Royal Jelly Proteins Research Articles

Novel T4 bacteriophage and royal jelly infused disinfectant versus E. coli compared to QACs

Antimicrobial resistance is an increasingly worsening phenomenon in the 21st century having resulted in thousands of deaths per year. Increases in the diffusion of antimicrobial resistance in gram-negative bacteria and prevalence of specific genes leading to resistance have been linked to an excessive usage of quaternary ammonium compounds (QACs) in gram-negative bacteria such as Escherichia coli. This experiment aims to develop a novel disinfectant solution (T4MRJP) that utilizes the entero-bacteriophage t4 and nine major royal jelly proteins (MRJPs) to inhibit the growth of E. coli on a MHA (Mueller Hinton Agar) growth medium. In the experiment, the Kirby Bauer Disk Diffusion Assay was first applied on six MHA plates inoculated with 36 evenly distributed susceptibility disks containing different concentrations of solution for three QAC groups, a positive t4 bacteriophage group, and two T4 groups at 90% and 98% dilution respectively. Results demonstrated a strong positive correlation between the increases of concentration to demonstrated inhibition but a lack of statistical significance between the T4 and QAC groups in regards to the proposed hypothesis. However, the diluted T4 group was just as effective as the QAC groups at inhibiting bacterial growth, especially at higher concentrations of the administered solution. In a second trial, however, the T4MRJP (Major royal jelly protein) cocktail was significantly less effective than the experimented QAC groups compared to the bacteriophage alone.

Antibacterial activity and bee-derived protein content of honey as important and suitable complementary tools for the assessment of honey quality

Honey possesses health-promoting properties; however, industrial processing and manipulation of raw honey can harm on its biological activities, including antibacterial activity. Therefore, this study aimed to compare the honey’s antibacterial activity, its total protein content, and the abundance of the most dominant bee-derived proteins in honey between raw (n = 92) and supermarket (n = 17) samples. We showed that tested raw honey samples were much more effective in inhibiting the growth of Staphylococcus aureus, a model bacterium, at a median minimal inhibitory concentration (MIC) value of 4.5 % compared to supermarket honey samples ceasing bacterial growth at median MIC value of 36 %. Moreover, raw honey samples, tested in this study, contained significantly higher amounts of total protein as well as the content of particular bee-derived proteins (major royal jelly protein 1 (MRJP1), glucose oxidase (GOX), and α-glucosidase) in contrast to supermarket honey samples. These data hint that some supermarket honey samples exhibited strikingly low protein content. Furthermore, α-amylase (diastase) from Aspergillus oryzae was found in the supermarket sample characterised with the lowest protein content. Our findings highlight the burning need to refine and monitor the specific quality parameters, ensuring the authenticity of honey and maintaining its reputation as a functional food.

Royal jelly: a predictive, preventive and personalised strategy for novel treatment options in non-communicable diseases.

Royal jelly (RJ) is a bee product produced by young adult worker bees, composed of water, proteins, carbohydrates and lipids, rich in bioactive components with therapeutic properties, such as free fatty acids, mainly 10-hydroxy-trans-2-decenoic acid (10-H2DA) and 10-hydroxydecanoic acid (10-HDA), and major royal jelly proteins (MRJPs), as well as flavonoids, most flavones and flavonols, hormones, vitamins and minerals. In vitro, non-clinical and clinical studies have confirmed its vital role as an antioxidant and anti-inflammatory. This narrative review discusses the possible effects of royal jelly on preventing common complications of non-communicable diseases (NCDs), such as inflammation, oxidative stress and intestinal dysbiosis, from the viewpoint of predictive, preventive and personalised medicine (PPPM/3PM). It is concluded that RJ, predictively, can be used as a non-pharmacological therapy to prevent and mitigate complications related to NCDs, and the treatment must be personalised.

