Investigating the antioxidant and antibacterial properties of propolis and its potential use in some food products

Propolis is a resinous bee hive product that has many biological activities. In this study, a total of 54 raw propolis samples were collected from various geographical areas and beekeepers in Turkey in 2013 (32 samples) and 2014 (22 samples). Phenolic compounds were extracted from all samples using 70% ethanol by ultrasound assisted extraction method. Total phenolics and total flavonoids were estimated by Folin-Ciocalteau and aliminium chloride colorimetric method, respectively. The total phenolic contents of ethanolic extracts of propolis (EEP) samples varied between 6.18±0.36 mg GAE/g EEP and 157.25±12.2 mg GAE/g EEP. Their total flavonoid contents were found to be between 10.24 ± 0.33 mg QE / g EEP and 261.61±13.6 mg QE/g EEP. All the samples showed high total phenolic and flavonoid contents. Total antioxidant capacities of EEP samples collected in 2013 and 2014 from various geographical locations and beekeepers in Turkey were measured using 4 different spectrophotometric methods including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) methods. The highest total antioxidant capacities of samples were found in sample #23 from Istanbul province of Turkey as 1392.27±131.33 µM/g EEP, 1510.64±55.4 µM TE/g EEP, 4220.24±161.75 µM TE/g EEP and 557.49±20.47 µM TE/g EEP measured by DPPH, ABTS, CUPRAC and FRAP methods, respectively. The results show significantly positive high correlations between total phenolic contents, total flavonoid contents, and total antioxidant capacities determined by four different spectrophotometric assays (p < 0.05). Moreover, in this study, the individual phenolic compounds of ethanolic extracts of Turkish propolis were identified using High Performance Liquid Chromatography with Photodiode Array Detection (HPLC-PDA) method. Nine phenolic compounds were identified and quantified in 54 propolis extracts including galangin, chrysin, pinobanksin, pinostrobin, pinocembrin, caffeic acid, ferulic acid, p-coumaric acid and t-cinnamic acid. Propolis samples collected in 2013 and 2014 (54 samples) were from 6 different geographical regions of Turkey including 4 samples from Aegean Region, 5 samples from Black Sea Region, 4 samples from Central Anatolia Region 6 samples from East Anatolia Region, 29 samples from Marmara Region and 6 samples from Meditteranean Region. According to their Univariate Analysis of GLM procedure, there were statistically significant differences between total phenolic contents and total flavonoid contents of propolis samples corresponding to different regions (p < 0.05), but the differences between total phenolic contents of propolis samples corresponding to 2013 and 2014 were insignificant (p > 0.05). In addition, the differences between total antioxidant capacities of propolis samples according to year and regions were statistically insignificant (p > 0.05). There were statistically insignificant differences (p > 0.05) between individual phenolic contents of propolis samples collected in 2013 compared to individual phenolic contents of propolis samples collected in 2014. However, statistically significant differences (p < 0.05) were observed between individual phenolic contents of propolis samples including chyrisin, galangin, p-coumaric acid, pinobanksin, pinocembrin, t-cinnamic acid depending on regions. Moreover, the amounts of total phenolics and flavonoids, phenolic profiles and antioxidant capacities (with 4 different methods) of propolis samples were analyzed using PCA to investigate the differences for the samples collected from 6 different regions of Turkey. The first three principal components (PCs) explained 82.8% of the total variance, where PC1 explained 65.5%, PC2 9.6% and PC3 7.7%, respectively. Additionally, Discriminant Analysis (DA) was used for classifying the propolis based on their sample collection regions. Accordingly, six of the nine phenolic compounds were found to be significant (p < 0.05) for the geographical discrimination of propolis samples. Those predictors were subjected to Linear Discriminant Analysis (LDA) if they can be reliable predictors to discrimate the geographical regions of propolis. The results show that there were strong statistical evidence of significant differences between means of geographical regions for all predictors with pinocembrin, t-cinnamic acid, p-coumaric acid, and chrysin. Furthermore, Quadratic Discriminant Analysis, which is to base the classification not on the combined covariance matrices but on the separate ones, was used by assuming unequal group variances. The first discriminant function (F1) accounted for 71.50% of total variance while the second and third accounted for 14.31% and 8.29%. In the present research, antiproliferative and proliferative effect of propolis extracts were also investigated on 2 different breast cancer cell lines; MDA-MB-231 and UACC-3199 and on 2 normal cell lines; fibroblasts and mouse mesenchymal stem cell lines. Propolis extract used in this study were a mixture of propolis samples collected from 54 different beekepers of various geographical regions of Turkey in years 2013 and 2014. They