Microbiological and physico-chemical characterization of khadi, a traditional non-cereal based alcoholic beverage of Botswana

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.