Major royal-jelly proteins intake modulates immune functions and gut microbiota in mice

In this study, we investigated the effects of major royal jelly proteins (MRJPs) on the estrogen, gut microbiota, and immunological responses in mice. Mice given 250 or 500 mg/kg, not 125 mg/kg of MRJPs, enhanced the proliferation of splenocytes in response to mitogens. The splenocytes and mesenteric lymphocytes activated by T-cell mitogens (ConA and anti-CD3/CD28 antibodies) released high levels of IL-2 but low levels of IFN-γ and IL-17A. The release of IL-4 was unaffected by MRJPs. Additionally, splenocytes and mesenteric lymphocytes activated by LPS were prevented by MRJPs at the same dose as that required for producing IL-1β and IL-6, two pro-inflammatory cytokines. The production of IL-1β, IL-6, and IFN-γ was negatively associated with estrogen levels, which were higher in the MRJP-treated animals than in the control group. Analysis of the gut microbiota revealed that feeding mice 250 mg/kg of MRJPs maintained the stability of the natural intestinal microflora of mice. Additionally, the LEfSe analysis identified biomarkers in the MRJP-treated mice, including Prevotella, Bacillales, Enterobacteriales, Gammaproteobacteria, Candidatus_Arthromitus, and Shigella. Our results showed that MRJPs are important components of royal jelly that modulate host immunity and hormone levels and help maintain gut microbiota stability.

Royal Jelly (Apis mellifera) As an Antimicrobial Activity Inhibition

Royal jelly (RJ) has lots of compounds such as protein, vitamins, and a variety of bioactive compounds (10-hydroxy-2-decanoic or 10-HDA acids). 10-HDA is a lipid fraction component in RJ content with antibacterial and immunomodulatory activity. RJ proteins that have antimicrobial, antifungal, anti-tumor, antidiabetic, and antihypertensive properties. The authors continue their research about the effect of RJ on the inhibitory strength of the given antibacterial activity. L. monocytogenes, S. aureus, and E. coli were among the microbes studied in this research. This study used an experimental method with a complete randomized design (CRD) of 5 treatments and 4 replications, if the treatment showed a noticeable effect (P<0.05) then Duncan’s multiple distance test was continued. According to the findings of this study, the royal jelly variations given had a significant inhibitory effect on antibacterial activity. The inhibitory ability of L. monocytogenes proved that the second treatment had a higher inhibitory power of 30.94±25.786b, in S. aureus, the second treatment gave a moderate inhibitory power of 9.013±1.841a, and in E. coli, the second treatment gave a weak inhibitory power of 3.24±3.748a. The conclusion of studies showed royal jelly with dilution variety could be antimicrobial agent and to be strongest inhibitor in L. monocytogenes and lowest in E. coli. The suggestion of studies needs management in the isolates of bacteria available and should be estimated before we do the research about antimicrobial activity inhibitors.

Evolution of a Cockroach Allergen into the Major Protein of Termite Royal Jelly.

Termites live in colonies, and their members belong to different castes that each have their specific role within the termite society. In well-established colonies of higher termites, the only food the founding female, the queen, receives is saliva from workers; such queens can live for many years and produce up to 10,000 eggs per day. In higher termites, worker saliva must thus constitute a complete diet and therein resembles royal jelly produced by the hypopharyngeal glands of honeybee workers that serves as food for their queens; indeed, it might as well be called termite royal jelly. However, whereas the composition of honeybee royal jelly is well established, that of worker termite saliva in higher termites remains largely unknown. In lower termites, cellulose-digesting enzymes constitute the major proteins in worker saliva, but these enzymes are absent in higher termites. Others identified a partial protein sequence of the major saliva protein of a higher termite and identified it as a homolog of a cockroach allergen. Publicly available genome and transcriptome sequences from termites make it possible to study this protein in more detail. The gene coding the termite ortholog was duplicated, and the new paralog was preferentially expressed in the salivary gland. The amino acid sequence of the original allergen lacks the essential amino acids methionine, cysteine and tryptophan, but the salivary paralog incorporated these amino acids, thus allowing it to become more nutritionally balanced. The gene is found in both lower and higher termites, but it is in the latter that the salivary paralog gene got reamplified, facilitating an even higher expression of the allergen. This protein is not expressed in soldiers, and, like the major royal jelly proteins in honeybees, it is expressed in young but not old workers.

Molecular mechanism of high-pressure processing regulates the aggregation of major royal jelly proteins

The aggregation behavior of major royal jelly proteins (MRJPs) significantly impacts their quality and nutrient bioavailability, yet studies on their aggregate regulation are limited. This study aimed to evaluate the effect of high-pressure processing (HPP) treatment on the aggregation behavior of food-derived proteins, like MRJPs, and to reveal the regulation mechanism at the molecular level. Results showed that HPP treatment, particularly at pressures of 100–200 MPa, effectively disrupted the filaments aggregation of MRJPs, resulting in smaller particle size and uniformity. Higher pressure (400–600 MPa) caused small particles to aggregate and form larger aggregates. Additionally, HPP treatment regulates aggregation behavior mainly by affecting the higher-order structure (tertiary and quaternary structures) of MRJPs, rather than by altering its secondary structure. Protein conformational dynamics based on high-pressure conditions reveal that HPP regulates MRJPs aggregation mainly by affecting interchain hydrogen bonding and hydrophobic interactions. In conclusion, this study provides valuable insights and theoretical support into the regulation mechanism of food-derived protein aggregation and supports the pre-treatment of MRJPs in the development of functional foods.

