IN VITRO ANTIBACTERIAL ACTIVITY OF ROYAL GELLY AGAINST PATHOGEN ESCHERICHIA COLI

In the study was used a pathogen strain of E. coli, caused septicemia for ducks, resistant for different antibacterial agents: Amoxicillin, Lincospectin, Chloramphenicol, Doxycyclin, Enrofl oxacin, Sulfonamides and Trimetoprim. Bacterial suspension of E. coli icontaminated each from test solutions in TSB of royal jelly (n=6), mixes of royal jelly and rape honey, and independent used rape honey (10–45% v/v). Have in mind exactly counts of colonies before and after incubation from each of test substances was calculated the percent of reduction up to 30 min, and after incubation (24 h and 48 h). In almost all concentrations of royal jelly (10–45 v/v), were found total inhibition effect to E. coli. Mixes from royal jelly and rape honey (1:100) possessed a higher antibacterial effect, compared with independent use of rape honey. Up to 45% (v/v), rape honey does not cause total antibacterial reduction. Royal jelly and mixes from royal jelly and rape honey have potential as alternative therapeutics agents against resistant for antibiotics pathogen strains of E. coli.

The antimicrobial activity of honey is attributed largely to osmolarity, pH, hydrogen peroxide production and the presence of other phytochemical components.In vivo, such activity may occur due to a synergistic relationship between any of these components rather than a single entity (Mavric et al., 2008).It was found, that the honey acids and low pH exert the main antibacterial factors (Bogdanov, 1997).for animals and humans.According spectrophotometric studies, the MIC 100 (the lowest concentration of test material which results in 100% inhibition of growth) value for E. coli to manuka honey was 12,5% (v/v), (Patton et al., 2006).Some authors found that growth of E. coli was completely inhibited by 30-100% honey concentrations (Noori and Al-Waili, 2004).In other study antibacterial activity of 13 types honeys were tested at four concentrations (10%, 5%, 2,5%, and 1% w/v), against E. coli.It was found that several honeys can inhibit E. coli and may have potential as therapeutic honeys (Wilkinson and Cavanagh, 2005).As the potential role for honey as a topical agent to manage surgical site or infections is increasingly acknowledged and other types of honeys need to be assessed and evaluated (Gethin and Cowman, 2008).

Pathogen strains of E. coli are often case agents for intestinal infections
The hypopharyngeal glands of the honeybee (Apis mellifera L.) produces royal jelly (RJ) that is essential to feed and raise broods and queens (Li et al., 2010).RJ may cause allergic reactions in humans, asthma, to even fatal anaphylaxis, thus this product remains unaffordable in most countries (Leung et al., 1997;Lombardi et al., 1998;Takahama and Shimazu, 2006).
From the other hand, it was found more positive effect of RJ: imunostimulating, activating vegetative and central neural systems etc.The main RJ acid, 10-hydroxy-2-decenoic acid (10-HDA), is known to have high antibiotic effect (Blum et al., 1959;Melliou and Chinou, 2005).
Research suggests that the 10-HDA found in RJ may inhibit the vascularization of tumors (Izuta et al., 2007).
Recently, it was found specific antibacterial peptide Royalisin in RJ, displayed certain antibacterial activities against Gram-positive bacteria (Shen et al., 2010).
It was found a few studies about antibacterial effect of RJ to Gram-negative microorganisms (Shirzad et al., 2007).E. coli have been used to determine the minimum inhibitory concentration (MIC) of a freshly reaped RJ.The MIC of RJ against E. coli was 2% (v/v), (Boukraa et al., 2009).In available references not found studies for effect of royal jelly for resistant for antibacterial agents strains of E. coli, case agents for septicemia for animals.
To avoid acid taste and alergic reactions after consumption of royal jelly many producers recommend mixing of this product with honey, mainly in proportin 1:100.In available references not found studies about exactly degrees of antibacterial activity from this mix for pathogen for animals strains of E.coli..

In many studies for detection of antibacterial activities of honey was used agar well diffusion method (Al Jabri et al., 2005).
Usefulness of agar well diffusion method must be interpreted with clear criterions.To the moment some authors used measuring the clear zone around the well, and expressed in phenol concentration possessing equivalent activity (Baltrusaityte et al., 2007).
On the other side standards for methods required for microbiological testing of foods for pathogens used incubation that make objective detection of pathogens.This arguments were used from as to investigate a new principle for antibacterial activity testing for bee products (Stratev et al., 2012).
In our study we use the rape honey because of fi ndings, that the antimicrobial activity of rape honey is higher, similar to that of honeydew honey (Bogdanov, 1997) -a little-known fact, which would be useful of consumers.The potential for production of the additionally processed fi nely crystallised rape honey, which is especially attractive for many consumers, is also substantial.Thus, the aims of our study were to found by microbiological method the Real Bactericidal Concentration (RBC) or 100% inhibition (0 CFU/ml), of rape honey, mixes from royal jelly with rape honey (10,20,30 and 45%v/v), and independent used royal gelly, to pathogen strain of E. coli, caused septicemia for ducks, resistant for different antibacterial agents.

