Major Royal Jelly Proteins Research Articles

SIRT3, a New Hope in Liver Diseases from Pathogenic Mechanisms to Therapeutic Strategies.

The liver, the largest internal organ in the human body, regulates multiple reactions and processes, including detoxification, regeneration, and immune defense. Liver diseases have emerged as a significant global public health issue. Numerous studies have indicated that the mitochondrial deacetylase SIRT3 has played various roles in the pathogenesis and pathological progression of liver diseases. Objectives: This review aims to explore the advances in the study of SIRT3 and liver disease and review possible mechanisms. Natural and chemical activators of SIRT3 are also discussed. The role of SIRT3 in the pathogenic mechanisms and therapeutic strategies of liver disease is summarized by reviewing Pubmed. SIRT3 alleviates liver diseases by regulating fatty acid metabolism, mitochondrial function, and immune-inflammatory response. Meanwhile, Withaferin A, lipoic acid, major royal jelly proteins, and berberine can activate SIRT3 or upregulate its expression, thereby alleviating liver damage. SIRT3 can effectively slow down the progression of liver disease and protect the liver from further damage. The use of SIRT3 as a pharmacological target for the treatment of liver disease is a potential therapeutic approach.

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

Royal Jelly: Biological Action and Health Benefits.

Royal jelly (RJ) is a highly nutritious natural product with great potential for use in medicine, cosmetics, and as a health-promoting food. This bee product is a mixture of important compounds, such as proteins, vitamins, lipids, minerals, hormones, neurotransmitters, flavonoids, and polyphenols, that underlie the remarkable biological and therapeutic activities of RJ. Various bioactive molecules like 10-hydroxy-2-decenoic acid (10-HDA), antibacterial protein, apisin, the major royal jelly proteins, and specific peptides such as apisimin, royalisin, royalactin, apidaecin, defensin-1, and jelleins are characteristic ingredients of RJ. RJ shows numerous physiological and pharmacological properties, including vasodilatory, hypotensive, antihypercholesterolaemic, antidiabetic, immunomodulatory, anti-inflammatory, antioxidant, anti-aging, neuroprotective, antimicrobial, estrogenic, anti-allergic, anti-osteoporotic, and anti-tumor effects. Moreover, RJ may reduce menopause symptoms and improve the health of the reproductive system, liver, and kidneys, and promote wound healing. This article provides an overview of the molecular mechanisms underlying the beneficial effects of RJ in various diseases, aging, and aging-related complications, with special emphasis on the bioactive components of RJ and their health-promoting properties. The data presented should be an incentive for future clinical studies that hopefully will advance our knowledge about the therapeutic potential of RJ and facilitate the development of novel RJ-based therapeutic opportunities for improving human health and well-being.

Exploring the effect of high-pressure processing conditions on the deaggregation of natural major royal jelly proteins (MRJPs) fibrillar aggregates

High pressure processing is a safe and green novel non-thermal processing technique for modulating food protein aggregation behavior. However, the systematic relationship between high pressure processing conditions and protein deaggregation has not been sufficiently investigated. Major royal jelly proteins, which are naturally highly fibrillar aggregates, and it was found that the pressure level and exposure time could significantly promote protein deaggregation. The 100–200 MPa treatment favoured the deaggregation of proteins with a significant decrease in the sulfhydryl group content. Contrarily, at higher pressure levels (>400 MPa), the exposure time promoted the formation of disordered agglomerates. Notably, the inter-conversion of α-helix and β-strands in major royal jelly proteins after high pressure processing eliminates the solvent-free cavities inside the aggregates, which exerts a 'collapsing' effect on the fibrillar aggregates. Furthermore, the first machine learning model of the high pressure processing conditions and the protein deaggregation behaviour was developed, which provided digital guidance for protein aggregation regulation.

The characterization of Apis cerana and Apis mellifera mrjp2 gene in Indonesia and the phylogeny relationship with mrjp family genes

The major royal jelly protein 2 (mrjp2) gene is one of the molecular markers that can discriminate between Apis cerana Fabricius and A. mellifera Linnaeus. Due to the lack of mrjp2 gene sequences registered in GenBank for Indonesian A. cerana and A. mellifera, DNA characterization and bioinformatics analysis were needed. This research aimed to characterize the exon-intron organization of mrjp2 genes for both Indonesian bee species and analyze the phylogenetic relationship with other mrjp family genes. DNA samples of Apis cerana and A. mellifera, collected from Bogor, were amplified using MF-MR primer at annealing temperatures of 47 oC and 50 oC, respectively. The length of A. cerana and A. mellifera DNA sequences were 579 and 597 bp, respectively. The DNA sequences of both species were comprised of partial exons 1, introns 1, exons 2, introns 2, and partial exon 3. The number of putative amino acids of A. cerana and A. mellifera mrjp2 genes were 111 and 123, respectively. We confirmed that the partial MRJP2 putative amino acids of both honey bees belong to the MRJP family and contained the peptide signal in the 14 first amino acid sites. Nucleotide variation of the mrjp2 gene in A. cerana was higher than A. mellifera. Phylogenetic tree construction showed that A. cerana and A. mellifera form a monophyletic clade with the A. mellifera mrjp7 gene and another mrjp family gene clustered as reported in a previous study.

