Katarina Bilikova

Towards functional proteomics of minority component of honeybee royal jelly: the effect of post-translational modifications on the antimicrobial activity of apalbumin2.

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with Mr of 49–87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing.

New anti-Paenibacillus larvae substances purified from propolis

Propolis plays an important role in the exogenous defense of honeybee colony against pathogens. However, the studies dealing with the activity of propolis against bee pathogens are scarce. Poplar propolis extracts demonstrated promising activity against Paenibacillus larvae, the causative agent of American foulbrood. From the same propolis, five individual components and a mixture of caffeates were isolated, and their structures confirmed by spectroscopic data. Among the isolated propolis constituents are flavonoids, ferulic acid esters, and the oxylipin 9-oxo-10(E)-12(Z)-octadecadienoic acid, newly identified as propolis component. These substances were tested for their activity against P. larvae strains. The most active constituents were pinocembrin, 3-O-acetyl pinobanksin, and the caffeate mixture. This is the first communication of antimicrobial activity of individual propolis constituents against P. larvae; their important advantage is the fact that they are naturally present in the hive.

Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera

Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in Escherichia coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra-disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin.

New Criterion for Evaluation of Honey: Quantification of Royal Jelly Protein Apalbumin 1 in Honey by ELISA

The 55 kDa major protein of royal jelly, named apalbumin 1, is an authentic protein of honey and pollen pellet, and for its quantification an enzyme-linked immunosorbent assay (ELISA) was developed using specific polyclonal anti-apalbumin 1 antibody. The limit of detection for apalbumin 1 was 2 ng mL(-1). The floral honeys contained apalbumin 1 as follows: acacia, 0.011%; linden, 0.010%; chestnut, 0.029%; rape, 0.010%; and dandelion, 0.014%. The saccharose syrup honey contained only 0.001% of apalbumin 1. The average amount of apalbumin 1 relating to the total protein content of analyzed honey samples was 23.39%, whereas in SCCH apalbumin 1 presented only 4.81% of total proteins of honey. Apalbumin 1 is thermostabile in honey at 80 degrees C incubated for 40 min. ELISA results show good precision in the evaluation of apalbumin 1 quantity in honey (CV ranged from 0.69 to 4.25%).

Stimulation of TNF-α Release by Fungal Cell Wall Polysaccharides

Carboxymethylated derivatives were prepared from the (1→3)-β-ᴅ-glucan isolated from the cell wall of baker’s yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-α by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-α release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1→3)-β-ᴅ-glucan, are potent macrophage stimulators and activators of TNF-α release, which implies their potential application in antitumor therapy.

Major royal jelly proteins as markers of authenticity and quality of honey / Glavni proteini matične mliječi kao markeri izvornosti i kakvoće meda

Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature. Do sada su svojstva meda bila definirana isključivo na temelju sadržaja komponenti nektara određene biljke. Mi smo pokazali da je apalbumin1, glavni protein matične mliječi, izvoran i uobičajeni sastojak meda. Apalbumin1, ostali proteini matične mliječi i peptidi odgovorni su za imunostimulatorna svojstva i antibiotsko djelovanje meda. Korištenjem poliklonalnog anti-apalbumin 1 protutijela osmišljen je imunoenzimski test (ELISA) za kvantifikaciju apalbumina 1. Metoda je ne samo prikladna za utvrđivanje izvornosti meda nego i korisna za detekciju meda, peluda i matične mliječi u medicinskim, farmaceutskim, kozmetičkim i prehrambenim proizvodima na kojima je naznačena prisutnost pčelinjih proizvoda. Rezultati analize prisutnosti i količine apalbumina 1 koristit će se za probir velike količine uzoraka meda diljem svijeta. Na temelju naših eksperimenata, koji pokazuju da su proteini matične mliječi uobičajene i fiziološki aktivne komponente meda, predlažemo izmjenu definicije meda (na temelju Direktive EU-a o medu 2001/110/EC): Med je prirodna slatka tvar koju pčele proizvode od nektara ili izlučevina biljaka ili izlučevina insekata koji sišu biljke. Nju pčele skupljaju, pretvaraju kombinacijom glavnih proteina matične mliječi i ostalih vlastitih specifičnih tvari, polažu, dehidriraju, pohranjuju i ostavljaju u saću da sazrije.