Test substances
The tested rape bee honey samples were obtained from beekeepers owning many hives (from 50 to 210), immediately after the flowering of rape (the centrifugation of honey was performed in June) in different regions of the Stara Zagora district, Bulgaria.During the honey collection period, bees were not supplemented with carbohydrate syrups or treated with antimicrobial drugs.Until the analysis, samples were kept at refrigerator conditions (0-4°С).
Water content, pH, free acidity, electrical conductivity, diastase and invertase activity, specific optical activity and hydroxymethylfurfurol (HMF) content were assayed as per the harmonized methods of the European honey commission (Bogdanov et al., 1997) Д. Динков и сар.: In vitro antibacterial activity of royal gelly against pathogen escherichia coli decomposition of biologically active proteins and thus royal jelly should be frozen as soon as it is harvested (Li et al., 2007).For our experiments royal jelly was stored prior to analyze in the dark bottle in frozen conditions (-20°C).
Immediately before conducting microbiological assays in order to aid pipetting during preparation of diluted honey solutions, all test substances were adjusted to 40°C in a water bath.Solutions containing 10, 20, 30, 40 and 45% (v/v) from each of test substances were prepared in sterile TSB.To prevent photodegradation of glucose oxidase, conected with antimicrobial activity in honey (Bogdanov, 1997), all honey samples and mix from royal jelly and rape honey, were stored in the dark and dilutions were prepared immediately prior to testing (Sherlock et al., 2010).

Microbiological survey
In the study was used a pathogen strain of E. coli, caused septicemia for ducks, resistant for different antibacterial agents: Amoxicillin, Lincospectin, C h l o r a m p h e n i c o l , D o x y c y c l i n , Enrofloxacin, Sulfonamides and Trimetoprim (NCCLS, 2002).
Bacterial suspension was with density 0,5 McFarland and be prepared from 24 h bacterial culture of E. coli, by taking 3-4 colonies and dissolving in 0,85% sterile saline solution.Received bacterial suspension was with approximate concentration 1,5x10 8 CFU/ml.From suspension were prepared tenfold dilutions with sterile Triptic Soy Broth (TSB), (Merck), at to 10 7 .For detection of exactly count of E.coli from each of dilutions (1 ml) was made cultivation with ChromoCult ® TBX Agar (Merck), followed by incubation with 37°С for 24 h.Used quantity of 0,6 ml for diluted test suspension of E. coli in each from test solutions in TSB (11.4 ml), maintain the mean fi nal concentration of log 1,9-2,11 CFU/ml (table 3).This microbiological survey was done up to 30 min after inoculation (without incubation), and after 24 h and 48 h incubation at 37°C from all dilutions in TSB of contaminated with bacterial suspense test substances.With a view to calculate percent of reduction we adopt as 100% the initial (up to 30 min) and bacterial count after 24 h incubation in positive controls.Have in mind exactly counts of colonies before and after incubation, from each of test substances was calculated the percent of reduction (table 1).
In almost all concentrations of royal jelly (10-45 v/v) were found total inhibition effect to E.coli.Only for sample 2 in 10% (v/v) after 24 h and 48 incubation was found higher content of E.coli.This could be connected with content of sugars in this sample.Five from samples of royal jelly have a higher content of glucose, but only sample 2 a higher content of fructose (table 2).A higher contamination with E.coli in this case also could explain a fi nal multiplication in 10% concentration of royal jelly (log > 8 and 7,4 CFU/ml) (table 1).Mixes from royal jelly and rape honey (1:100) possessed a higher antibacterial effect compared with independent use of rape honey.In 40 and 45 % (v/v) were found 100% inhibition after 24 and 48 h, but in rape honey after 24 h -100% inhibition, but after 48 h were found different log reduction of Veterinary Journal of Republic of Srpska (Бања Лука-Banja Luka), Вол/Vol.XIV, бр/No.1,1-142, 2014 Д. Динков и сар.: In vitro antibacterial activity of royal gelly against pathogen escherichia coli Д. Динко , 1 Д.Страте , 1 Р.Балканска 2 Оригинални рад IN VITRO АНТИБАКТЕРИЈСКА АКТИВНОСТ МАТИЧНОГ МЛЕЧА ПРОТИВ ПАТОГЕНА ЕSCHERICHIA COLI Кратак са ржа Ветеринарски журнал Републике СрпскеVeterinary Journal of Republic of Srpska (Бања Лука-Banja Luka), Вол/Vol.XIV, бр/No.1,1-142, 2014 Д. Динков и сар.: In vitro antibacterial activity of royal gelly against pathogen escherichia coli Only alive cells of microorganisms could survive and made colonies.This is the principle, required for microbiological testing of foods in Europe (Commission regulation (EC) No 1441/2007).
maximal values of the main components of royal jelly samples are in the limits proposed from Sabatiny et al. (2009).They are comparable with those of royal jelly samples produced in other countries (Balkanska et al., 2012).

Table 1 .
Antibacterial activity of royal jelly (J), mix from royal jelly and rape honey (1:100), (JH) and rape honey (H), against resistant pathogen strain of E. coli.

Table 2 .
Quality parameters of rape honey