Protein and phenol content in honey samples as indicators of the influence of stress factors on honey bee colonies

Predmet rada ove doktorske disertacije je razvoj analitičkih postupaka za praćenje relativnog sadržaja ukupnih proteina i ukupnih fenola u različitim uzorcima meda radi procene uticaja biotičkog stresa na pčelinja društva usled zaraze parazitima Varroa destructor i Nosema ceranae. Pored proizvodnje meda, pčele imaju veoma važnu ulogu u oprašivanju biljaka. Poslednjih godina u svetu dolazi do znatnog smanjenja pčelinjih društava, a jedan od razloga zbog kog dolazi do ove pojave su bolesti pčela. U literaturi je malo podataka o kvalitetu meda koji potiče iz pčelinjih zajednica izloženih biotičkom stresu. U cilju nalaženja indikatora u medu za procenu stepena zaraženosti pčelinjih društava, prikupljeni su uzorci meda iz košnica zaraženih varoom i nozemom, a zatim analizirani odabranim biohemijskim i fizičkohemijskim metodama. Određena je vrsta i stepen infekcije košnica pčelinjih društava iz kojih su prikupljeni uzorci meda. Polenskom analizom je određena botanička vrsta meda i nije utvrđena direktna veza između prisustva i koncentracije polena u uzorcima meda i stepena zaraženosti košnica. Uzorci meda su okarakterisani fizičkohemijskim metodama. Određeni su specifična optička rotacija, električna provodljivost, sadržaj vlage, slobodna kiselost i sadržaj pojedinačnih šećera. Profil šećera u uzorcima meda određen je visokoefikasnom jonskom hromatografijom sa elektrohemijskom detekcijom (HPAEC-PAD). Analiza glavnih komponenata (PCA) je upotreblјena za utvrđivanje razlika između medova koji potiču iz košnica različitog stepena zaraženosti na osnovu dobijenih fizičkohemijskih parametara. Rezultati analize glavnih komponenata su pokazali da sa porastom infekcije košnica u uzorcima meda raste sadržaj vode dok slobodna kiselost, apsolutna vrednost specifične optičke rotacije i električna provodljivost opadaju. Veći sadržaj fruktoze, melbioze i melezitoze izmeren je u uzorcima meda iz košnica višeg stepena infekcije u odnosu na uzorke meda koji potiču iz košnica manje zaraženosti. Sadržaj glukoze i izomaltoze bio je veći u uzorcima meda koji potiču od pčelinjih društava manje zaraženosti u odnosu na uzorke meda iz košnica veće zaraženosti. U cilju ispitivanja varijacije sadržaja biljnih i pčelinjih proteina u medu primenjene su elektroforetska analiza i određivanje aktivnosti enzima. Pomoću natrijum dodecilsulfat poliakrilamid gel elektroforeze (SDS PAGE) nije utvrđena značajna razlika u proteinskom profilu analiziranih uzoraka. Stoga je primenjena spektrofotometrijska metoda određivanja aktivnosti specifičnih enzima, katalaze i dijastaze, kao biljnog odnosno pčelinjeg marker enzima. Rezultati korelacione analize su pokazali da je stepen zaraze košnica u pozitivnoj korelaciji sa aktivnošću katalaze i može biti potencijalno koristan indikator za skrining meda poreklom iz košnica zaraženih varoom i nozemom. Pored toga, nije utvrđena promena u aktivnosti dijastaze u uzorcima meda čije je poreklo od društava koja su zaražena. Fluorescentna spektroskopija u kombinaciji sa naprednim statističkim metodama multivarijaciona rezolucija krivih – naizmenični najmanji kvadrati (MCR-ALS) i paralelna faktorska analiza (PARAFAC) je korišćena za određivanje relativnog sadržaja proteina i fenola u uzorcima meda...Protein and phenol content in honey samples as indicators of the influence of stress factors on honey bee colonies Abstract The subject of this doctoral dissertation is the development of analytical procedures for monitoring the relative content of total proteins and total phenols in different honey samples to assess the impact of biotic stress on honey bee colonies due infected with parasites Varroa destructor and Nosema ceranae. In addition to honey production, bees have very important role in pollinating plants. In recent years, there has been a significant decrease in bee colonies in the world and one of the reasons why this phenomenon occurs is bee diseases. In the literature, the data are not abundant on the honey quality change related to bee colonies exposure to biotic stress. In order to find markers in honey to assess the degree of infection of bee colonies, honey samples were collected from hives infected with parasites V. destructor and N. ceranae parasites and analyzed by selected biochemical and physicochemical methods. The type and degree of infection of bee colonies related to the analyzed honey samples were determined. Pollen analysis has proved the botanical type of honey samples and it was observed that the presence and concentration of pollen in honey samples were not correlated with the infestation of hives. Honey samples were characterized by physicochemical methods. Specific optical rotation, electrical conductivity, moisture content, free acidity, and sugar content were determined to obtain additional characteristics of honey samples. Sugar profile in honey samples was determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Principal component analysis (PCA) was used to determine differences between honey samples originating from the hives with different infection levels. The obtained results showed that in hives with the increase of infection, in honey samples the water content increased, while the free acidity, the absolute value of the specific optical rotation, and the electrical conductivity decreased. The higher content of fructose, melbiosis and melezitose was observed in honey samples from the hives with a higher degree of infection in comparison with honey samples from the hives with the lower infection degree. Glucose and isomaltose content was higher in honey samples originating from the bee colonies less infected compared to honey samples from the hives with the higher infection. In order to investigate variations in the content of plant and bee protein markers in honey, biochemical methods such as electrophoretic analysis and determination of enzyme activity were applied. Using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE), no significant difference was found in the protein profile of the analyzed honey samples. Therefore, the spectrophotometric method was applied for determination of the activity of specific enzymes, catalase and diastase, as plant and bee marker enzymes, respectively. The results of correlation analysis showed that the colonies’ infestation level was positively correlated with the activity of catalase which can be a useful indicator for screening of honey originating from beehives infested by V. destructor and N. ceranae. On the other hand, no change in diastase activity was found in honey samples obtained from infected societies. Fluorescence spectroscopy in combination with advanced statistical methods Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) and Parallel Factor Analysis (PARAFAC) was used to determine the relative protein and phenol content in honey samples..

