Silk Gel Medium for Probiotics Growth

Based on prior research, it has been found that silk cocoons from the Bombyx mori silk worms are known to produce a silk protein polymer which could be made into various forms such as gels, films, tubes, etc. for each unique qualities. Therefore, this exploration aimed to discover whether the silk film form specifically could be used as an effective medium for a biodegradable alternative to a wound healing patch, alongside checking for the effectiveness of silk film for probiotic growth. It was theoretically devised that the silk film would provide the probiotics with a source of protein to help with its growth, as the antibacterial agent combating an open wounded area. However, from the comparison between Paper Dish and Silk Film Probiotic testing, it was found that silk probiotic films had somewhat of a better potential to act as a growth medium for probiotics, but in the presence of additional moisture (not independently growing on the silk). This was because prior, another test was conducted to solely attempt growing probiotics on silk film, and very little growth was visible, that is why a moisture/ additional growth medium is required alongside the silk film for optimal probiotic growth.

Design Setup and Methods (Silk, Probiotics and Aloe vera Gel)

First of all, it is known that to extract the silk gel from the raw cocoons, that the sericin protein must first be removed in order to avoid allergenic reactions when used within/on the body. The method used to remove it involves first cutting the cocoon silk into small pieces, boiling, drying, then combining with a 9.3M LiBr solution, dialyzing the mixture to remove LiBr, then finally storing the remaining silk fibroin solution in refrigeration temperatures. Since the first few steps require lots of repeated strenuous cycles, pre-bought silk cloth fibers were selected to be the prerequisite materials to be created into silk solution.

To further create the silk film, the silk solution was first let to dry out in thin layers placed alongside the shape of round petri dishes (in room temperature). After being left to dry/set for a few hours, they would then be stored in a dry place for later use. The silk films are also autoclaved prior to being used for in-vivo testing as the preferred sterilization technique. Additionally, the other form of silk known as silk gel, can be created by extending the period of refrigerated storage of the liquid silk solution until it sets into a firm rubber-like solid under highly humid conditions.

To select which probiotics were best suited to combat pathogenic strains of bacteria bacteria, namely S. Aureus (gram positive) and E. Coli (gram negative), a Paper Dish vs. Silk Film test was preliminarily conducted to select which probiotic was best suited to combat those pathogenic strains (via inhibition circle and growth radius) while determining their effectivity to grow on silk film against the control paper dishes. The probiotics tested were Lactobacillus Plantarum, Bacillus Amyloliquefaciens, Lactobacillus Fermentum, Pediococcus Pentasaceus, Bacillus Subtilis.

As for the Aloe Vera gel, the Aloe leaves were sourced from local Indonesian markets, and was prepared by first washing it thoroughly with running tap water. Next, the base of the leaf was cut to remove as much of the yellow ‘latex’ sap found in between the rind and the parenchymatic cells/layer as the sap is known to act as a potent laxative and possibly causes kidney disease in high enough doses. Then, after the sap removal, the gel blocks are separated from the rind, blended, then strained of its fibers via use of metal/plastic strainers. As for the rind and fibrous residue, they were left to air dry in room temperature until forming the paper-like mesh (suggested to be done in dry area to avoid microbial growth/decomposition while drying).

Prebiotic Effect of Aloe vera Gel Extract on Probiotic Growth

While discovering that Aloe vera gel extract indeed holds prebiotic properties from previous well diffusion tests, the extent to which the probiotic grows was also tested and monitored. An experiment was devised in which two probiotics L. casei Shirota Strain (sourced from Yakult probiotic drink) and Bacillus Amyloliquefaciense, would be grown in nutrient broth solution and checked from growth concentration/density to measure the rate of growth. There were 5 concentrations of Aloe gel extract being used, 10, 5, 2.5, 1.25% for both probiotics, and it was found that the Optical Density reading via spectrophotometer at wavelength of 640nm was at its peak in 5% and 2.5% Aloe gel concentration for L. casei and Bacillus A. respectively (48 and 72 hours after bacterial inoculation). These results indicated that bacterial growth density was highest during these time periods and also that demonstrates the optimal concentration for the prebiotic effect of Aloe gel extract. Lastly, it is important to note that formation of insolulable bacterial plaque was visible due to lack of high enough concentration of EDTA to break down plaque formation to allow for consistent optometric readings, and that the main control were test tubes indicated signs of infections, so only day to day comparisons between concentrations are viable since the controls were used as the baseline optical density readings.

Qualitative Observation of Incorporating Vitamin C to Aloe Gel

Vitamin C/Ascorbic acid is a well known antioxidant which has multiple uses as an reducing agent. In particular, the process of extracting Aloe vera gel was best conducted via blending the gel blocks. However, this highly vigorous process incorporates a high amount of oxygen into the gel which also contain high viscosity, trapping the oxygen molecules. As such, oxidation of the gel occurs at a much faster rate causing increased rate of deterioration. Thereby, powdered vitamin C was added in concentrations of 5 g/100 mL to reduce radical oxygen molecules from further damaging the gel’s contents and prolong shelf-life. 4 conditions were tested: 2 in room temperature conditions, 2 in refrigerated conditions, 2 with ascorbic acid, 2 control (without ascorbic acid). The results showed that the gel kept in room temperature without ascorbic acid quickly turned pink/brown compared to with the ascorbic acid. Similarly, the gel even turned pink inside the refrigerated/frozen state without the ascorbic acid, and visually remained the same grayish white gray color with the ascorbic acid condition.

Well Difussion Test with Aloe Vera Gel

Several well diffusion test was conducted to test and confirm the supposed antibacterial properties of Aloe vera gel. What occurred was that the antibacterial ring of inhibition was only demonstrated on one of many dozen attempts and could not be replicated even after several different attempts/adjustments. In the end, the original published method used to demonstrate effectiveness of Aloe vera gel extract was replicated to create Aloe vera gel extract (through freeze drying, extracting with ethanol, and distillation). This extract was then tested to result in a surprising result, that the probiotic cultures grown on the Aloe extract region, signifying that the extract contains a prebiotic effect. Meaning that Aloe vera gel instead of being innately antimicrobial, it helps probiotics grow at a faster rate, therefore indirectly having antimicrobial effects towards pathogens.