Utilising yeasts to encourage the bioconversion of mesembrine-type alkaloids? Fermentation of Sceletium is traditionally considered a vital part of the preparation of Kougoed, tribes considering the unfermented plant material 'lacks power', authors stating "the psychoactive effect of this plant is greatly enhanced" by fermentation [1]. This has been studied by various authors with some varying findings: The first studies by Patnala (2016) noted the requirement of an aqueous environment together with the presence of light to facilitate transformation on the crushed plant material. The fermentation of Kanna accomplishes the following primary outcomes: ⦁ Lowers oxalic acid ⦁ Lowers 4′-O-demethylmesembrenol ⦁ Significantly converts mesembrine to mesembrenone and ∆ 7-mesembrenone ⦁ May increase total alkaloid levels (by a very small measure) Chen and Viljoen (2019) noted "only a slight change in the mesembrenol and mesembranol content during fermentation, [but] the mesembrine content increased significantly from not detected – 1.6 μg/mL to 7.40–20.8 μg/mL. A corresponding decrease was found in the initial mesembrenone content of 8.00–33.0 μg/mL to 1.30–32.7 μg/mL after fermentation. The total alkaloid content also increased as a result of fermentation." [2] The effects of various fermentations on Sceletium [3] It is proposed that the efficacy of D. bosseranum may also be improved via a similar fermentation process. 1 Interestingly, traditional use of another putative mesembrine-type alkaloid containing plant Karee Moer ( Trichodiadema stellatum syn. barbatum ) involves utilising the yeasts (or other constituents) present on the root for preparation of active fermented products including a "potently inebriating beer". As yeasts are often capable of interesting bioconversions, it was hypothesised adding a yeast source to the standard Kanna fermentation process may encourage a higher level of yeast-induced bioconversion of the alkaloids. Strains of S. cerevisiae have been particularly shown to catalyse reduction of compounds with carbonyl groups or carbon–carbon double bonds [4]. It was thus proposed added yeast could encourage reductions such as mesembrenone to mesembrine, and to mesembranol Some bioconversions encouraged by yeast [3] It was also hypothesised that an improved Sceletium product could also potentially be obtained via prolonged fermentation into a functional beverage, once again the yeasts potentially encouraging a beneficial bioconversion. Experimental: Fermentation of D. bosseranum Several whole (incl. root) small D. bosseranum plants were pulverised fresh in a mortar and pestle and a small quantity of Saccharomyces cerevisiae (EC-1118) added. The plant material was left to ferment in light for several days. Within hours, slight visible fermentation (and CO 2 production) was noted. The fermented material was then dried, extracted with basified isopropanol (aq. ammonia), concentrated to a small sample and subjected to TLC (Silica, 0.2mm, glass backed, acetone elution, I 2 visualisation). The following was obtained: 2 Rf's obtained were 0.14, 0.36, 0.70 and a visible pigmented spot at 0.89. The band at 0.36 was quite broadly intense on visualisation Previous TLC analyses of unfermented material noted Rf's of 0.18 and 0.33 as major constituents. Due to differences in sample sizes quantitative analysis was not possible but it seems a similar profile of actives was obtained, the structural similarity of mesembrine-type alkaloids potentially making it hard to narrow down the composition. Fermentation of S. tortuosum into a functional beverage A yeast starter of sugar (10g), dried S. tortuosum powder (~3g), diammonium phosphate [DAP] (1g) and Saccharomyces cerevisiae (EC-1118) yeast in 50mL water was started. This was added to a stock 800mL sucrose solution with 1g/L DAP and allowed to ferment to completion under air-lock. References [1] Srinivas Patnala and Isadore Kanfer (2016) Sceletium Plant Species : Alkaloidal Components , Chemistry and Ethnopharmacology, Alkaloids - Alternatives in Synthesis , 3 Modification and Application DOI : 10 5772 / 66482 https :// www intechopen com / chapters / 53426 [2] W Chen , A M Viljoen , To ferment or not to ferment Sceletium tortuosum – Do our ancestors hold the answer ?, South African Journal of Botany , Volume 122 , 2019 , Pages 543 - 546 , ISSN 0254 - 6299 , https :// doi org / 10 1016 / j sajb 2018 10 011 [3] Kaylan Reddy, Gary I. Stafford and Nokwanda P. Makunga (2023) Skeletons in the closet? – Using a bibliometric lens to visualise phytochemical and pharmacological activities linked to Sceletium, a mood enhancer https://doi.org/10.1101/2023.08.11.552916 [4] Guat Kheng Khor, Mohamad Hekarl Uzir (2011) Saccharomyces cerevisiae: a potential stereospecific reduction tool for biotransformation of mono- and sesquiterpenoids https://onlinelibrary.wiley.com/doi/10.1002/yea.1827 4