Sweet Tea and Symbiosis – Exploring Kombucha’s Detoxifying Effects

Hi! My name is Stacey Heaver and I am a senior BMB major spending my second summer in the X-SIG program with Drs. Krukonis and Delesalle. Last summer I worked on a bacteriophage bioinformatics project, but this summer I decided to mix it up and start on a new project that I’ll continue into the upcoming year – studying kombucha!

What’s kombucha? It’s sweet tea that’s been fermented by a symbiotic culture of bacteria and yeasts (SCOBY) into a slightly vinegary and effervescent drink. It’s thought to have originated in the Chinese Tsin dynasty in 212 BC and has been touted since by cultures worldwide as a health tonic – doing everything from preventing or curing cancer to treating arthritis and easing symptoms of depressive or anxiety disorders. Of course, we want scientific evidence before we can accept any of those claims!

Today, you can stop by the Gettysburg Giant and buy a commercial version of kombucha – and across the country, dozens of artisanal kombucha brewers are popping up. Each of these companies uses a different community of bacteria and yeasts to ferment their tea. While Acetobacter and Gluconacetobacter bacteria and Schizosaccharomyces and Saccharomyces yeasts are known to predominate in kombucha, there is no standardization of species in different fermentations – and many will have small amounts of very unique strains. Because the proposed health benefits of kombucha are expected to result from the metabolic products of the species present within it, variations in kombucha composition can change any expected health outcomes from drinking the tea. Knowing what specific strains make the healthiest kombucha would be a great asset.

kombucharainbow

Newly inoculated black, rooibos, and green tea beginning fermentation in the flow hood. The whitish solid seen floating on the top is the SCOBY – it’s a thick biofilm with cellulose scaffolding. Cutting into it, it has the same texture as raw chicken.

 

newSCOBY

A newly forming SCOBY.

As sweet tea ferments after inoculation with a kombucha SCOBY and starter tea, the first major metabolite produced is ethanol. As the ethanol concentration builds, the acetic acid bacteria grow more active, increasing the drink’s acidity and giving it a vinegar kick. The growing acidity helps make the tea broth inhospitable to a number of pathogenic bacteria that might otherwise threaten to overtake the fermentation. Lactic acid and glucuronic acid can also be produced in smaller amounts, the latter being involved in the body’s detoxification system.

Glucuronates are very polar, and by binding to toxins they solubilize and target them for expulsion from the body. They can bind both to toxins produced in the body, like endogenous reactive metabolites or the breakdown products from heme catabolism, or to external toxins introduced via environment or diet. The body also takes advantage of their solubilizing nature to send chemicals to their target tissues – like steroid hormones, fat-soluble vitamins, essential unsaturated fatty acids, and dietary polyphenols. We don’t have to produce brand-new glucuronates each time. Instead, we have an enzyme called β-glucuronidase widespread throughout our bodies, produced both by our own cells and the bacteria that inhabit it. This enzyme catalyzes the dissociation of the glucuronate moiety off of whatever compound it bound to.

B-glucuronidase reaction

Diagram via BRENDA database. The left compound is the bound glucuronate; β-glucuronidase catalyzes its dissociation and frees it to bind again.

 

When β-glucuronidase frees a toxin instead of a harmless compound, these toxins can interact negatively with the surrounding cells. In patients with colon cancer, the enzyme has been found to be 12 times more expressed in feces than in healthy patients. In patients with bladder cancer, β-glucuronidase has been found to be overexpressed in urine. However, the enzyme’s presence can also be used therapeutically, with glucuronide prodrugs targeted to tumor sites for activation and expression via the cleaving action of β-glucuronidase.

Because our gastrointestinal tracts are so frequently exposed to potential carcinogens (think heterocyclic amines in the black crust on a grilled steak), it seems that consuming these in tandem with a dose of glucuronic acid ready to bind them up would be a smart thing to do. But kombucha appears to take that even a step further, producing a dynamic duo of compounds – in addition to glucuronic acid, some kombucha samples also produce D-saccharic acid 1,4-lactone (DSL), which is a competitive inhibitor against β-glucuronidase.

DSL

DSL, a competitive inhibitor against β-glucuronidase. Structure via BRENDA database.

In my project, I am exploring how to change kombucha production conditions to maximize DSL content. The bacteria and yeasts present in each fermentation produce a complex interacting network of metabolites depending on which initial substrates they have to work with, and we’re able to change the initial type of tea or sweetener or ferment with an entirely new media, often with a unique bacterial population all its own (think kvass, kefir, wine, and beer). Kombucha is also traditionally fermented with an 8-14 day initial aerobic ferment followed by anaerobic conditions for 1-3 days, during which the acetic acid bacteria shut down and the tea becomes carbonated. Often different extracts are added for this secondary ferment, and any of these additions may have the power to change the DSL content of the final product.

Besides comparing different media for fermentation, I’m also looking at enhancing DSL production through the addition of the bacteria Gluconacetobacter saccharivorans. This bacteria is genetically closely related to Gluconacetobacter A4, which has been shown to produce large amounts of DSL. After collecting kombucha samples at different time points and in different cultures, I can filter sterilize the samples to remove any bacteria and yeasts but keep their metabolites. Then, by inoculating with G. saccharivorans, I will be able to observe how interactions with the previously made metabolites affect DSL production. By first sterilizing the samples, I will be able to see more specifically the effect this bacteria has without worrying about the unknown identities of the bacteria and yeasts in the different initial cultures. A large number of different starter samples will offer a level of reproducibility despite the unstandardized microbial composition of each culture.

My largest struggle this summer has been reliably quantifying the amount of DSL in each sample, learning how to use the capillary electrophoresis machine, and optimizing a protocol specific to this compound. I’m currently able to tell when I have more or less DSL, but I haven’t yet minimized the variation between runs of the same samples to have reliable enough data. I am grateful that I will have the next two semesters to continue working out the kinks and collecting data!

7_7_14_DSL_standards

An example of three overlayed runs of DSL standards of known amounts on the high performance capillary electrophoresis machine. The largest peak around 17 min in the top run represents the larger quantity of DSL in the sample, compared to the smaller peak when the compound is more dilute or the absence of a peak at the same timepoint when no compound in injected (bottom run).

 

I feel so fortunate to be able to spend my summer studying something as fascinating as kombucha. Our microbiome and its interactions with our environment are so wonderfully interesting. Plus, you can make this out of kombucha SCOBYs:BioDenim_jacket

image source

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