Bee Propolis, Bee Bread, and Royal Jelly: Proximate Analysis, Fatty Acid Composition, Nutritional Quality, and Anti-Amylase Activity

This work explores the proximate composition, fatty acid pro�le, nutritional quality, and anti-amylase activity of propolis, royal jelly, and bee bread. The differential FTIR patterns of propolis, royal jelly, and bee bread re�ect these products have different proximate compositions and nutritional properties. The values for carbohydrate, fat, and protein values of be products are similar to egg and soya. The primary fatty acids bee bread and bee propolis are palmitic, linolenic, oleic, linoleic, myristic, and docosanoic acids. The primary fatty acids in royal jelly are 2-dodecenedioic, 10-hydroxy-2-decenoic, decanedioic, linoleic, 10-hydroxydecanoic acid 3-hydroxy-decanoic acids, respectively. The propolis, bee bread, and royal jelly have well-balanced saturated, unsaturated, monounsaturated, polyunsaturated, omega-3, and omega-6 fatty acids. Their nutritional quality, including omega-6/omega-3, thrombogenicity, atherogenicity, hypocholesterolemic, nutritional value, and peroxidizability indexes, are similar to egg and soya. Fatty acids inhibit amylase by increasing Km/Vmax and decreasing Vmax and Km through an un-competition or non-competition strategy. Molecular docking, ultraviolet absorption, and �uorescence quenching analysis reveal that fatty acids interact with amino acid residues of amylase through Van der Waals and hydrogen bonds interactions. Functional fatty acids from bee products can be used in a number of food supplements, food ingredients, and medications to provide carbohydrate-degrading enzymes.


Introduction
The increasing shortage of world food and pharmaceutical sources and the growing world population compels us to search for alternative resources such as bee products.Bee propolis, royal jelly, pollen, and bee bread possess nutritional value and health-protecting properties and recommend a superfood for human food and feed.Propolis, bee bread, and royal jelly are applied in the management of different illnesses since they have several biological properties including, antioxidant, anti-fungal, anti-bacterial, antiseptic, anti-cancer, and anti-in ammatory, depending on chemical composition and region of collection.Current research demonstrated the advantageous properties of propolis in the management of obesity, diabetes mellitus, and dyslipidemia 1 .Bee propolis is a sticky mixture of wax, resin, protein, fatty acid, polysaccharides, and exudate from the plant's ower produced by mixing with the bee salvia.Commonly, propolis contains resins (50-60%), waxes (30-40%), essential oil (5.0-10%), and other constituents, including proteins, amino acids, free fatty acids, vitamins, and micronutrients, depending on plant sources 2 .Bee bread is a mixture of plant ower pollen, wax, honey, and salivary gland secretion of bees compacted into honeycombs.Bee bread mixture is a perfectly complete food, has high energy, and is a nutrient resource for bee larvae and adult bees 3 .The components of bee bread encompass proteins and amino acid (8.0-28%), carbohydrates (15-45%), lipids and fatty acids (7.0-15%), vitamins, minerals, and antioxidant compounds such as avonoids, and phenolic acids, depending on the plant's sources and collationed region 4 .
Royal jelly is a yellowish and creamy emulsion of protein (8.0-22%), amino acid (1.5-3.5%),sugars (7.0-52%, primarily consists of fructose and glucose), lipids (7-33%, short hydroxyl fatty acids, and dicarboxylic acids), and polyphenols, avonoids, and vitamins 5 .This high-energy emulsion is a valuable food for the queen in adult and larvae stages and young bee larvae.One of the main characteristics of royal jelly emulsion is hydroxylated fatty acids and dicarboxylic acids.The primary hydroxylated fatty acids in royal jelly are 10-hydroxydecanoic and 10-hydroxy-2-decanoic acids with various biological activities 6 .Proximate and nutrient analysis of bee products plays a crucial role in assessing their nutritional signi cance.As various bee products are also used as food and medicinal ingredients, evaluating their nutritional effectiveness can help understand the worth of these products.As far as herbal drug standardization is concerned, WHO also emphasizes the need and importance of determining proximate and macronutrients analysis.Soybean 7 and egg 8 are signi cant sources of protein and fatty acid for the human diet, recommended by WHO/FDA.
The fundamental goal of current work is to determine the nutritional properties of bee bread, bee propolis, and royal jelly through examining their proximate composition and comparing them with egg (as an animal resource) and soybean (as a plant resource).The lipid nutritional quality and fatty acid composition of bee bread, bee propolis, and royal jelly were determined and compared with egg and soybean fatty acids.The data matrix of the fatty acid composition and nutritional value in the samples were subjected to principal component analysis to illustrate the apparent discrepancy of distribution of these components among the samples.The researcher examines the anti-amylase activity of fatty acids from bee bread, bee propolis, and royal jelly.Double reciprocal Lineweaver-Burk plots will investigate the mechanism of inhibition of fatty acid on the amylase activity.The interactions between amylase and fatty acids were analyzed using ultraviolet absorption spectroscopy and uorescence quenching spectroscopy.Furthermore, molecular modeling does use to determine the interaction between fatty acids and amylase.

