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 utvrđ...ena 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...