Proučavanje raznovrsnosti bakterije Pseudomonas syringae poreklom sa različitih voćaka u Srbiji

Pseudomonas syringae is a widespread and economically important plant pathogen, one found on a number of hosts, including fruit trees, field crops, vegetables, and ornamental plants. This bacterium has been experimentally identified as a parasite of pear, apple, apricot, peach, cherry, sour cherry, plum, and raspberry. The present study was designed to establish differences between strains isolated from fruit trees in Serbia. The pathogenic and biochemical characteristics of isolates were studied. The BOX-PCR method was used to generate genomic fingerprints of Pseudomonas syringae isolates and to identify strains that were previously not distinguishable by other classification methods. Different Bacillus sp. strains were tested for in vitro inhibitory activity against Pseudononas syringae isolates. Bacillus sp. strains show inhibitory activity only against P. syringae isolates that originated from peach. The obtained results demonstrate that the population of the bacterium Pseudomonas syringae from the fruit trees in Serbia is very diverse.Pseudomonas syringae je široko rasprostranjena i ekonomski značajna fitopatogena bakterija, sa širokim krugom domaćina koji uključuje voćke, ratarske, povrtarske i ukrasne biljke. Pseudomonas syringae u Srbiji je eksperimentalno potvrđen kao parazit kruške, jabuke, kajsije, breskve, trešnje, višnje, šljive i maline. Cilj ove studije bio je da utvrdi postojanje eventualnih razlika između sojeva izolovanih sa različitih vrsta voćaka u Srbiji. Proučavane su patogene i biohemijske osobine sojeva. BOX-PCR je korišćen za dobijanje profila izolata Pseudomonas syringae u cilju identifikacije sojeva koji se ne mogu utvrditi drugim metodama. Različiti sojevi roda Bacillus su testirani u cilju utvrđivanja njihove in vitro inhibitorne aktivnosti. Sojevi roda Bacillus su pokazali inhibitornu aktivnost samo na P. syringae izolovanih sa breskve. Dobijeni rezultati pokazali suda je populacija bakterije Pseudomonas syringae poreklom sa voća u Srbiji vrlo raznovrsna