Proximate composition of bee product
Bee products including bee propolis, bee bread, and royal jelly are prepared from beekeepers in Marvdasht region of Fars province (Fars, Iran).The predominant vegetation in this area is wild almond.To evaluate the differential chemical components of bee propolis, bee bread, and royal jelly, Fourier transforms infrared spectroscopy (FT-IR), in the range between 4000 − 400 cm -1 , performed utilizing Bruker FTIR spectrophotometer (Germany).The moisture content of bee products determines by drying the samples at 105°C for two h through a vacuum oven (model: BM55E, Fan Azma Gostar, Iran).The bee samples were heated at 550°C for ve h in electric ovens (model: FM2P, Fan Azma Gostar, Iran) for ash determination.The phenol-sulfuric acid method applies to assessing total carbohydrate content 9 .According to the Bradford method, total protein content in the bee products evaluates through Coomassie brilliant blue G-250 10 .The sulfuric acid-phosphoric acid-vanillin reagent uses to quantify total lipid and fatty acids 11 .The following formula measured the total energy content of the bee product: Total energy = (4.0kcal/g × g protein) + (9.0 kcal/g × g fat) + (4.0 kcal/g × g carbohydrate).
Bee products (1.0 g) mix with pepsin (2 mg/100 ml) in 7.5 mM HCl (100 ml).These mixtures incubate at 40°C for 15 h with continuous shaking.The undigested protein content in the samples before and after pepsin treatment was determined as mentioned above.The ratio of digested protein to the original protein re ects the digestibility level.The protein digestibility level expresses as the digested protein (g) per 100 g of total protein in the raw materials.

Fatty acid preparation
The bee products biomass wash digested for 48 hours using hydrochloric acid: normal saline: methanol (2:1:1) solution at 60°C.Hexane was applied to the digestion solution and vortexed for 10 minutes to separate fatty acids.The fatty acids in the hexane phase separate from the bottom phase.The fatty acid chemical composition was examined by gas chromatography/mass spectrometry (GC-MS).A rotary evaporator uses to evaporate the hexane phase at ambient temperature.The residual fatty acids dissolve in ethanol for further experiments 12 .

Fatty acid pro ling by GC-MS
Fatty acid pro le attains by means the gas chromatograph (Agilent 7890B GC 7955A MSD) equipped with a single quadrupole mass spectrometer.The standard column for fatty acid determination was the silica capillary HP-5MS column (30 m × 0.25 mm, 0.25 µm).Helium at a ow rate of 1.0 ml/min applies as carrier gas.Interface and ion source temperatures were 300°C and 250°C, respectively.The oven temperature program was 80-250°C as follow: 80°C for four min, rise at 20°C/min to 140°C, grow at ten °C/min to 250°C, then hold at 250°C for 10 min.The types of fatty acids are determined by comparison the fragmentation patterns of the associated peaks with these suggested in the libraries of Wily and NIST 13 .