Major royal jelly proteins as markers of authenticity and quality of honey

Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature. Do sada su svojstva meda bila definirana isključivo na temelju sadržaja komponenti nektara određene biljke. Mi smo pokazali da je apalbumin1, glavni protein matične mliječi, izvoran i uobičajeni sastojak meda. Apalbumin1, ostali proteini matične mliječi i peptidi odgovorni su za imunostimulatorna svojstva i antibiotsko djelovanje meda. Korištenjem poliklonalnog anti-apalbumin 1 protutijela osmišljen je imunoenzimski test (ELISA) za kvantifikaciju apalbumina 1. Metoda je ne samo prikladna za utvrđivanje izvornosti meda nego i korisna za detekciju meda, peluda i matične mliječi u medicinskim, farmaceutskim, kozmetičkim i prehrambenim proizvodima na kojima je naznačena prisutnost pčelinjih proizvoda. Rezultati analize prisutnosti i količine apalbumina 1 koristit će se za probir velike količine uzoraka meda diljem svijeta. Na temelju naših eksperimenata, koji pokazuju da su proteini matične mliječi uobičajene i fiziološki aktivne komponente meda, predlažemo izmjenu definicije meda (na temelju Direktive EU-a o medu 2001/110/EC): Med je prirodna slatka tvar koju pčele proizvode od nektara ili izlučevina biljaka ili izlučevina insekata koji sišu biljke. Nju pčele skupljaju, pretvaraju kombinacijom glavnih proteina matične mliječi i ostalih vlastitih specifičnih tvari, polažu, dehidriraju, pohranjuju i ostavljaju u saću da sazrije.

Standard methods for Apis mellifera royal jelly research

Royal jelly, a honey bee secretion, plays a critical role in caste determination in honey bees because it serves as the source of nutrition for young larvae destined to become queens. It is also fed to adult queens. Royal jelly possesses numerous functional properties and thus has been used as a medication, health food, and cosmetic in many countries. In this paper, we first introduce a traditional method for producing royal jelly by artificial larvae grafting and a newly developed method that does not require grafting of larvae. We describe protocols for the storage and freeze-drying of royal jelly to preserve its biological properties. Routine methods for determination of two important quality criteria, water content and trans-10-hydroxy-2-decenoic acid content, are outlined. On a dry basis, protein, carbohydrate, and fatty acids were found to be the 3 most abundant components of royal jelly. Methods for their isolation, identification, and quantification are described. Because royal jelly is susceptible to contamination with veterinary drugs and acaricides, we also describe methods for detection and quantification of some veterinary drugs and acaricides in royal jelly.

Glavni proteini matične mliječi kao markeri izvornosti i kakvoće meda

Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature. Do sada su svojstva meda bila definirana isključivo na temelju sadržaja komponenti nektara određene biljke. Mi smo pokazali da je apalbumin1, glavni protein matične mliječi, izvoran i uobičajeni sastojak meda. Apalbumin1, ostali proteini matične mliječi i peptidi odgovorni su za imunostimulatorna svojstva i antibiotsko djelovanje meda. Korištenjem poliklonalnog anti-apalbumin 1 protutijela osmišljen je imunoenzimski test (ELISA) za kvantifikaciju apalbumina 1. Metoda je ne samo prikladna za utvrđivanje izvornosti meda nego i korisna za detekciju meda, peluda i matične mliječi u medicinskim, farmaceutskim, kozmetičkim i prehrambenim proizvodima na kojima je naznačena prisutnost pčelinjih proizvoda. Rezultati analize prisutnosti i količine apalbumina 1 koristit će se za probir velike količine uzoraka meda diljem svijeta. Na temelju naših eksperimenata, koji pokazuju da su proteini matične mliječi uobičajene i fiziološki aktivne komponente meda, predlažemo izmjenu definicije meda (na temelju Direktive EU-a o medu 2001/110/EC): Med je prirodna slatka tvar koju pčele proizvode od nektara ili izlučevina biljaka ili izlučevina insekata koji sišu biljke. Nju pčele skupljaju, pretvaraju kombinacijom glavnih proteina matične mliječi i ostalih vlastitih specifičnih tvari, polažu, dehidriraju, pohranjuju i ostavljaju u saću da sazrije.