الفهرس 
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Abstract
In the present investigation, comparative studies were carried out between different bees races [Carniolan, Carniolan hybrid (F1), Italian and Italian hybrid (F1)] and their propolis products, brood and stored pollen under temperature and relative humidity. These studies adult with efficiency of these colonies in propolis gathered according to bees race type. Certain biological aspects were considered concerning propolis gathering activity and their relationship to brood rearing activity and amounts of stored pollen. Detection of bioactive organic acids were also measured by means of GC-MS. These compounds were 20, 26, 17 and 16 for EECP, EECH, EEIP and EEIH, respectively. These compounds were identified as esters, organic acids, aromatic compounds, ketones, hydrocarbons, cyclic hydrocarbons, terpenes, alcohols, amines, amides, alkaloids and phenols. In addition, some different biological effects of propolis on different microorganisms within ethanolic extract of propolis were also performed.
The experiments of this study were carried out at Monshat El-Gharby, Temai-El-Amdeid, Dakahlia Province for one year, beginning in March 2010 and ending in February 2011. The ethanolic extracts of propolis was prepared and the microbiological experiments were carried out at Microbiol. Dept., Faculty of Agric., Damietta Univ., Damietta, Egypt.
The study dealt with the following points:
1- Propolis gathering activity
2- Sources of propolis
3- Worker brood activity and its relation with propolis gathering
4- Amounts of stored pollen and their relations with propolis gathering
5- Correlation coefficients between propolis, brood, stored pollen, temperature and relative humidity.
6- Chemical analysis of propolis by means of organic compounds.
7- Antibacterial activity by means of pathogenic G+ and G- bacteria
8- Antiyeasts activity both benefit and pathogenic ones
9- Antifungal activity using phytopathogenic fungi in liquid and solid cultures.
10- Determination of fungal growth reduction in both liquid culture and solid one.
The obtained results can be summarized as follows:
1. Propolis gathering activity
1.1. The total amount of propolis gathered during whole year by Carniolan hybrid (F1), Carniolan, Italian and Italian hybrid (F1) races were 118.28, 96.45, 87.73 and 86.56 g/colony, respectively.
1.2. The total amount of propolis collected during summer in different bees races was slightly higher than the amount gathered during spring ranged between 38.63, 28.55, 27.18 and 24.31 g/colony in Carniolan hybrid (F1), Carniolan, Italian and Italian hybrid (F1) races, respectively.
2. Sources of porpolis
Nine propolis sources were found in the experimental area; Camphor (Eucalyptus rostrata), Casurian (Casurina eguistifolia), willow (Salix babylonica), willow (Salix safsafa), Royll poinciana (Delonixregia pegiapoinciana regia), Green geen (Ficus nitidae), Acacia (Acacia Arabica), Guava (Psidium gugava) and Fig (Ficus carica).
3. Worker brood activity and its relation with propolis gathering
3.1. Activity of worker brood rearing
3.1.1. The major peak of sealed worker brood occurred during March recorded in Italian, Carniolan and Italian hybrid (F1) colonies ranged between 622, 574 and 437 sq. in/colony, respectively. While, the lowest rates of worker brood were found during February being 222, 128, 119 and 110 sq. in/colony in Italian, Carniolan hybrid (F1), Carniolan and Italian hybrid (F1), respectively.
3.1.2. Significant differences were found between different bee races during spring and summer, while insignificant differences were found between them during autumn and winter.
3.2. The relationship between worker brood activity and propolis gathering
3.2.1. An obvious relationship was found between rearing activity and the amounts of propolis gathering according to race type.
3.2.2. High level of propolis gathered was found in summer while, high level of brood rearing was found in spring. These results were occurred in all different races.
3.2.3. Carniolan hybrid (F1) colonies recorded largest amount of propolis a whole year with lowest amount of worker brood (118.28 g/colony and 3383 sq. in/colony), respectively.
3.2.4. The lowest amounts of propolis and worker brood were recorded during winter season in all bee races.
4. Amounts of stored pollen and their relation with propolis gathering activity
4.1. Amounts of stored pollen
4.1.1. Largest amounts of stored pollen were recorded during spring, summer, autumn and winter in Italian colonies (236, 134, 91 and 90 g/colony), respectively. While, lowest amounts were recorded during the same seasons in Carniolan hybrid (F1) (172, 120, 102 and 103 g/colony), respectively.
4.1.2. Carniolan colonies were more active in collecting pollen than other bee races.
4.2. The relation between amounts of stored pollen and propolis gathering activity
4.2.1. Lowest amounts of propolis and stored pollen were found in December and January and recorded in Carniolan hybrid (F1) to be 5.7 and 5.1 g/colony and 25 and 42 g/colony, respectively.
4.2.2. Largest amounts of stored pollen were recorded during spring in Italian colonies (236 g/colony) with amount of propolis recorded to be 17.23 g/colony.
