Results

System Selection and Initial Expression

To produce our proteins of interest, an E. coli expression system was selected due to its high protein yield, rapid expression time, and cost-effectiveness. The BL21(DE3) strain, a standard host for recombinant protein production, was chosen for its reliability and scalability—allowing initial optimization in small-scale cultures before scaling up for larger applications. Transformed BL21(DE3) cells were cultivated at 37°C with shaking at 180 rpm, and protein expression was induced with 1 mM IPTG at an OD600 of 0.6, followed by a 4-hour incubation under the same conditions.

Small-scale expression and solubility tests, analyzed by SDS-PAGE, confirmed that the target protein was successfully expressed. However, the majority of the protein was found in the insoluble fraction, indicating the formation of inclusion bodies. This outcome prompted the need to optimize expression conditions to enhance soluble protein yield.

Optimization of Expression Conditions

Based on literature and empirical evidence, we hypothesized that reducing the expression temperature and IPTG concentration could slow protein synthesis, potentially improving solubility. The protocol was adjusted as follows: cells were grown at 37°C to an OD600 of 0.4–0.5, after which the temperature was lowered to 20°C, and induction was performed with half the original IPTG concentration (0.5 mM). Protein production was then carried out overnight to allow for slower, more controlled synthesis.

Evaluation of Optimized Conditions

Solubility and expression efficiency were reassessed by SDS-PAGE (Fig.1). While the modified protocol increased overall protein yield, likely due to prolonged expression at a lower temperature, the target protein - α-S1-casein - remained predominantly insoluble.

Additional Optimization and Evaluations

Even after primary expression condition optimization, bovine α-S1-casein produced was primarily insoluble with a relatively small fraction of soluble protein. To address this, we used the Rosetta 2 strain of E. coli, which is optimized for eukaryotic protein expression. We tested protein production efficiency and solubility using SDS-PAGE analysis. The results indicated that while Rosetta 2 provided a similar overall protein yield compared to the BL21(DE3) strain, it did not significantly improve the solubility of α-S1-casein. These results suggest that additional strategies, such as co-expression with chaperones, use of solubility-enhancing tags, or alternative host strains, may be required to achieve soluble expression of α-S1- casein in E. coli [1].

Since the bovine α-S1- casein protein exhibited partial solubility, it was sufficient to enable the purification of the soluble fraction using HisTrap FF Ni²⁺ affinity chromatography. This finding is significant as it indicates that, despite the partial insolubility, a functional portion of the protein can be obtained for further study. The successful purification of the α-S1- casein protein using HisTrap Ni²⁺ affinity chromatography was confirmed by SDS-PAGE analysis of the eluate fractions, which revealed that functional 6xHisTag has been successfully integrated into the protein amino acid sequence (fig. 2). The presence of the 6xHisTag is crucial as it allows for the specific binding and elution of the protein from the affinity column, facilitating its purification. The efficiency of the purification process was demonstrated by the clear presence of the α-S1- casein protein in the eluate fractions, with minimal contamination from other cellular proteins. This successful purification opens the door for possible further applications of the α-S1- casein in the overall context of our project.

Conclusion and Next Steps

The optimization of temperature and inducer concentration improved total protein production but did not resolve the solubility issue for α-casein. Future efforts will focus on exploring alternative expression systems, such as Saccharomyces cerevisiae or Kluveromyces marxianus, or employing fusion tags (e.g., MBP, GST) to enhance solubility. Additionally, refining lysis and refolding protocols could be considered to recover functional protein from inclusion bodies.

Results: Attempted Preparation of Casein-Genipin Hydrogels

The objective of this study was to prepare hydrogels by cross-linking casein proteins with genipin, following established protocols and optimizing reaction conditions. Despite systematic experimentation, hydrogel formation was not achieved, though chemical interaction between genipin and casein was confirmed by a characteristic color change.

Experimental Approach and Observations

Initial Protocol

The first protocol, adapted from [doi:10.1016/j.ijpharm.2009.02.005], specified a final casein concentration of 8% (w/w) and genipin concentrations of 2.5 mM, 5 mM, and 10 mM, with crosslinking at 50°C. After preparing the hydrogel mixture and incubating for the suggested duration, vial inversion tests revealed no increase in viscosity; the mixture remained liquid, albeit with a dark blue color indicative of genipin-casein interaction. Extending the incubation to 24 hours did not yield a hydrogel, suggesting that either the crosslinking time or other protocol details (e.g., solvent choice, mixing order) were insufficient for gelation.

Revised Protocol

A second protocol was implemented, using a lower casein concentration (25 g/L) dissolved in HEPES buffer with calcium chloride (pH 7.10) and genipin prepared in HEPES buffer with absolute ethanol (final genipin concentrations: 5 mM, 10 mM, 20 mM). After 24 hours at 50°C, the mixture again failed to gel, despite the persistent blue color change.

Crosslinking Assessment

SDS-PAGE under non-reducing conditions was performed to evaluate potential crosslinking. While some protein staining was observed at the gel origin, likely due to high-molecular-weight aggregates, the results were inconclusive. Such staining could indicate extensive crosslinking preventing protein migration, but this interpretation remains speculative without further evidence.

Discussion

The consistent color change from transparent to dark blue confirmed that genipin reacted with casein primary amine groups, as reported in the literature. However, the absence of gelation suggests that sodium caseinate—used in both protocols—may not be suitable for hydrogel formation under these conditions. Casein s native structure, typically present as micelles in milk, is disrupted in sodium caseinate, potentially hindering the formation of a stable three-dimensional network required for hydrogel formation. Successful hydrogel preparation with genipin often relies on intact protein structures or assembled micelles, which provide the necessary physical interactions for gelatination.

