Introduction

Kombucha constitutes a ‘tea fungus’ or SCOBY (symbiotic culture of bacteria and yeast) fermented in sweetened black tea. Specifically, the type of bacteria present in the ‘tea fungus’ is acetic acid bacteria, which produces a floating cellulosic pellicle as it ferments [1]. This culture of acetic acid bacteria commonly includes Komagataeibacter xylinus (formerly known as Gluconacetobacter xylinus, Acetobacter xylinum), Acetobacter xylinoides, and Bacterium gluconium. The production of bacterial cellulose by Komagataeibacter xylinus further supports the symbiosis between the bacteria and yeasts found in kombucha[2]. Through nucleotide-activated glucose situated in the cell membrane, bacterial cellulose is synthesized as fibrils containing D-glucose units linked with β-1,4-glycosidic bonds. Then, the unidirectional chains form further intra- and inter-chain hydrogen bonding with the hydroxyl groups, which merge into insoluble nanofibres. These nanofibres then continue to merge and aggregate, forming wider fibrils which contribute to the characteristics of bacterial cellulose[3].

Piper betel (betel), classified in the family Piperaceae, belonging to order Piperales, superorder Nymphaeflloraea, is a vine plant endemic to most Asian countries, such as Indonesia, India, Srilanka, and Malaysia. Some of its well-known characteristics include its aromatic scent and burning taste when consumed. Other than its aromatic purposes, betel leaves are also proven to have antimicrobial, antioxidant, antihemolytic, radioprotectant, chemopreventive, and chemotherapeutic properties.

Collagen is a glycoprotein responsible for cellular morphogenesis and tissue repair, which plays a vital role in protecting tissue from pathogens[8]. In response to collagen and laminin, α2β1 integrins mediate the cellular growth, adhesion, and morphogenetic capacity of epithelial cells, which is important for tissue regeneration.

Analysis

Two main colony characteristics were: round, opaque colonies and cellulose/skin-like, irregular, and translucent colonies. The potential was seen in the cellulose/skin-like, irregular, and translucent colonies as it matches the colony characteristics of the target strain K. xylinus. However, cultured colonies that were transferred into nutrient broths did not show any cellulose activity or form a bacterial cellulose layer on the surface. Thus, the kombucha pellicle was directly extracted from the pellicle and sterilized. All samples were able to absorb more than twice their weight but retain their shape. The samples did not exhibit any expansion in size. For gram-negative bacteria, betel leaf extract has a relatively higher antimicrobial activity compared to the liquid antibiotic. Additionally, it is observed that the kombucha pellicle did not affect the efficacy of the active ingredients (extract). In comparing the results between well diffusion and disc diffusion, it is observed that in disc diffusion assays the 50% extract had less microbial activity compared to 25% extract. This can be caused by the absorption of the kombucha pellicle as 50% extract is more viscous and denser compared to 25% extract. Thus, the pellicle was only able to absorb a minimal amount of 50% extract, which reduced its efficacy. As a result, the well diffusion will inevitably show relatively higher antimicrobial activity compared to disc diffusions because they contain more extract.

Conclusion and Future Work

• Although isolation of colonies showed potential strains of K. xylinus, the investigation was unable to identify and isolate the BC-producing strand of bacteria as cultured broths did not show any cellulose growth.

• By using the alternative method of directly acquiring the pellicle from the kombucha culture, a prototype bandage is produced.

• It can absorb twice its initial weight without a significant expansion of size.

• Through well diffusion, it is observed that betel leaf extract has substantial antimicrobial activity against both B. subtilis and E. coli, shown through the inhibition zones of larger than 0,5cm.

• Through disc diffusion, it is concluded that the pellicle did not significantly reduce the efficacy of the betel leaf extract (or any liquid it is immersed in)—as factors such as less active ingredient and well size contribute to the reduced results.

• However, betel leaf extract infused in kombucha discs shows higher efficacy against E. coli compared to B. subtilis.

It is possible for the pellicle to be utilized in various other medical prototypes as a drug medium, to improve efficacy and drug delivery to patients. As kombucha pellicle is more bioavailable, biocompatible, and environmentally sustainable, it is hoped to be commercially available as an alternative to healing scarred tissue.