Determination of Niclosamide and its Metabolites in Liver and Muscles of Common Carp (Cyprinus carpio) Fingerlings

Background: Niclosamide is a medication used to treat tapeworm infestation in animals and humans. It is also lampricide and molluscicide, and can be used in in agriculture as a pesticide. In the treatment of parasitic diseases in fish, niclosamide can be used as bath or mixed with the feed. Its most important use in common carp (Cyprinus carpio) is for the treatment of Bothriocephalus acheilognathi, which is a very common parasite in this fish species. The aim of this study was to determine the concentrations of niclosamide (NIC) and its metabolite 2-chloro 4-nitro aniline (CNA) and 5-chloro salycilic acid (CSA) in the liver and muscles of common carp fingerlings.Materials, Methods & Results: The fish for the experiment were obtained from Kapetanski Rit fish pond, and were acclimated to test conditions at 20.5 ± 1°C. Common carps with an average mass of 60 ± 10 g were treated with niclosamide in concentration of 2 g/kg of feed during five consecutive days. The experiment was performed in two treatments: one control and niclosamide, in three replications.  Each group contained of 30 fish, in 120 L polyethylene tanks. At the end of the treatment, the levels of niclosamide residues were determined using a high performance liquid chromatography (HPLC) analysis during over 13 days. The mean values of niclosamide and CNA concentrations in the muscles ranged from 27.7 µg/kg starting from the first day to <0.5 µg/kg on the 11th day and 14.2 µg/kg from the first day to <1 µg/kg on the 9th day. The CSA metabolite in muscles were <1 µg/kg during throughout the entire study. The niclosamide concentration in the liver were found to be 51.5 (30.2-61.8) µg/kg the first day and decreased proportionally to <0.5 µg/kg on the13th day. CNA level in the liver of treated Common Carps amounted to 170.1 (157-181) µg/kg on the first day and continuously declined until the 13th day when recorded values were <1 µg/kg. The CSA concentrations in the liver reached a maximum level of 11.5 (10.1-12.8) µg/kg on the 7th day and fell to <1 µg/kg on the 13th day.Discussion: Niclosamide use in fish is questionable, primarily due to the possible toxic effects on some aquatic organisms. In Serbia, niclosamide preparation for use in aquaculture, has been produced by Veterinarski zavod Subotica since 1984 when it was registred for the first time. Niclosamid degradation mechanism showed that the metabolism of niclosamide resulted in two main metabolites CNA and CSA. Withdrawal of niclosamide and its residues in the liver and muscle in the present investigation lasted from 9 to 13 days. This decrease in residues concentrations is expected and depends primarily on several factors such as the length and concentration of drug with which the fish is treated, biotransformation, excretion and decomposition of used drug. Niclosamide and CNA were proportionally decreased during the withdrawal time, while the CSA value increased to the seventh day although the fish during this period no longer consumed food with niclosamide, after which the value then decreased until the end of its elimination. This is also not unexpected because it is known that liver and gallbladder is a major organ for collection, storage and elimination of chemical residues. Although the treated fish received 2 mg of the niclosamide per g of feed for five consecutive days results obtained in this study indicate that the maximal residues concentrations were much lower than doses of niclosamide that each fish absorbed into the body. Data obtained during this study provided information about the concentration and withdrawal times of niclosamide and its residues CNA and CSA in the liver and muscles of common carp treated orally

Intrinsic Fluorescence Markers for Food Characteristics, Shelf Life, and Safety Estimation: Advanced Analytical Approach