Amylase activity inhibition
To the porcine pancreatic α-amylase (EC: 3.2.1.1,5.0 U/mg) solution (1.0 Unit/ml), different concentrations (0.3.0.6, 0.9 mg/ml) of fatty acid and acarbose solutions was added, then incubated at 37°C for 20 minutes.To the amylase-inhibitor, starch solution (5.0 mg/ml in 100 mM phosphate buffer, pH 7) add.The amylase-inhibitor-starch mixture incubates for 20 min at 37°C.By adding 0.02 ml of 1.0 M HCl to the mixture, the amylase reaction stops.To assess undigested starch, iodine solution pours on the amylase reaction.The amylase reaction dilutes with distilled water, and the absorbance of samples is recorded at 580 nm.The activity (velocity) of amylase in the absence and presence of acarbose or fatty acid was calculated via monitoring decomposition of starch per min by measuring light absorbance at 580 nm for 30 min at an interval of 3.0 min.The slope of starch decomposition versus time plot represented the amylase activity and expressed as µg/min.The kinetic parameters of amylase activity were obtained from the Lineweaver-Burk curve that plotted based on 1/V versus 1/[starch] in the absence and presence of fatty acid or acarbose (0.3.0.6, 0.9 mg/ml).The kinetic parameters like Km/Vmax, Km, and Vmax and the type of inhibition attained from the Lineweaver-Burk plot 12 .

Fluorescence and ultraviolet spectroscopy analysis
The interactions between amylase and fatty acid investigated using ultraviolet absorption spectroscopy (UV1280, Shimadzu, Japan) and uorescence quenching spectroscopy (Varian Cary Eclipse, Agilent, USA) 16 .Amylase solution (5.0 ml of 500 µg/ml) was prepared in phosphate buffer (100 mM, pH = 7).To the enzymatic solutions, 5.0 ml of different concentrations (0.3.0.6, 0.9 mg/ml) of acarbose or fatty acid were added.The reaction mixtures incubate at ambient temperature for 10 min.The UV absorption spectrum of the amylase-inhibitor mixture was recorded from 200 to 400 nm using quartz cuvettes at room temperature.The intrinsic uorescence absorption spectrum of the amylase-inhibitor solutions records at 280 nm for excitation wavelength and 290-500 nm for emission wavelengths.

Molecular dicking
Three-dimensional structure of the amylase downloads from the protein data bank (5U3A, PDB).Threedimensional structures of fatty acid and acarbose download from the chemspider website.AutoDock vina platform was used to examine interactions between amylase and ligands.The evaluation of the docked positions and amylase-ligand interactions and docking scores (binding free energy) was attained employing PYMOL software 12 .

Statistical analysis
All results show mean values ± standard deviations.The data were analyzed with a one-way analysis of variance (ANOVA) using SPSS software (SPSS Inc., Chicago, IL, USA).The signi cant differences between samples were examined by the Tukey test at P < 0.05.Minitab software was used to perform the principal component analysis (PCA).