were all blended and ground to powder. Ethanolic extract of propolis was prepared using 30% propolis tincture and removing ethanol using rotary evaporator. According to the tetrazolium dye (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium), XTT, assay results, Turkish propolis sample showed significant antiproliferative effect on MDA-MB-231 and UACC breast cancer cell lines. Interestingly, Turkish propolis sample had proliferative effect on both fibroblasts and mouse mesenchymal stem cells. These results suggest that Turkish propolis can be considered as a potent agent on breast cancer treatment for further investigations. Furthermore, in this thesis study, heat-treated dry fermented sucuk samples were produced at pilot scale with or without addition of propolis and nitrites to investigate its preservative effects based on its antimicrobial attributes. The sucuk samples were produced with 9 different formulations, as follows: (R) reference sample with 150 ppm nitrite; (A) 150 ppm nitrite and 0.25% propolis extract; (B) 150 ppm nitrite and 0.50% propolis extract (C) 50 ppm nitrite and 0.25% propolis extract; (D) 50 ppm nitrite and 0.50% propolis extract; (E) 75 ppm nitrite and 0.25% propolis extract; (F) 75 ppm nitrite and 0.50% propolis extract; (G) 100 ppm nitrite and 0.25% propolis extract; (H) 100 ppm nitrite and 0.50% propolis extract. Those samples were stored for 200 days. In samples, the physicochemical analyses (pH, moisture content, fat content, salt content, moisture/protein content, collagen content, color, thiobarbautiric acid reactive substances) and microbiological analyses (total aerobic bacteria, Eschericia coli, total coliform, total mold and yeast, Lactobacillus spp., Salmonella spp. Listeria monocytogenes) were performed starting from day zero to the end of storage. Furthermore, sensory analysis was also performed to detect any statistically significant differences in flavor perceptions of panel when propolis was added to the sucuk samples. The results showed that all samples were acceptable according to their physicochemical characteristics stated in Turkish Food Codex during 200 days of storage time. TBARS values were significantly affected (p < 0.05) by the addition of propolis extracts. Besides, increasing the concentration of propolis extract from 0.25% to 0.50% concentration change (p < 0.05) the TBARS values. The highest TBARS values were observed at control sample during 200 days of storage. Reference sample has reached to a maximum TBARS value of 1.08±0.012 mg/kg product at the end of 200th day where off-odors were produced above 1 mg/kg product level. Heat-treated sucuks containing propolis extract had significantly lower (p < 0.05) TBARS value when compared to the control sample. It was observed that propolis extracts appeared to be more effective on retarding lipid oxidation (p < 0.05) than sodium nitrate because of its high antioxidant capacity. According to the microbiological analysis including total aerobic bacteria, E. coli, total coliform, total mold and yeast, Lactobacillus spp., Salmonella spp. L. monocytogenes all samples are considered as acceptable according to the food safety rules. Therefore, it can be stated that propolis is a strong antibacterial agent and can be effective at very small amounts when used in heat-treated dry fermented sucuk formulations to ensure the food safety throughout 200 days of storage time. Furthermore, a General Lineer Model (GLM) of UNIVARIATE analysis was applied to selected variables where nitrite and propolis are accepted as independent two factors. Such analysis was aimed to understand the effects of those parameters and the presence of any interaction at their various levels on all physicochemical and microbiological analysis. Propolis addition was found to be significant (p < 0.05) in physicochemical properties including moisture, fat, collagen contents, TBARS, color (b* value) and microbiological properties of only total mold count (p < 0.05). Nitrite concentration was found to be significant (p < 0.05) in physicochemical characteristics including fat, protein, collagen contents, TBARS values, color (a* and b* values) and microbiological characteristics of only total mold counts. The differences of physicochemical characteristics including pH, moisture, fat, collagen contents, TBARS values and color characteristics were found to be dependent on interactions between propolis % and nitrite concentration (p < 0.05). Besides, sensory analysis were applied to samples according to duo-trio test procedure to determine whether product differences result from a change of propolis extract content in their formulations and an overall difference exist between samples. As a result of this study it was observed that sample A, sample E, sample F results are below the significance level. It can be concluded that using half of the amount of nitrite than reference sample and addition of propolis as a powerful antimicrobial agent to dry-fermented sucuk formulations result in no statistically significant sensory difference. It would be healthier alternative to use propolis as an antimicrobial agent to decrease the nitrite amount in production of heat-treated dry fermented sucuk.