, the ratio of FAs n-3/n-6 is also an important criterion for evaluating the health-beneficial properties of bee pollen. The value of n-3/n-6 ratio ranged from 0.06 to 3.09 in Indian pollen and from 0.17 to 0.52 in Portuguese pollen. Based on the review of the conducted research, the studies show that the diversity and differences of FAs present in bee-collected pollen can be attributed to their origin and the influence of geographical conditions, pollen harvest season and pollen quality, as well as the methodologies used for their identification [
35,
38,
39].
Sea buckthorn oil is extracted from the berries, seeds and peel of the sea buckthorn plant (
Hippophae rhamnoides) [
40]. Usually, the oil obtained from the pulp/peel fraction is blended due to their separation difficulties [
41]. The berries’ pulp and the seed yielded a nutritious oil of different FAs composition [
42]. The main FAs present in seed oil are linoleic (C18:2 n-6), α-linolenic (C18:3 n-3), oleic (C18:1 n-9) and palmitoleic (C16:1 n-7) acids, constituting approximately 40%, 30%, 16% and 0.5% of the total FAs in seed oil, respectively [
42,
43]. Palmitoleic fatty acid is more abundant in peel and pulp oil, accounting for 24% to 36% of the total FAs, respectively [
44]. Palmitoleic acid is known to be valuable for skin care as it supports cellular tissue and wound healing [
45]. In pulp oil, the major saturated fatty acids are palmitic acid (C16:0) and stearic acid (C18:0), [
42]. Palmitic and stearic acids accounted for 11.23–19.12% and 2.15–2.88%, respectively, in the oil of the Indian sea buckthorn seed species [
46]. The presented data on sea buckthorn oil show that the difference between seed and pulp oil lies in the relatively high amount of C16 fatty acids in the pulp oil and the relatively high content of C18 fatty acids in the seed oil. The FAs composition of sea buckthorn oil can be used in plant breeding to assess the nutritional value of their production [
46].
The aim of the study was to (1) evaluate the composition of FAs in sea buckthorn oil, royal jelly and bee-collected pollen, as well as in the mixtures of honey with pollen and with pollen and royal jelly; (2) evaluate the changes in the FAs content and composition in the studied products stored for two years under specified conditions; (3) determine the influence of bee pollen FAs content on the FAs content in the pollen mixture with honey after a two-year period of storage.
4. Discussion
Royal jelly (RJ) is a secretion produced by the hypopharyngeal and mandibular glands of worker honeybees. Bee queens’ larvae are fed with RJ constantly and therefore develop into reproductive queens; while workers’ larvae are fed only for the first three days [
17]. Subsequently, worker bee larvae are fed with worker jelly (WJ), which has a different composition than RJ and is also known as bee queen’s jelly. Significant differences in levels of moisture, protein, 10-hydroxy-2-decenoic acid (10-HDA), fructose (F) and glucose (G) were found between the RJ and WJ samples [
52]. RJ is used in health foods and traditional medicines [
53].
Research indicates that RJ contains a set of C8, C10 and C12 FAs [
54,
55]. We identified and quantified caprylic (octanoic) acid (C8:0) in RJ. Medium-chain hydroxy caprylic FAs (C8:0-OH) in the form of 8-hydroxyoctanoic acid are also found in Polish herbal honey [
56]. Medium-chain FAs are natural components of coconut oil, palm kernel oil and milk, e.g., goat milk, and characterized by saturated forms of caproic C6:0, caprylic C8:0 and capric C10:0 FAs [
57,
58].
The lipid content of RJ ranged from 7.0% to 18.0% and was mainly composed of hydroxy FAs with 8–12 carbon atoms, which accounted for 90% of the total lipid content [
59]. It has long been considered that the main FA in royal jelly is 10-hydroxy-2-decenoic acid (10-HDA) [
21]. The content of 10-HDA in RJ varies depending on the plant source that the bees forage during the production of royal jelly, as well as the bee strain and other factors [
60]. However, it is doubtful whether this acid can be used to evaluate the freshness of royal jelly or as a marker with which to assess the quality of royal jelly [
61]. According to the requirements of international trade, fresh royal jelly must contain no less than 1.8% of 10-HDA [
60].