Proximate composition
The differential FTIR patterns from bee bread, royal jelly, and bee propolis re ect the different components in these products (Fig. 1).The bands in the 3700 − 3100 cm -1 might connect to stretching vibration of OH groups of water or aromatic compounds.The peaks in the 2900 − 2700 cm -1 may link to the stretching vibration of CH, CH2, and CH3 groups of lipid and fatty acids.The bands in 1300 − 900 cm -1 related to the stretching of C = O of amides, C = C of aromatic, N-H of amines, or carboxyl groups in proteins.The bands at 1100 − 500 cm -1 are due to vibration of polysaccharides, including symmetric stretching of C-O-C and OH groups 17 .
The chemical composition (moisture content, ash, carbohydrate, fat, and protein ad energy values) of bee bread, bee propolis, and royal jelly product were compared with egg and soya and summarized in Table 1.Principal component analysis showed that the sum of the rst and second major components accounted for 94.4% total variance of the changes, with the rst major component (PC1) accounting for 65.7% (eigenvalue = 4.60) and the second major component (PC2) for 28.7% (eigenvalue = 2.00) (Fig. 2).The rst principal component (PC1) is positively correlated with energy (0.458), ash (0.444), fatty acid (0.351), and protein (0.318).Therefore, increasing energy, ash, fatty acid, and protein values increase the value of the rst principal component.PC1 is negatively correlated with digestibility (-0.421) and moisture (-0.414).The second principal component (PC2) is positively correlated with sugar (0.670).Therefore, increasing the values of sugar increase the value of the PC2.PC2 is negatively correlated with digestibility (-0.215), moisture (-0.279), protein (-0.462) and fatty acid (-0.456).Royal jelly in the rst quarter is not closely related to the measured parameters.The bee bread and propolis in the second quarter are the most related to sugar and energy.Egg in the 3rd quarter is most associated with digestibility and moisture.In the 4th quarter, soya is most associated with a fatty acid, protein, and ash 18 .

Fatty acid composition
The fatty acid composition of bee bread, bee propolis, and royal jelly product were stated in Table 2.
Table 3 Lipid nutritional quality of fatty acid from bee bread, bee propolis, and royal jelly in comparison with egg and soya.The high omega-3 diet causes a decrease in triglycerides, increases mitochondrial biogenesis, prevents in ammation, and restores insulin sensitivity.Besides, an omega-3 rich diet reduces chronic diseases such Bee propolis, bee bread, and royal jelly inhibit amylase with a lower level than acarbose.Kinetic parameters of amylase in the presence of acarbose and bee products measure through kinetic analysis.Figure 5a demonstrates plots of amylase activity at varying concentrations of acarbose.According to the Lineweaver-Burk plot, acarbose inhibits with a competitive inhibition trend.Since Vmax of amylase remains constant, in contrast, the Km/Vmax and Km increase (Table 4).Acarbose is a competitive inhibitor of amylase activity, and as the concentration of acarbose increases, the Vmax values do not change, but the Km values increase (Fig. 5A).Fatty acid from bee bread (Fig. 5B), royal jelly (Fig. 5C) and, bee propolis (Fig. 5D), on the other hand, are mixed un-competitive or non-competitive inhibitors.The Km/Vmax increases while Km and Vmax value decrease.By binding fatty acid to the allosteric site of the enzyme, this rises in the Km/Vmax ratio links to the reduction in the Vmax and functional enzyme 24 .The active compounds in fatty acid attach to allosteric sites, avoiding starch degradation.The enzyme conformation varies when inhibitors bind to the enzyme, and the a nity of the enzyme active site for starch reduces 25 .

Ultraviolet and uorescence spectroscopic analysis
Ultraviolet-visible absorption is one of the effective methods for enzyme conformation changes during inhibitor binding 26 .This study examines the absorption spectra of amylase in the presence of acarbose and fatty acids from bee products (Fig. 6).The ultraviolet absorption peak around 210-220 nm relates to the carbonyl group ππ transition of the peptide bond.The ultraviolet absorption peak around 255-280 nm attributes to the ππ shift of aromatic groups in the protein sequence like tryptophan, tyrosine, and phenylalanine 16 .Fatty acid and acarbose at these wavelengths did not have ultraviolet absorption.By increasing the concentration of acarbose and fatty acid, the ultraviolet absorption of amylase regularly increases.Accordingly, ligands such as fatty acid and acarbose can establish complexes with amylase, alter their conformation and expose the aromatic group to ultraviolet light in this manner, increasing the ππ transition of aromatic groups 27 .
Another effective technique to monitoring the interactions between amylase and inhibitors such as acarbose and fatty acid is uorescence quenching analysis (Fig. 7).The uorescence emission spectra of the enzyme contribute to aromatic amino acids such as tyrosine, tryptophan, and phenylalanine 28 .
Acarbose and fatty acid did not have a uorescence emission at this condition.The amylase a uorescence emission peak at 360 nm after excitation at 280 nm.By increasing the concentrations of acarbose and fatty acid, the uorescence emission of amylase declines.
Furthermore, the uorescence intensity slightly shifts toward the blue region (Fig. 7).The uorescence quenching and shifting of amylase directly con rm the interaction between the enzyme and its inhibitors that re ect the changes in the structural architecture, conformation, surrounding environment, and polarity of the enzyme 26 .The molecular interactions between fatty acid or acarbose with amylase generate a nonuorescent amylase-inhibitor complex and change the enzyme architecture and microenvironment.These effects increase the collision between uorescent groups in the enzymes (aromatic amino acid) and quenching agents (inhibitors), leading to a decrease in the intrinsic uorescence intensity 28 .