Genome-wide identification of yellow gene family in Hermetia illucens and functional analysis of yellow-y by CRISPR/Cas9.

The yellow gene family plays a crucial role in insect pigmentation. It has potential for use as a visible marker gene in genetic manipulation and transgenic engineering in several model and non-model insects. Sadly, yellow genes have rarely been identified in Stratiomyidae species and the functions of yellow genes are relatively unknown. In the present study, we first manually annotated and curated 10 yellow genes in the black soldier fly (BSF), Hermetia illucens (Stratiomyidae). Then, the conserved amino acids in the major royal jelly proteins (MRJPs) domain, structural architecture and phylogenetic relationship of yellow genes in BSF were analyzed. We found that the BSF yellow-y, yellow-c and yellow-f genes are expressed at all developmental stages, especially in the prepupal stage. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) system, we successfully disrupted yellow-y, yellow-c and yellow-f in the BSF. Consequently, the mutation of yellow-y clearly resulted in a pale-yellow body color in prepupae, pupae and adults, instead of the typical black body color of the wild type. However, the mutation of yellow-c or yellow-f genes did not result in any change in color of the insects, when compared with the wild type. Our study indicates that the BSF yellow-y gene plays a role in body pigmentation, providing an optimal marker gene for the genetic manipulation of BSF.

Investigating the Protein-Based Therapeutic Relationship between Honey Protein SHP-60 and Bevacizumab on Angiogenesis: Exploring the Synergistic Effect through In Vitro and In Silico Analysis.

Honey is a natural product produced by honeybees, which has been used not only as food but also as a medicine by humans for thousands of years. In this study, 60 kDa protein was purified from Pakistani Sidr honey named as SHP-60 (Sidr Honey Protein-60), and its antioxidant potential and the effect of Bevacizumab with purified protein on in vitro angiogenesis using human umbilical vein endothelial cells (HUVEC) were investigated. We further validated the molecular protein-protein (SHP-60 with Bevacizumab) interactions through in silico analysis. It showed very promising antioxidant activity by reducing 2,2-diphenyl-1-picrylhydrazyl free radicals with a maximum of 83% inhibition at 50 μM and an IC50 of 26.45 μM statistically significant (**p < 0.01). Angiogenesis is considered a hallmark of cancer, and without it, the tumor cannot grow or metastasize. Bevacizumab, SHP-60, and both in combination were used to treat HUVEC, and the MTT assay was used to assess cell viability. To demonstrate in vitro angiogenesis, HUVEC was grown on Geltrex, and the formation of endotubes was examined using a tube formation assay. HUVEC viability was dose-dependently decreased by Bevacizumab, SHP-60, and both together. Bevacizumab and SHP-60 both inhibited angiogenesis in vitro, and their combination displayed levels of inhibition even higher than those of Bevacizumab alone. We investigated the protein-protein molecular docking interactions and molecular dynamics simulation analysis of MRJP3 (major royal jelly protein 3) similar to SHP-60 in molecular weight with both the heavy chain (HC) and light chain (LC) of Bevacizumab. We found significant interactions between the LC and MRJP3, indicating that ASN468, GLN470, and ASN473 of MRJP3 interact with SER156, SER159, and GLU161 of LC of Bevacizumab. The integration of experimental data and computational techniques is believed to improve the reliability of the findings and aid in future drug design.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Jelleine, a Family of Peptides Isolated from the Royal Jelly of the Honey Bees (Apis mellifera), as a Promising Prototype for New Medicines: A Narrative Review.

The jelleine family is a group of four peptides (jelleines I-IV) originally isolated from the royal jelly of honey bee (Apis mellifera), but later detected in some honey samples. These oligopeptides are composed of 8-9 amino acid residues, positively charged (+2 to +3 at pH 7.2), including 38-50% of hydrophobic residues and a carboxamide C-terminus. Jelleines, generated by processing of the C-terminal region of major royal jelly proteins 1 (MRJP-1), play an important biological role in royal jelly conservation as well as in protecting bee larvae from potential pathogens. Therefore, these molecules present numerous benefits for human health, including therapeutic purposes as shown in preclinical studies. In this review, we aimed to evaluate the biological effects of jelleines in addition to characterising their toxicities and stabilities. Jelleines I-III have promising antimicrobial activity and low toxicity (LD50 > 1000 mg/Kg). However, jelleine-IV has not shown relevant biological potential. Jelleine-I, but not the other analogues, also has antiparasitic, healing, and pro-coagulant activities in addition to indirectly modulating tumor cell growth and controlling the inflammatory process. Although it is sensitive to hydrolysis by proteases, the addition of halogens increases the chemical stability of these molecules. Thus, these results suggest that jelleines, especially jelleine-I, are a potential target for the development of new, effective and safe therapeutic molecules for clinical use.