Proteomic Analysis of Honey: Peptide Profiling as a Novel Approach for New Zealand Mānuka (Leptospermum scoparium) Honey Authentication.

New Zealand mānuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to "not true to label" marketed mānuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in mānuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen mānuka and twenty six non-mānuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve mānuka-specific tryptic peptide markers were present in all mānuka honeys with minor regional variation. By comparison, they had negligible presence in non-mānuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Mānuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for mānuka honey authentication.

Zinc methionine or zinc sulphate supplementation modulate the development of the hypopharyngeal gland and expression of major royal jelly protein genes in Apis mellifera L. bees

AbstractWe analysed whether the source of zinc (inorganic and organic) influences the morphology of the hypopharyngeal gland (HPG) and the relative expression of major royal jelly protein (MRJP) genes in nursing bees at 6 days of age. For this, 15 colonies of Apis mellifera bees were subjected to the following treatments: Zn0 (control), zinc methionine (50 ppm) and zinc sulphate monohydrate (50 ppm) during the off‐season (characterized by food reduction). MRJP1 and MRJP5 gene expression was analysed using real‐time PCR and HPG sections under a microscope. Bee mortality and population development were also evaluated for 30 days. It was observed that the honeybees consumed all sugar syrup containing zinc both in its organic and inorganic forms, which did not promote mortality or affect population development. Regarding HPG morphometry, it was observed that zinc, regardless of the source, promoted the acini area significantly greater than that of the control. Zn methionine promoted a significant increase in HPG area compared with that of an inorganic source. The relative expression of MRJP1 and MRJP5 was not significantly affected compared with the control. We conclude that the mineral Zn promotes an increase in the HPG area and does not alter the pattern of expression of the MRJP1 and MRJP5 genes, and that the organic source of the mineral presents the best results.

Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin–hypopharyngeal gland axis in honeybees

Honeybee diseases are a serious threat to beekeeping and pollination. Transgenerational immune priming (TGIP) has been attracting increasing attention as a promising strategy to protect honeybee colonies from infections. This study investigated whether feeding honeybees (Apis mellifera) with a heat-killed pathogen cocktail can provide them with transgenerational immunity to these pathogens. We found that vitellogenin (Vg) and defensin-1 were highly upregulated in nurse bees upon feeding them with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P cocktail). Pathogen-pattern–recognition receptor genes in the Toll signaling pathway were upregulated in nurse bees upon ingestion of the A + P cocktail. In the nurse bees of the hives supplied with the A + P cocktail, Vg was upregulated in the fat body, and the defensin-1 expression and Vg uptake in the hypopharyngeal glands were induced. Consequently, the major proteins in royal jelly were upregulated. In addition, defensin-1 was upregulated in the queen larvae and young worker larvae in these hives. In correlation, the young worker larvae showed high pathogen resistance to P. larvae infection. Thus, our findings imply that introduction of a heat-killed pathogen cocktail into hives is an efficient strategy for conferring honeybees with social immunity through TGIP.

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein (RJP) at different degrees of hydrolysis (DH) by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34 %, 11.65 %, 15.19 %, 21.38 % and 23.91 %, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20 %. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Analysis of Royal Jelly Apis Cerana as Therapeutic Candidate in Cbavd Based on Bioinformatics Studies

Background: Apis cerana honey bee is a honey bee native to Asia that spreads from Afghanistan, China, and Japan to Indonesia. The productivity of Apis cerana honey bees can produce as much as 2-5 kg of honey per colony in a year. Royal jelly is the queen's nourishment, and it is produced by larvae, adult bees, young worker bees, and male bee larvae. CBAVD contributes to 1–2% of the 20–25% of males with subfertility. Aim: The objective of this study was to evaluate Royal Jelly Apis Cerana as a potential therapeutic candidate for CBAVD using a bioinformatics approach. Method: This research method consisted of converting nucleotides into amino acids, analyzing the three-dimensional structure of Apis Cerana Royal Jelly Protein, Ramachandran Plot Analysis, Analysis of Epitope and Allergen Proteins, and Analysis of Proteins that were antigens and toxins. Results: The research results were conducted on six three-dimensional Apis Cerana Royal Jelly bee protein structures and had very good validity based on the Ramachandran plot, GQME value, and QmeanDisCo value. In addition, this study also obtained the results of proteins that are epitope, antigenic, non-allergenic, and non-toxic. Conclusion: The findings of this study can be used as a basis for therapy against CBAVD.