Food is a complex matrix of proteins, fats, minerals, vitamins, and other components. Various analytical methods are currently used for food testing. However, most of the used methods require sample preprocessing and expensive chemicals. New analytical methods are needed for quick and economic measurement of food quality and safety. Fluorescence spectroscopy is a simple and quick method to measure food quality, without sample preprocessing. This technique has been developed for food samples due to the application of a front-face measuring setup. Fluorescent compounds–fluorophores in the food samples are highly sensitive to their environment. Information about molecular structure and changes in food samples is obtained by the measurement of excitation–emission matrices of the endogenous fluorophores and by applying multivariate chemometric tools. Synchronous fluorescence spectroscopy is an advantageous screening mode used in food analysis. The fluorescent markers in food are amino acids tryptophan and tyrosine; the structural proteins collagen and elastin; the enzymes and co-enzymes NADH and FAD; vitamins; lipids; porphyrins; and mycotoxins in certain food types. The review provides information on the principles of the fluorescence measurements of food samples and the advantages of this method over the others. An analysis of the fluorescence spectroscopy applications in screening the various food types is provided

Characterization of Mung bean (Vigna radiata L.) seeds: antioxidant activity, chlorophyll and carotenoid content

Mung bean (Vigna radiata L.) is a leguminous plant with high nutritional value, traditionally known as a functional food. Legume seeds are a rich source of proteins, vitamins, minerals, and essential amino acids but also contain bioactive components and polyphenols which possess a high antioxidant capacity. Pigments content (chlorophyll a and b, carotenoids) was determined as good parameter for estimation of seed quality and an indicator of tolerance to different types of stress. The antioxidant activity of the seeds was determined using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. The concentration of chlorophile a and b and carotenoids were determined by a spectrophotometric method. Obtained results indicate a higher content of chlorophyll a than chlorophyll b, 0.352 μg/ml and 0.220 μg/ml respectively, while total carotenoids content was 0.108 μg/ml and DPPH radical scavenging activity was 54.52% ± 1.77. The advancement in this research lies in collecting information about bioactive compounds, such as chlorophylls and carotenoids, that are useful in improving the functional and antioxidant properties of quality seeds used in daily diet

Characterisation of Mung bean (Vigna radiata L.) seeds using fluorescence spectroscopy and multivariate analyis

Mung bean (Vigna radiata L.) is a leguminous plant cultivated mainly in south-east Asia and used as an ingredient in local cuisine. Its principal nutritional value is contained in its constituents such as starch, proteins, (poly)phenols, and natural antioxidants. Fluorescence spectroscopy is increasingly used as a method of choice for food analysis; due to the presence of different fluorophores originating from aromatic amino acids and secondary metabolites, it is useful for proteins and phenolics detection. In this study, the total protein and phenolic contents of mung bean seed extracts were determined using the Bradford method and Folin–Ciocalteu (FC) reagent, respectively. Antioxidant activity was determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Fluorescence spectra were recorded for a series of excitation-emission wavelengths. Further, we used the multivariate analysis on the recorded excitation-emission fluorescence matrix of the studied samples. The results showed the presence of three different fluorescence components, with the position of the emission maximum corresponding to the fluorophore of proteins (component 1 with excitation/emission peak maxima at Ex 290/Em 345 nm) and phenolics (component 2 - Ex 295/Em 395 nm and component 3 - Ex 350/Em 450 nm). This fluorescence-based method could be a useful approach for estimating the nutrient properties of leguminous food

Fluorescence spectroscopy and Multivariate Analysis for the assessment of stability of the cereal flours during storage and thermal processing

In this work, we used Fluorescence spectroscopy in combination with the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm for the assessment of stability of the cereal flours, based on stability of their individual fluorescence components, which can be altered by shelf life or heating. The tested samples were commercial maize flour (Maize flour for human consumption),wheat (T-500) and graham flour, purchased directly from a local market. The fluorescence emission spectra of the flour samples were measured in the range of 280 nm to 660 nm with excitation wavelength varing from 250 nm to 360 nm in 10-nm steps. Resulting from the analysis, the four flourescence components were derived from the emission spectra of every analyzed sample. Our results showed that the components' positions were unchanged for all flours after 2 months storage, whereas for the samples with thermal processing at 180 °C during 1 h, the positions were shifted. This method may be useful and simple for screening of a large number of flour samples

