Khadi is a traditional fermented alcoholic beverage popular in Botswana. There is little documented information about the brewing processes, the microorganisms responsible for fermentation as well as the safety despite its popularity in local markets. Such information is vital for production of a standardized beverage with a consistent quality to aid its entrance into the modern commercial markets. This thesis explored the brewing processes, microbiological quality and safety, as important aspects towards a bioprocess development of khadi. Firstly, the methods of brewing khadi were investigated by administering survey questionnaire to traditional brewers in 1 town and 5 villages in Central and Northern Botswana. The findings suggest that the beverage is produced from the spontaneous fermentation of Grewia flava fruits supplemented with brown table sugar. Next, the microbial biodiversity was investigated by isolating microbes from G. flava fruits and subsequently genotyping through sequence analysis of D1/D2 domain of the 26S rDNA for yeasts and molds whereas 16S rDNA was used for bacteria. Microbiota responsible for fermentation were further characterized by an in-silico restriction fragment length polymorphism of sequenced amplicons (D1/D2 PCR-RFLP). Findings show that fruits predominantly harbored bacteria (77.8 %) with a surprisingly low abundance of yeasts and molds (22.2 %). Saccharomyces and non-Saccharomyces yeasts dominated the fermented product despite the dominance of bacteria in the fruits before fermentation. Specifically, Saccharomyces cerevisiae was the most predominant species (50%) in khadi followed Candida ethanolica (12.2%) and Saccharomycodes ludwigii (10.8%). On average khadi contained ethanol concentrations of 5.27 ± 2.02 % (v/v) with an average pH of 3.1 ± 0.31. Further probing of the identity of the fermenting yeasts using in-silico PCR-RFLP, surprisingly, suggested that the S. cerevisiae isolates were more genetically identical to the commercial baker’s yeast, instead of either the brewing or wine yeasts as the most common fermentation strains for production of alcoholic beverages. Isolation of spoilage molds such as Cladosporium sphaerospermum and Aspergillus ochraceus prompted further investigation using the Randox Evidence Investigator and Myco-10 biochip to ascertain the presence of mycotoxins that could render the beverage unsafe for consumption. A variety of mycotoxins were detected although at concentrations below the acceptable limits. Of notable concern, was deoxynivalenol (11.10 ± 5.82 ppb) and diacetoxyscirpenol (22.94 ± 8.15 ppb) with their concentrations being higher than the acceptable limits for a beverage to be deemed safe for consumption. This work is the first to expand the knowledge of the microbial and physiochemical properties of a popular beverage, khadi. In addition, this work highlights that a consortium of yeasts is responsible for fermentation of khadi. Such findings are critical for recommendation of potential starter cultures for possible commercialization of khadi with a consistent quality from standardized processes. The work further characterizes the brewing attributes of yeast isolates such as the ability to utilize different carbon sources, and tolerance to brewing-associated stresses such as high temperature, low pH and high ethanol concentrations. Overall, the results from this work provide perspectives on the strategies for production and improvement of the overall quality and safety of khadi.

Thesis (MSc Biological Sciences) --Botswana International University of Science and Technology, 2021