Fresh RJ is sensitive to light, heat and air. Therefore, special precautions must be taken to preserve the biological properties of RJ during the shelf period [
62]. As a result, recommendations are given regarding the storage conditions of RJ and its preparation for longer storage. The requirements are as follows: after collection, RJ should be immediately transferred into a dark and airtight container. If RJ is intended for quick use, it can be stored in refrigerator at 0–5 °C, while for longer storage, RJ should be placed in a freezer at the temperature of −18 °C or below. It is also stated that since there are no criteria for determining the “safety” limits for product activity, storage and shelf-life should be as short as possible. During storage, RJ must be packed in dark, air-tight containers that are tightly closed.
Our study indicated that during the RJ storage for 1.5 years, out of 30 identified FAs, the amount of 13 FAs had been reduced significantly. The remaining content for two FAs was higher than 50.0%. However, for nine FAs, the content reduced from 39.3% to 3.14%. The smallest amount of FA remained for the following acids: C22:5n-3; C16:1n-7trans; and C20:3n-3. The residual content of the latter acids was less than 10.0%.
We identified and quantified 8 saturated and 22 unsaturated FAs in fresh RJ. The six long-chain saturated fatty acids are as follows: C14:0; C15:0; C16:0; C18:0; C22:0; and C24:0 (
Table 1 and
Table 2). The analysis revealed a variety of long-chain USFAs in RJ (
Table 2). The α-linolenic acid (ALA; 18:2n-3) and linoleic acid (LA; 18:2n-6
cis) were found in contents of 4.35% and 5.83%, respectively. The ratio of n-3/n-6 for these long-chain polyunsaturated fatty acids (PUFAs) accounted for 0.75. It is recommended to use food containing PUFAs in the n-6/n-3 ratio, which is 4–5/1 [
63]. According to our data, the total content of n-6 PUFAs exceed n-3 by 1.8 times (
Table 3). The following USFA from the n-3 family were also identified in RJ: eicosapentaenoic acid (EPA); docosahexaenoic (DHA); eicosatrienoic; and docosapentaenoic. This indicates a proper FAs balance in RJ. The major n-3 FA in the Western diet is ALA [
64]. The author confirms that Western diets lack eicosapentaenoic and docosahexaenoic acids (EPA and DHA). According to our previous research, EPA and DHA were determined in bee bread [
65].
When storing RJ in the prescribed conditions for 18 months, two FAs had more than 50.0% remaining content, and the other two were close to 50.0%. However, nine FAs were reduced from 39.3% to 3.14%. The smallest amount remained for the following FAs: C22:5n-3; C16:1n-7trans; and C20:3n-3. The residual content of these FAs was less than 10.0%.
The buckthorn oil we used in this study contained 94.5-fold higher amounts of linoleic acid (LA; 18:2n-6) than n-3 PUFAs (
Table 3). In order to maintain a balanced ratio of n-3/n-6 FA of SBO, which is produced with high content of linoleic acid, it should be mixed with other types of oil that are higher in n-3 PUFAs. The balanced ratio of LA to ALA fatty acids can be improved by using flaxseed oil, which is the richest source of α-linolenic acid (C18:3, omega–3), accounting ˃50.0% of all edible oils. In order to obtain high-quality oil with 1:1 or 1:2 ratios of n-3 and n-6 FAs, selected flax varieties can be sown. [
66].
EPA and DHA are nutritionally significant long-chain PUFA produced by the microalgal species [
67]. Therefore, microalgal are considered as aquaculture plant feed [
68]. These FAs, sometimes called marine omega-3s, are found in seafood (especially fatty fish) and are also used in pharmaceutical products. EPA and DHA are physiologically active acids [
69]. Our studies have shown that these acids are also present in some bee products.