4.2.3. Carniolan hybrid (F1) colonies collected more amounts of propolis than other races, while Italian colonies collected more amounts of stored pollen than others.
5. Correlation coefficients between propolis, brood, stored pollen, temperature and relative humidity
5.1. Italian colonies
5.1.1. Strong positive correlation between propolis and temperature, while no correlation was found between propolis and relative humidity.
5.1.2. Negative correlation was found between brood and both temperature and relative humidity, while positive correlation was found between brood and stored pollen.
5.2. Carniolan hybrid colonies (F1)
The correlation in these colonies took the same trend as in Italian colonies.
5.3. Carniolan colonies
5.3.1. A strong positive correlation was found between propolis and temperature, while no correlation was found between propolis and relative humidity.
5.3.2. A strong negative correlation was found between brood and temperature, while a positive correlation was found between brood and propolis.
5.4. Italian hybrid colonies (F1)
5.4.1. The correlation between propolis, temperature, brood and relative humidity in these colonies took the same trend as in other races.
5.4.2. A negative correlation was found between stored pollen and relative humidity.
Generally, temperature was more effective factor on some bees activities than relative humidity in all bee races in study.
6. Bioactive compounds containing ethanolic extracts
2.1. Ethanolic extracts of the four porpolis samples were prepared; EECP, EECH, EEIP and EEIH to analyse using GC-MS technique.
2.2. The chemical analyses indicated the presence of bioactive organic compounds in the above mentioned extracts. The total number of detected bioactive organic compounds were 20, 26, 17 and 16 for EECP, EECH, EEIP and EEIH, respectively. The main recorded components were esters, organic acids, aromatic compounds, ketones, hydrocarbons, cyclic hydrocarbons, terpene hydrocarbons, alcohols, amines, amides, alkaloids and phenols.
7. Antibacterial activity
7.1. Inhibition of bacterial growth
Two G+ bacterial strains were used in these experiments. Results illustrated that Staphylococcus aureus DSM 1104 that causes boils and sepsis was more resistant than Streptococcus mutans DSM 1159 that causes dental caries regarding the all tested ethanolic extracts of propolis by means of diameter of inhibition zone of growth.
In case of G- bacteria, E. coli ATCC25922 was more resistant than Pseudomonas aeruginosa ATCC 27853 towards the all tested ethanolic extracts of propolis by means of diameter of growth inhibition zone.
7.2. MIC and MBC values
The MIC values were found to be 600, 500, 400, and 400 mg ml-1 using EECP, EECH, EEIP and EEIH for Staph. aureus DSM 1104. These values were 500, 400, 300 and 300 mg ml-1 for Streptococcus mutans DSM 1159 using the same above extracts.
The MBC values were 700, 600, 500 and 500 mg ml-1 and 600, 500, 400 and 400 mg ml-1 using EECP, EECH, EEIP and EEIH for Staph. aureus DSM 1104 and Strept. mutans DSM 1159, respectively.
The same trend was found with G- bacteria since MIC values were 600, 600, 300, 300; 600, 600, 300, 300 for E. coli and P. aeruginosa. While MBC were 700, 700, 400; 700, 700, 400, 400 for the two tested bacteria using EECP, EECH, EEIP and EEIH, respectively. These results proved that all tested extracts presented cidal effect beside the static action.
7.2. Reduction of bacterial number
For G+ bacteria the reduction (%) expressed in bacterial cells number by means of cfu × 104 ml-1 were 13.2, 17.8, 22.3, 26.4%; 17.2, 19.0, 24.6, 27.9% and 20.5, 23.9, 26.7, 29.2% in case of Staph. aureus DSM 1104 after 24, 48 and 72 h of incubation at 37°C using EECP, EECH, EEIP and EEIH, respectively. These values were 16.0, 18.3, 24.7 and 28.8%; 20.8, 25.9, 29.4, 35.8% and 22.0, 28.5, 32.6, 38.8% under the above temp., time and extracts using Streptococcus mutans DSM 1159, respectively.
For G- bacteria, the reduction (%) were 12.4, 16.8, 21.6%; 17.7, 20.8, 27.5%%; 24.8, 25.3, 33.7 and 28.8, 29.2, 36.9% after 24, 48, 72 h of incubation at 37°C using E. coli ATCC 25922 with EECP, EECH, EEIP and EEIH, respectively. In case of Pseudomonas aeruginosa ATCC 27853, these values were 14.6, 16.9, 18.6%, 18.4, 22.4, 23.2%; 24.1, 27.8, 40.4% and 28.8; 33.1, 39.5% with the same above parameters, respectively.
8. Antiyeast activity
8.1. Inhibition of yeast growth
Results proved that Saccharomyces cerevisiae ATCC 287 was more resistant than Candida albicans ATCC 14053 against the ethanolic extracts of propolis by means of diameter of growth inhibition zone. Obtained results also showed that the propolis extract of hybridized Carniolan was more effective than that of Carniolan race in the growth inhibition. The same phenomenon was found with hybridized Italian propolis extract than that of Italian race.