Conclusions
In summary, the differential FTIR patterns of bee bread, royal jelly, and bee propolis re ect the variations in proximate compositions and discrepancy in nutritional properties of bee bread, bee propolis, and royal jelly.
The fat of bee propolis, bee bread, and royal jelly, to some extent, is identical and equal to egg fat and lower than soya.The protein of royal jelly and bee bread was higher than egg, while the propolis had lower protein than an egg.Bee propolis had the most elevated sugar, followed by bee bread and royal jelly and then soy and egg.The highest energy was found in the soya, followed by bee bread, bee propolis, royal jelly, and then egg.The higher energy of soya may be related to the higher level of lipid and protein contents.The primary fatty acid in bee propolis and bee bread were palmitic, oleic, linolenic, linoleic, behenic, and myristic acids.
The primary fatty acid in royal jelly was 2-dodecenedioic, 10-hydroxy-2-decenoic, decanedioic, 10hydroxydecanoic, linolenic, 3-hydroxy-decanoic, palmitic, linoleic, 11-hydroxy-dodecanoic, oleic, palmitoleic, and γ-linolenic acids.The bee products have a well-balanced omega-6 to omega-3 ratio, thrombogenicity index, atherogenicity index, PUSA/SFA, and hypocholesterolemic/hypercholesterolemic index.Besides, kinetic analyses and molecular docking reveals that fatty acids inhibit amylase activity through nucompetition or non-competition strategy.Fatty acid similar to acarbose increases ultraviolet absorption and quenches the intrinsic uorescence intensity of amylase by forming a complex with the enzymes.In conclusion, bee products can consider as purposeful sustenance with nutritive values like egg and soya, and valuable fatty acids are essential as the non-competitive and un-competitive inhibitor for amylase intended for diabetes management.

Declarations Figures
Fourier transform infrared spectrum of bee bread, royal jelly and bee propolis.The differential FTIR patterns of bee bread, royal jelly and bee propolis re ect the different components in these products.
Principal Component Analysis (PCA) biplot illustrating the relationships among the biochemical composition of bee bread, bee propolis, royal jelly, egg and soyabean.For the abbreviation of the analyzed samples and the compounds see Table 1.
Principal Component Analysis (PCA) biplot illustrating the relationships among the fatty acid composition of bee bread (BB), bee propolis (BP), royal jelly (RJ), egg and soyabean.For the abbreviation of the analyzed samples and the compounds see Table 2.
Principal Component Analysis (PCA) biplot illustrating the relationships among the fatty acid nutritional quality of bee bread, bee propolis, royal jelly, egg and soyabean.For the abbreviation of the analyzed samples and the compounds see Table 3.
Figure 6 Ultraviolet spectra of amylase in the absence and presence of varying concentrations of acarbose (A) and fatty acid from bee bread (B), royal jelly (C) and propolis (D).

Table 1
Proximate analysis of bee propolis, bee bread and royal jelly in comparison with egg and soya.

Table 4
The values of kinetic parameters (Km /Vmax, Km, Vmax) of -amylase in response to acarbose bee bread, bee propolis and royal jelly fatty acid extract.