ESTIMATION OF THE ANTIFUNGAL ACTIVITY OF THE TWO DIFFERENT CARBON DOTS AGAINST Aspergillus flavus

Environmental occurrence of the pathogenic fungus Aspergillus flavus (A. flavus) has hazardous effects on the health status of plants, humans, and animals. It is important to explore novel antifungal agents to control the growth of this fungus. In this study, we aimed to evaluate the antifungal contact activity of two different types of carbon dots (CDS) nanomaterials; the one was coated with vitamin B12 (CDS@VB12) and the other was obtained from folic acid (FA@CDs). We used fluorescence spectroscopy to obtain their fluorescence fingerprinting profile. The CDs were pipetted on the PDA medium with sterilised filter paper placed in the center of the Petri dish. Then, pure A. flavus cultures were subcultured on PDA and incubated at 25°C for 7 days, and the diameters of mycelium growth were daily evaluated. The percentage of inhibition by CDS@VB12 ranged from 38.09% to 60.9% and from 46.03% to 71.39% for the amounts of 30 μl and 50 μl, respectively. On the other hand, FA@CDs did not show any inhibitory effect on the first day, and after that more progressive growth of A. flavus was noticed. The obtained results showed that CDS@VB12 could be considered as a potential new antifungal material against toxic and pathogenic A. flavus

CHARACTERISATION OF MUNG BEAN (VIGNA RADIATA L.) SEEDS USING FLUORESCENCE SPECTROSCOPY AND MULTIVARIATE ANALYIS

Mung bean (Vigna radiata L.) is a leguminous plant cultivated mainly in south-east Asia and used as an ingredient in local cuisine. Its principal nutritional value is contained in its constituents such as starch, proteins, (poly)phenols, and natural antioxidants. Fluorescence spectroscopy is increasingly used as a method of choice for food analysis; due to the presence of different fluorophores originating from aromatic amino acids and secondary metabolites, it is useful for proteins and phenolics detection. In this study, the total protein and phenolic contents of mung bean seed extracts were determined using the Bradford method and Folin–Ciocalteu (FC) reagent, respectively. Antioxidant activity was determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Fluorescence spectra were recorded for a series of excitation-emission wavelengths. Further, we used the multivariate analysis on the recorded excitation-emission fluorescence matrix of the studied samples. The results showed the presence of three different fluorescence components, with the position of the emission maximum corresponding to the fluorophore of proteins (component 1 with excitation/emission peak maxima at Ex 290/Em 345 nm) and phenolics (component 2 - Ex 295/Em 395 nm and component 3 - Ex 350/Em 450 nm). This fluorescence-based method could be a useful approach for estimating the nutrient properties of leguminous food

Characterization of colored maize seed fractions using fluorescence spectroscopy and multivariate analysis

Application of fluorescence spectroscopy combined with chemometrics algorithms provides rapid and non-destructive screening method in seed quality estimation, widely used in the agricultural industry and crop breeding. Fluorescence spectroscopy is a technique capable of detecting differs fluorophores among various colored maize seed cultivars and through different seed fractions. In the present study, we used the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm to analyse the excitation-emission matrices (EEMs) of various cultivars of colored maize (Zea mays L.) seeds and its fractions. The EEMs were recorded as a set, with the excitation ranging from 280 nm to 330 nm and the emission spectra ranging from 300 nm to 550 nm. The MCR-ALS analysis yielded two major fluorescence components for all of the analysed samples. Both position and shape of component 1 (C1) varied among the samples. On the other hand, the position and shape were similar for component 2 (C2). C1 could be used as a marker for the discrimination of colored seeds and their fractions. The observed variations in C1 between the analysed seeds may be due to the presence of their individual fluorophores, assigned to anthocyanins, proteins, and phenolics. In conclusion, the MCR-ALS analysis of the seed emission spectra has a great potential for the rapid and non-expensive characterization of various cultivars of colored seeds