8.2. MIC and MYC values
Results illustrated that Saccharomyces cerevisiae ATCC 287 was sensitive towards the tested ethanolic extracts of porpolis while Candida albicans ATCC 14053 was resistant. The values of MIC for Sacch. cerevisiae ATCC 287 were 500, 400, 500 and 400 mg ml-1 when using EECP, EECH, EEIP and EEIH, respectively. These vaues became 600, 500, 600 nad 500 with Candida albicans ATCC 14053 for the same extracts, respectively. The values of MYC were 600, 500, 600 and 500 mg ml-1 for Saccharomyces cerevisiae ATCC 287 while they were 700, 600, 700 and 600 in case of Candida albicans ATCC 14053. Results also showed that all tested extracts presented cidal effect beside the static action.
8.3. Reduction of yeast number
Obtained results proved that all the tested extracts caused reduction (%) in yeast cells number expressed in colony forming unit (cfu × 106 ml-1). Data also showed that EEIP and EEIH were more effective than the other extracts of EECP and EECH. Results showed that Saccharomyces cerevisiae ATCC 287 gave reduction value of 19.4, 21.1; 22.6, 27.5; 24.3, 27.7 and 29.5, 32.0% using EECP, EECH, EEIP and EEIH, respectively. In case of Candida albicans ATCC 14053 were 23.8, 25.5; 26.3, 30.8; 28.0, 30.5 and 34.1, 38.4% using the same extracts after 24 and 48 h, respectively.
9. Antifungal activity
9.1. Inhibition of fungal growth
Obtained results exhibited that A. flavus DSM 1830 was the most sensitive strain followed by R. nigricans DSM 1105 then M. miehei DSM 1330. On the other hand, Penicillium notatum DSM 1250 showed to be most resistant strain by means of diameter of growth inhibition zone (mm). This phenomenon was observed with the tested ethanolic extracts to be EECP, EECH, EEIP and EEIH.
9.2. Germination of fungal spores
Results illustrated that both EECH and EEIH were effective than EECP and EEIP by means of spores germination inhibition for all examined fungi.
9.3. MIC and MFC values
Regarding the MIC values, the relative order of sensitivity of the fungal strains was A. flavus DSM 1830, R. nigricans DSM 1105, M. miehei DSM 1330 and then P. notatum DSM 1250 which was more resistant strain. The values of MIC were 580, 1000, 900, 950 mg ml-1 for EEIP and EEIH for the tested fungi, respectively. These values were increased in case of MFC being 1000, 1100, 1050 and 1050 with EECP and EECH while they were 850, 950, 900 and 950 mg ml-1 with EEIP and EEIH with all the tested phytopathogenic fungi. These results illustrated that all examined extracts presented cidal effect beside static action.
9.4. Reduction of fungal growth on solid medium
Results showed that A. flavus DSM 1830 was the most sensitive fungus since the inhibition percentages were higher than other tested fungi within the two incubation period. On the other hand, penicillium notatum DSM 1250 was the most resistant. The reduction (%) of the fungal growth were 47.1, 51.8, 49.4, 54.1; 21.9, 24.7, 24.7, 28.8; 30.6, 33.3, 33.3, 36.1; 30.6, 29.2, 33.3, 36.1% after 10 days of incubation at 28°C on PDA medium using EECP, EECH, EEIP and EEIH with A. flavus DSM 1830, P. notatatum DSM 1250, R. nigricans DSM 11.0 and M. miehei DSM 1330, respectively. These values were 54.1, 57.7, 57.7, 58.8; 31.5, 30.1, 34.3, 37.0; 34.7, 37.5, 38.9, 41.7; 31.9, 34.7, 36.1, 37.5 after 15 days of incubation at 28°C on PDA medium with the same extracts and fungal strains, respectively.
9.5. Reduction of fungal biomass on liquid medium
Great effect of the tested ethanolic extracts of propolis on the fungal growth on liquid culture after 10 and 15 days at 28C was found. Results showed that the effectiveness of the bioactive organic compounds containing extracts not the same with the fungal genera but the same order of sensitivity found in solid medium was also found in case of liquid culture. The reduction (%) of fungal biomass after 10 days were 53.9, 55.3, 55.3, 56.9; 19.6, 21.4, 21.3, 22.5; 29.6, 30.8, 31.2; 26.8, 28.0, 29.4, 30.3% for EECP, EECH, EEIP and EEIH with A. flavus DSM 1830, P. notatum DSM 1250, and M. miehei DSM 1330, respectively. These values after 15 days incubation at 28°C; became 65.9, 58.5, 58.5, 59.9; 21.9, 23.5, 23.1, 23.9; 31.9, 33.5, 32.4, 33.8; 29.5, 30.4, 30.2, 31.2 for EECP, EECH, EEIP and EEIH, respectively.