Measuring the antimicrobial efficacy of betalain based hydrogel against Staphylococcus Aureus, and its colour change due to change in pH level.

By Zachary Yan, Singapore

Introduction:

Given that untreated infected wounds are a significant reason for the formation of chronic wounds (Williams, 2021), which lead to death in 28% of outpatient cases (Escandon et al., 2011), it is important that a wound infection can be detected early so that urgent medical attention can be provided. Currently, smart dressings which indicate temperature, pH, and warn of an infection are used to alert individuals of an infection. However, they are difficult to find commercially. Furthermore, commonly found dressings that stick to wounds often create a dry wound environment, which is not beneficial to the healing process (Nuutila & Eriksson, 2021). Hence, Betalain based hydrogel dressings are an alternative due to its antimicrobial nature, eco-friendliness, and pH-dependent chromogenic properties. Hence, this research project aims to measure the antimicrobial properties of betalain based hydrogel wound dressing against Staphylococcus Aureus Bacterium, through the Kirby Bauer method, as well as its colour change due to change in pH level.

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Literature Review:

With regards to its eco-friendliness, betalains are nitrogen-containing plant pigments that can be betacyanins or betaxanthins, found in plants of the order Caryophyllales. It is shown that they are a group of antimicrobial secondary metabolites (González-Lamothe et al., 2009) which have antimicrobial, antioxidant, and anti-inflammatory properties (Georgiev et al., 2010; Patel et al., 2023). As betalains can be extracted from common plants such as the red beetroot (Fernando et al., 2021), they can be produced naturally at low cost, allowing the dressing to be available to individuals in various income ranges while its production methods are kept sustainable.

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Furthermore, Betalain disrupts the cell membranes of gram-positive and gram-negative bacteria (Wijesinghe & Choo, 2022), which is supported by several studies that have shown its efficacy in killing ESKAPE pathogens commonly found in wound infections. These pathogens include Acinetobacter baumannii (Mukhopadhyay et al., 2025) and Staphylococcus aureus (Mbae et al., 2023), among others. For example, a study conducted in 2011 measured the antimicrobial activity of betalain, which is found within Beta vulgaris L. This was tested using the Kirby-Bauer disc diffusion method with the Staphylococcus aureus bacteria, whereby clear zones of inhibition of 12.5 ± 0.55 mm appeared when using 15 µL of the extract (Velicanski et al., 2011), indicating antimicrobial activity. Similarly, another study discovered its efficacy in killing Klebsiella pneumoniae bacteria (Wijesinghe & Choo, 2022).

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Betalains change color under varying pH as they are indole-derived pigments found in plants (Fernando et al., 2021), and their conjugated double-bond systems undergo protonation and deprotonation (Sadowska-Bartosz, 2021). Hence, under varying pH levels, the distribution of electrons varies, which causes the absorption maximum to shift and the color of the extract to change (Sadowska-Bartosz, 2021). A study found that open wounds exist in the pH range of 6.5 to 8.5 while chronic wounds exist from 7.2 to 8.9 (Tarricone et al., 2020); hence, there is likely to be a color change between a red-colored dressing in regular wounds which shifts toward a blue hue in infected wounds (Calva-Estrada et al., 2022).

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Therefore, as the literature review proves that betalain is an effective antimicrobial and indicator of infection, this study proceeds to investigate its usage in a hydrogel dressing, which previous studies have not conducted before. It is hypothesized that there will be a visible color change of the dressing as the pH of the solution increases from 6.5 to 9, and that there will be visible zones of inhibition displayed against the bacterium.

Methodology:​ 

Firstly, betalain is extracted from beetroot pumice through centrifugation with a 30% ethanol solution at a solid:liquid ratio of 1:100. The homogenate is placed in a centrifuge at 6000 rpm for 10 min, and the supernatant is collected, with this process being repeated twice (Ravichandran et al., 2013). It is infused into a simple sodium alginate hydrogel, which acts as a mock wound dressing. There will be 8 different mock wound dressings created, ranging from 0 to 35 µL of extract in each wound dressing, in intervals of 5 µL.

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The first set of experiments aims to determine the antimicrobial activity of the betalain-based hydrogel dressing through the Kirby-Bauer disk diffusion method. Two separate Mueller-Hinton agar plates of pH 6, the optimal pH for the growth of Staphylococcus aureus (Fetsch, 2017), are prepared. Each plate is swabbed with the bacteria and divided into four quadrants. A small ring of the hydrogel dressings of equal radius will be cut out and placed in the centre of the quadrants. The first quadrant is the control, whereby the hydrogel dressing used does not contain any betalain extract. Quadrants 2 to 8 contain dressings of increasing amounts of extract, and the agar is incubated at 37°C for 18 hours (Son & Taylor, 2012) with a lid to prevent contamination from airborne particles. Then, using a digital vernier caliper, the zone of inhibition can be measured, which is a reliable indicator of antimicrobial activity. This is repeated 4 times, and the average zone of inhibition for all the hydrogel dressings will be calculated. To ensure that the rate of bacterial growth is the same in setups, the controlled variables include the temperature of the incubator, pH of the agar plate, and the duration in the incubator. To determine the optimal amount of extract in the dressings, the amount of extract in each dressing versus the zone of inhibition is analysed.

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The second experiment will find the relationship of the color changes of betalain hydrogel dressings across pH ranges of 6.5 to 9.0, which stimulates normal to infected wound conditions (Tarricone et al., 2020). Dressing samples of uniform size are immersed in sterile buffers of 6.5 to 9.0 pH levels in 0.5 increments at 37°C for 60 minutes. Next, color transitions will be noted and put into numbers using spectrophotometry (Calva-Estrada et al., 2022). The variables controlled include the time soaked and the size of the sample, to ensure sufficient time for color change and uniform change in color. The dependent variable is the peak absorption measured by the spectrophotometer, and the independent variable would be the pH level. Through qualitative observations supported by data from the spectrophotometer, an association could be drawn between the colour of the dressing, caused by the Betalain pigment, and the change in pH. Hence, we can determine if it is a reliable indicator of an infection.

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Conclusion:

This study aims to determine the optimal amount of betalain which should be infused into the hydrogel based bandages so as to maximise its antimicrobial properties. Furthermore, it aims to determine its change in colour due to a change in pH, hence whether or not it is a reliable indicator of an infection. Future studies could discuss and quantify the antimicrobial efficacy of betalain in the hydrogen based dressings against other forms of pathogens commonly found in wounds, and explore other natural antimicrobial agents.

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