Seeds of flax (
Linum usitatissimum) and camelina (
Camelina sativa) contain high amounts of ALA, while linoleic acid (C18:2n-6
cis) was found in its highest concentration in hemp (
Cannabis sativa) seeds [
70]. In modern agriculture, animal feeds have been largely replaced by plant-based feeds to enhance production. As a result, the ratio of FAs required by the human body has changed in many food products. A suitable FA ratio for human health is considered to be n-3/n-6 FA of 1:1 or 1:2, and it can be used as a supplement for a balanced diet [
27]. According to other data, it is indicated that an FA ratio of n-6/n-3 from 1:1 to 5:1 is optimal for human health [
71]. Three known EFAs—C15:0, α-linolenic (omega-3) and linoleic acid (omega-6)—must be obtained from the human diet because human body does not produce them naturally [
72].
Our study demonstrates differences in the concentration of USFA in BP and BPH compared to prepared mixtures. Total n-3 FAs content was found to exceed n-6 content by about three times in BP as well as BPH in the first year and after two years of storage (
Table 6). The data show that the level of n-3 FAs was slightly lower in BPH + 1% SBO samples compared to BPH + 1% (
w/
w) SBO + 2% (
w/
w) RJ samples, where the n-6/n-3 ratio varied in the range 0.79–0.91. The results suggest that by adding the SBO to the mixtures, the content of n-3 FAs can be reduced.
Honeybee products contain different components, and the activity of a honeybee product results from synergic effects of all its components [
73]. Research suggests that stingless bees produce honey with antibacterial properties that has no peroxide activity [
74]. The antibacterial properties of honey are related to its peroxide and non-peroxide activity. Peroxide-related honey activities were described previously [
73]. The most specific components of honey are proteins of honeybee origin. Authentic honeybee proteins found in honey are royal jelly proteins, which include nine members and are designated as MRJP 1–9. The most abundant protein of this family is oligomer MRJP1 in complex with apisimin or its monomer, also known as apalbumin1 or royalactin. MRJP1oligomer protein has also been identified in bee bread [
75]. MRJP1 oligomer in complex with apisimin has molecular mass of 280–420 kDa, and MRJP1 monomer has a 55 kDa [
8,
76].
The monomer of MRJP1, known as royalactin, is reported to be the major factor inducing differentiation in queen bees and honeybees [
77]. Several studies have demonstrated the antibacterial properties of royalisins and suggested their uses as potential antimicrobial natural peptides. RJ proteins and peptides have been proposed to be responsible for the immunostimulating properties and antibiotic activity of honey. [
78,
79]. Previously, we identified the main proteins of plant and the honeybee
Apis mellifera carnica origin using different mass spectrometry (MS) techniques. The data revealed a range of RJ proteins MRJP1–MRJP9 with a molecular mass of 45.0 kDa to 70.24 kDa in buckwheat honey [
80]. The activities of enzymes catalase and glucose oxidase were estimated on the native PAGE in buckwheat honey. Therefore, we can hypothesize that the RJ proteins present in buckwheat honey supplemented its antibacterial properties. The same RJ proteins (MRJP1–MRJP2) were also detected in rape seed (
Brassica napus L.) honey by our research group [
81]. Each RJ protein found in rape seed honey has a different molecular weight than in buckwheat honey. Differentiation among RJ proteins indicates their relationship with the botanical origin of honey [
82].
The honey produced by
A. mellifera contains minor components derived from the nectar collected from plants, including different antimicrobial peptides of bee origin; therefore, honey cannot be considered simply a carbohydrates-rich food [
83]. When used for preserving bee products, honey protects the activity of glucose oxidase during storage [
13]. The quality of honey is associated with its authenticity. Therefore, one of the RJ proteins, identified as Apalbumin1 and also known as Apa1 or Major Royal Jelly Protein 1 (MRJP1), has been proposed as a marker for honey authenticity and quality [
84]. The evaluation of protein diversity and the use of these ingredients as markers to assess honey naturalness is the subject of modern research [
84]. Consumers usually prefer mostly monofloral honey and seek information about its quality. Therefore, various studies of honey components and all other bee products can be informative in order to evaluate their quality. Biomarkers such as EPA and DHA are proposed to assess the authenticity and biological value of bee products for functional food.