Effects of Major Royal Jelly Proteins on the Immune Response and Gut Microbiota Composition in Cyclophosphamide-Treated Mice.

Increasing evidence suggests that royal jelly (RJ) has exceptional biological properties, and that major royal jelly proteins (MRJPs) are the key active factors in RJ. The objective of this study was to compare the difference in the protein content between RJ and MRJPs using non-labeled, quantitative proteomics technology, and to investigate the adjustment features and mechanisms of MRJPs on murine immune functions and the composition of intestinal flora in cyclophosphamide-treated mice. Results showed that, during the process of extracting MRJPs, the ratio of the protein types in the main protein and other proteins decreased significantly, except for MRJP1 and MRJP7, which demonstrated that an enriching effect of MRJP1 and MRJP7 was present during the extraction process. Cyclophosphamide-induced mice were orally administered MRJPs. Results showed that the middle-dose group, which received 0.25 g/(kg·bw) of royal jelly main protein, demonstrated a clear impact on the development of the spleen and liver, the quantity of peripheral blood leukocytes, immunoglobulin content, immune factor level, and the proliferation ability of spleen lymphocytes. A 16S rRNA high-throughput sequencing technology analysis showed that MRJPs could improve the component and richness of intestinal flora and raise the immunity of mice. The above-mentioned results indicated that the application of MRJPs is very likely to have an advantage effect on murine immune functions.

A review of different members of Major Royal Jelly Proteins (MRJPs) and their function revised title Overview of different members of MRJPs genes in Honeybee (Apis mellifera) and its biological Activities

A review of different members of Major Royal Jelly Proteins (MRJPs) and their function revised title Overview of different members of MRJPs genes in Honeybee (Apis mellifera) and its biological Activities

Quantitative comparison of manuka and clover honey proteomes with royal jelly.

Royal jelly and honey are two substances produced successively by the worker bee caste. Modern proteomics approaches have been used to explore the protein component of each substance independently, but to date none have quantitatively compared the protein profile of honey and royal jelly directly. Sequential window acquisition of all theoretical fragment-ion spectra mass spectrometry (SWATH-MS) was used to compare protein quantities of bee origin in mānuka and clover honey to royal jelly. Two analysis techniques identified 76 proteins in total. Peptide intensity was directly compared for a subset of 31 proteins that were identified with high confidence, and the relative changes in protein abundance were compared between each honey type and royal jelly. Major Royal Jelly Proteins (MRJPs) had similar profiles in both honeys, except MRJP6, which was significantly more abundant in clover honey. Proteins involved in nectar metabolism were more abundant in honey than in royal jelly as expected. However, the trend revealed a potential catalytic role for MRJP6 in clover honey and a nectar- or honey-specific role for uncharacterised protein LOC408608. The abundance of MRJP6 in mānuka honey was equivalent to royal jelly suggesting a potential effect of nectar type on expression of this protein. Data are available via ProteomeXchange with identifier PXD038889.

Authentication of Apis Cerana and Apis Mellifera Derived Honey Using Major Royal Jelly Protein 2 Gene and Spectroscopy

Honey, as a natural health product, encounter great compositional influence by its entomological, botanical and geographical origin. In Bangladesh, apiculture relies on the honey production by mostly the European honeybee, Apis mellifera. However, the Asian honeybee Apis cerana honey typically costs three times as much as honey from A. mellifera in honey market due to the production limit, variation in nutrient composition, being prone to adulteration by mislabeling or incorporation of two honey. A. mellifera honey may be mislabeled as A. cerana honey or mixed with A. cerana honey by dishonest businesses and beekeepers who are motivated by substantial earnings. The current study used the MRJP2 (Major Royal Jelly Protein 2) gene and spectroscopic analysis to differentiate honey from A. mellifera and A. cerana. Primers for two different species were designed. A. cerana and A. mellifera exhibited amplification products of 212 and 560 bp, respectively. Duplex PCR was able to identify the presence of as little as 1% A. mellifera honey in the mixture. A. mellifera and A. cerana originated honey was differentiated using MRJP2 gene and UV-VIS spectroscopy. The resulted banding pattern can be used to identify adulterated honey. UV-VIS spectroscopy has been used to determine the concentration of DNA at different wavelength for standardizing the corresponding honey types. Amplifications MRJP2 gene fragments in gel electrophoresis and responsiveness in screening for two honey types with their probable admixtures provided an effective tool for discriminating the honey types. DNA Spectrophotometric Spectrum analysis in different wavelengths and ratios also provided a clear-cut discrimination protocol of these two honey types. The findings of these two methods can be used efficiently apply towards authentication of the honey originated by A. mellifera and A. cerana in the honey market of Bangladesh.