Proteome analysis of propolis deciphering the origin and function of its proteins

Propolis is a resinous honeybee product, rich in polyphenolic compounds and with high economic value. Although extensive studies regarding the chemical composition of different propolis extracts have been carried out, the propolis proteome remained unknown. The present study aimed at characterizing the proteome of two geographically-distinct propolis originating from Belgium and Iran. Propolis extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) followed by Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) and Liquid chromatography-electrospray ionization MS (LC-ESI-MS) analyses. Our comprehensive proteomic analysis led to the identification of honeybee venom and royal jelly proteins, and numerous plant-defense proteins in propolis, extending our understanding of the antimicrobial properties of this complex natural substance. The proteome of both propolis was mainly made up of proteins belonging to the poplar tree. Pathogenesis-related (PR) proteins, poplar PR-2 (β-1,3-glucanases) and PR-8 (acidic endochitinases) were found to be the major components of both propolis extracts, while the major bee venom allergen (Api m 1) and royal jelly proteins (MJRP 1, 2, 3 and 5) were identified in the propolis from Belgium. Overall, our proteome analyses revealed that propolis extracts from Belgium and Iran shared a core protein composition originating from the poplar proteome. The majority of identified proteins are involved in the plant defense against pathogens, most belonging to well-known pathogenesis-related families. Proteomics could thus represent a viable approach for origin authentication and allergen identification in poplar-type propolis.

Transcriptome analysis of Apis mellifera antennae reveals molecular divergence underlying the division of labour in worker bees.

The olfactory system plays a fundamental role in mediating insect behaviour. Worker bees exhibit an age-dependent division of labour, performing discrete sets of behaviours throughout their lifespan. The behavioural states of bees rely on their sense of the environment and chemical communication via their olfactory system, the antennae. However, the olfactory adaptation mechanism of worker bees during their behavioural development remains unclear. In this study, we conducted a comprehensive and quantitative analysis of antennal gene expression in the Apis mellifera of newly emerged workers, nurses, foragers and defenders using RNA-seq. We found that the antenna tissues of honey bees continued developing after transformation from newly emerged workers to adults. Additionally, we identified differentially expressed genes associated with bee development and division of labour. We validated that major royal jelly protein genes are highly and specifically expressed in nurse honey bee workers. Furthermore, we identified and validated significant alternative splicing events correlated with the development and division of labour. These findings provide a comprehensive transcriptome profile and a new perspective on the molecular mechanisms that may underlie the worker honey bee division of labour.

A new approach for the treatment of bleomycin-induced rat pulmonary injury by combined protein fraction of major royal jelly protein 2 and its isoform X1

Nowadays, royal jelly (RJ) has gained great interest as a functional food due to its valuable pharmacological effects. We investigated the therapeutic potency of combined protein fraction (PF50) of major RJ protein 2 and its isoform X1 on bleomycin (Bleo)-induced pulmonary injury in rats. Our study examined the impact of PF50 on pulmonary oxidative and inflammatory stress as well as smooth muscle alpha-actin (α-SMA). In addition, the predicted impacts of this PF on the activity of matrix metalloproteinase (MMP)− 8 and 15-prostaglandin dehydrogenase (15-PGDH) and the E-type prostanoid 2 (EP2) and IL-13 α2 subunit (IL13α2R) receptors, were evaluated using molecular docking. The results showed that PF50 reduced pulmonary inflammatory cells and their secreted pro-inflammatory mediators, including NF-κB, IKK, IL-4, IL-6, and NO. Additionally, the levels of IgE and mucin were diminished after treatment with PF50. Moreover, PF50 treatment improved pulmonary oxidative stress indices such as lipid peroxidation, GSH, SOD, and GPX. The histopathological findings, chest conventional X-ray, and immunohistochemistry of α-SMA confirmed the ameliorating effect of PF50. The docking outcomes reported the probable competitive inhibitory influence of PF50 on MMP-8 and a postulated blocking effect on EP2 and IL13α2R. Thus, PF50 could be a novel approach for treating pulmonary injuries.

Mandibular glands secrete 24-methylenecholesterol into honey bee (Apis mellifera) food jelly

Honey bee (Apis mellifera) workers feed their larvae with food jelly that is secreted by specialized glands in their heads – the hypopharyngeal and the mandibular glands. Food jelly contains all the nutrients the larvae need to develop into adult honey bees, including essential dietary sterols. The main sterol in food jelly, 24-methylenecholesterol (24MC), is pollen-derived and delivered in food jelly to the larvae in a complex with two proteins, major royal jelly protein 1 (MRJP1) and apisim. Whereas the proteins are synthesized in the hypopharyngeal glands, the sterol-secreting gland has not been identified. We here identified the mandibular glands as sterol-secreting gland for food jelly production by direct detection of the four main honey bee sterols (24MC, campesterol, β-sitosterol and isofucosterol). Furthermore, 24MC seems to be specifically enriched in the mandibular glands, thereby ensuring that food jelly contains the amounts of 24MC necessary for complex formation with MRJP1 and apisimin.

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.

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.