Conclusion

Propolis extract demonstrated antimicrobial activity in liquid culture, but its efficacy was weaker than anticipated. The 96-well plate assay proved more reliable for quantifying growth inhibition, though extract opacity constrained the usable volume. Future work should focus on producing more concentrated extracts or employing alternative detection methods (e.g., viability staining) to overcome these limitations.

Results: Assessment of Propolis Antimicrobial Activity

We aimed to evaluate the antimicrobial efficacy of propolis extract, prepared using PEG 400 to avoid ethanol-related interference, against Escherichia coli and Saccharomyces cerevisiae. Despite iterative adjustments to the experimental design, consistent inhibition was only observed using a 96-well plate assay, which allowed for quantitative analysis of growth inhibition.

Experimental Approach and Observations

Agar Well Diffusion Method

Initially, the agar well diffusion method was employed, following a published protocol. Propolis extract (assumed phenolic concentration: 10.7 ± 1.2 mg/mL) was applied to wells in dual-layer agar plates at volumes of 100 μL, 150 μL, and 200 μL. After 48 hours of incubation, no clear zones of inhibition were visible, though slight growth reduction was noted near the wells. Repeating the experiment with slightly dried plates and a 20% PEG 400 positive control yielded similar results, suggesting limited diffusion or insufficient antimicrobial activity under these conditions.

Colony Assay Stamp Method

A 3D-printed colony assay stamp was introduced to standardize inoculation and improve diffusion. Plates were stamped with microorganisms, and propolis extract was added to a central well. Post-incubation, colonies closer to the well appeared smaller, indicating a potential localized effect. However, nutrient competition among colonies could not be ruled out as a contributing factor.

96-Well Plate Assay

To enhance repeatability and quantifiability, a 96-well plate assay was implemented. Optical density measurements over time revealed a dose-dependent inhibitory effect: higher propolis concentrations correlated with reduced microbial growth. Controls with 20% PEG 400 confirmed that the solvent alone did not contribute to inhibition. Nevertheless, the effect was modest, likely due to the extract s opacity limiting accurate optical density readings at higher volumes.

Discussion

The lack of visible inhibition zones in agar-based methods may stem from insufficient extract diffusion or low active compound concentration. The 96-well plate assay provided clearer evidence of antimicrobial activity, though the opacity of the extract posed a technical challenge. These findings suggest that while propolis exhibits antimicrobial properties, the extract’s potency or the method’s sensitivity may need optimisation for robust detection.

Conclusion

Propolis extract demonstrated antimicrobial activity in liquid culture, but its efficacy was weaker than anticipated. The 96-well plate assay proved more reliable for quantifying growth inhibition, though extract opacity constrained the usable volume. Future work should focus on producing more concentrated extracts or employing alternative detection methods (e.g., viability staining) to overcome these limitations.

Results: phage virulency in proximity to propolis

We evaluated the ability of phages to infect and lyse bacterial host cells in the presence of propolis extract. The propolis extract was prepared using a 20% aqueous PEG 400 solution, to which propolis powder was added at a 1:10 (w/v) ratio. The mixture was heated at 70°C for 15 minutes to facilitate extraction, resulting in an assumed phenolic compound concentration of 10.7 ± 1.2 mg/mL, as previously described by Koo et al.(2015). PEG 400 was selected as the extraction solvent to avoid the bacterial growth inhibition associated with ethanol-based extracts.

To assess the compatibility of phages with propolis, a 96-well plate assay was designed. Host bacterial cultures (Escherichia coli) were first grown in LB media to an optical density (OD600) of 0.4–0.6, corresponding to the exponential growth phase. Once the target OD was achieved, the measurement cycle was paused, and the plate was removed under aseptic conditions. Propolis extract at six different concentrations, along with an active phage culture, was then added to the wells. The plate was reinserted into a TECAN Infinite® M Nano microplate reader, and OD600 measurements were resumed to monitor bacterial lysis over time. The assay included a negative control (propolis extract + media, without bacterial inoculum or phages) and a positive control (host bacteria + propolis, without phages), both following the same propolis concentration gradient as the test wells.

Results and Analysis

Analysis of the data revealed that phages retained their lytic activity across all propolis concentrations tested. Although propolis exhibited a slight inhibitory effect on bacterial growth, this did not significantly impair the phages ability to infect and lyse their host cells. The results indicate that propolis, at the concentrations used, does not interfere with the phages capacity to target and destroy bacteria.

Discussion and Implications

These findings suggest that phages and propolis can coexist without antagonistic effects, supporting the feasibility of their combined use in antimicrobial strategies. The mechanisms underlying phage infection—including receptor recognition, genome injection, and host cell takeover—appear to remain functional in the presence of propolis. This compatibility is particularly valuable, as it opens the door to synergistic applications where propolis could provide broad-spectrum antimicrobial coverage, while phages offer targeted elimination of specific bacterial pathogens.

Conclusion and Future Directions

In conclusion, we have demonstrated that phages can effectively survive, infect, and lyse bacterial cells in the presence of propolis extract. While propolis alone exerts a mild inhibitory effect on bacterial growth, it does not compromise phage activity, making their combined use a viable strategy for enhanced antimicrobial efficacy. Future work could explore the optimization of phage-propolis combinations, particularly against multidrug-resistant bacterial strains, and investigate their stability and activity in more complex environments.