The Potential Effect of Royal Jelly on Biomarkers Related to COVID-19 Infection and Severe Progression.

Royal jelly is a yellowish to white gel-like substance that is known as a "superfood" and consumed by queen bees. There are certain compounds in royal jelly considered to have health-promoting properties, including 10-hydroxy-2-decenoic acid and major royal jelly proteins. Royal jelly has beneficial effects on some disorders such as cardiovascular disease, dyslipidemia, multiple sclerosis, and diabetes. Antiviral, anti-inflammatory, antibacterial, antitumor, and immunomodulatory properties have been ascribed to this substance. This chapter describes the effects of royal jelly on COVID-19 disease.

Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

For reasons of high transmissibility and virulence, Alpha (UK, B.1.1.7) and Beta (South African, B.1.351) SARS-CoV-2 variants are classified with other types as variants of concern. Here we report on the influence of royal jelly (RJ) protein fraction (PF)50 (major RJ protein 2 and its isoform X1) on the entry of these variants into the ACE2-human embryonic kidney (HEK) 293 cells using the lentiviral system. The efficiency of PF50 on SARS-CoV-2 replication (RNA-dependent RNA polymerase “RdRp” activity), as well as its impact on bleomycin-induced lung injury in vitro, were also assessed. The PF50 efficiently inhibited infection of kidney cells with the UK and S. African variant spikes of pseudotyped lentivirus particles (IC50 = 7.25 μM and 16.92 μM, respectively) and suppressed the RdRp activity (IC50 = 29.93 μM). Moreover, PF50 displayed protective and therapeutic efficacy against lung injury due to its antioxidant, anti-inflammatory, and angiotensin II blocking activities. The current findings, taken together, offer a novel perspective on PF50 as a promising agent against COVID-19.

A comparative transcriptome analysis of the head of 1 and 9 days old worker honeybees (Apis mellifera).

The role of bees in the environment, economic, biodiversity and pharmaceutical industries is due to its social behavior, which is oriented from the brain and hypopharyngeal gland that is the center of royal jelly (RJ) production. Limited studies have been performed on the head gene expression profile at the RJ production stage. The aim of this study was to compare the gene expressions in 9 and 1-day-old (DO) honeybee workers in order to achieve better understanding about head gene expression pattern. After sequencing of RNAs, transcriptome and their networks were compared. The head expression profile undergoes various changes. 1662 gene transcripts had differential expressions which 1125 and 537 were up and down regulated, respectively, in 9_DO compared with 1_DO honey bees. The day 1th had more significant role in the expression of genes related to RJ production as major RJ protein 1, 2, 3, 5, 6 and 9 encoding genes, but their maximum secretion occurred at day 9th. All process related to hypopharyngeal glands activities as CYP450 gene, fatty acid synthase gene, vitamin B6 metabolism and some of genes involved in fatty acid elongation and degradation process had an upward trend from 1_DO and were age-dependent. By increasing the age, the activity of pathways related to immune system increased for keeping the health of bees against the chemical compound. The expression of aromatic amino acid genes involved in Phenylalanine, tyrosine and tryptophan biosynthesis pathway are essential for early stage of life. In 9_DO honeybees, the energy supplying, reducing stress, protein production and export pathways have a crucial role for support the body development and the social duties. It can be stated that the activity of honeybee head is focused on energy supply instead of storage, while actively trying to improve the level of cell dynamics for increasing the immunity and reducing stress. Results of current study identified key genes of certain behaviors of honeybee workers. Deeper considering of some pathways will be evaluated in future studies.

Detection and pH-Thermal Characterization of Proteinases Exclusive of Honeybee Worker-Fate Larvae (Apis mellifera L.)

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation.