Khadi is a popular traditional alcoholic beverage in rural households in Botswana. The product is produced by fermentation of ripened sun-dried Grewia flava (Malvaceae) fruits supplemented with brown table sugar. Despite its popularity, its growing consumer acceptance, its potential nutritional value, and its contribution to the socio-economic lifestyle of Botswana, the production process remains non-standardized. Non-standardized production processes lead to discrepancies in product quality and safety as well as varying shelf life. Identification of unknown fermentative microorganisms of khadi is an important step towards standardization of its brewing process for entrance into commercial markets. The aim of this study was to isolate and identify bacteria and yeasts responsible for fermentation of khadi. Yeasts and bacteria harbored in 18 khadi samples from 18 brewers in central and northern Botswana were investigated using classic culture-dependent techniques and DNA sequencing methods. Additionally, we used the same techniques to investigate the presence of bacteria and yeasts on six batches of ripened-dried G. flava fruits used for production of the sampled brews. Our results revealed that Saccharomyces cerevisiae closely related to a commercial baker’s yeast strain sold locally was the most predominant yeast species in khadi suggesting a possible non-spontaneous brewing process. However, we also detected diverse non-Saccharomyces yeasts, which are not available commercially in retail shops in Botswana. This suggests that spontaneous fermentation is partially responsible for fermentation of khadi. This study, presenting the first microbiological characterization of a prominent traditional alcoholic beverage in Botswana, is vital for development of starter cultures for the production of a consistent product towards the commercialization of khadi.

Abstract:Fermentation remains an important food preparation technique of health, cultural and economic importance throughout the world. In Sub-Saharan Africa, traditional alcoholic fermentation of cereal and non-cereal based substrates into alcoholic beverages is deeply rooted in the society. Although a multitude of traditional alcoholic beverages from cereal substrates are well researched and documented, their non-cereal based counterparts, mostly produced from indigenous, inexpensive substrates, remain less well studied. In addition, reports of health problems associated with non-cereal based alcoholic beverages produced from spontaneous fermentation are a major cause of concern. This review aims to highlight the microbiological and chemical profiles of these non-cereal based alcoholic beverages with a focus on the Sub-Saharan region. Here, we underscore the importance of the microbial repertoire and the substrates thereof in attaining aromatic complexity and a characteristic taste in these beverages. These aspects are an important starting point towards the potential commercialization of these complex aromatic non-cereal based traditional beverages.

Nine morphologically distinct halophilic yeasts were isolated from Makgadikgadi and Sua pans, as pristine and extreme environments in Botswana. Screening for biosurfactant production showed that Rhodotorula mucilaginosa SP6 and Debaryomyces hansenii MK9 exhibited the highest biosurfactant activity using Xanthocercis zambesiaca seed powder as a novel and alternative inexpensive carbon substrate. Chemical characterization of the purified biosurfactants by Fourier Transform Infra-Red spectroscopy suggested that the biosurfactant from R. mucilaginosa SP6 was a rhamnolipid-type whereas the biosurfactant from D. hansenii MK9 was a sophorolipid-type. The two biosurfactants exhibited antimicrobial activities against eight pathogenic bacteria and fungal strains (Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Micrococcus luteus, Cryptococcus neoformans, Candida albicans and Aspergilus niger). The sophorolopid-type biosurfactant was found to be the most potent among the antimicrobial drug resistant strains tested. The findings open up prospects for the development of environmentally friendly antimicrobial drugs that use an inexpensive source of carbon to reduce the costs associated with the production of biosurfactants.

Mycotoxin contamination is a major food safety drawback towards the commercialization of food products. The commercialization of khadi, a popular fermented alcoholic beverage of Botswana necessitates the investigation of the presence of mycotoxins. Khadi brewing involves the uncontrolled and unstandardized spontaneous fermentation of sun-dried Grewia flava fruits, which could be a source of mycotoxin-producing filamentous fungi (molds). This study sought to investigate the presence of mycotoxins producing fungi and mycotoxins in 18 samples of khadi collected in Central and Northern Botswana. Periconia thailandica, Cladosporium cladosporioides, Aspergillus ochraceus, Phoma eupyrena, Setosphaeria turcica, Cladosporium sphaerospermum, Chaetomium longiciliata, and Flavodon ambrosius were identified in 10 out of 18 khadi samples. Mycotoxins were detected using the Myco-10 Randox Evidence Investigator biochip kit and confirmed using a UPLC-ESI-MS/MS. Mycotoxins such as paxilline, ochratoxin A, ergot alkaloids, aflatoxin G1/G2, and zearalenone were detected using the Myco-10 Randox Evidence Investigator biochip kit. The Myco-10 results revealed that the mycotoxins in the khadi samples were lower than the regulatory limits set by FDA or European Commission. Confirmation of results using an UPLC-ESI-MS/MS system involved confirming selected mycotoxins (AFB1, DON. ZEA, FB1, FB2, FB3, NIV, and OTA) from selected khadi samples (Palapye 1, Palapye 2, Letlhakane 2, Maun 3, Mmashoro 3, and Tonota 3). The UPLC results demonstrated that the aforementioned mycotoxins in the selected khadi samples were below the detection thresholds. The study shows that while fungal isolates were present, there is no to minimal danger/risk of exposure to toxic mycotoxins after consumption of khadi. Towards commercialization endeavors, the production process would necessitate minimal mycotoxin monitoring and product preservation but no detoxifying steps.

Yeasts play an important role in spontaneous fermentation of traditional alcoholic beverages. Our previous study revealed that a mixed-consortia of both Saccharomyces and non-Saccharomyces yeasts were responsible for fermentation of khadi, a popular, non-standardized traditional beverage with an immense potential for commercialization in Botswana. Functional characterization of isolated fermenting yeasts from mixed consortia is an indispensable step towards the selection of potential starter cultures for commercialization of khadi. In this study, we report the characterization of 13 khadi isolates for the presence of brewing-relevant phenotypes such as their fermentative capacity, ability to utilize a range of carbon sources and their ability to withstand brewing-associated stresses, as a principal step towards selection of starter cultures. Khadi isolates such as Saccharomyces cerevisiae, Saccharomycodes ludwigii and Candida ethanolica showed good brewing credentials but Lachancea fermentati emerged as the isolate with the best brewing attributes with a potential as a starter culture. However, we were then prompted to investigate the potential of L. fermentati to influence the fruity aromatic flavor, characteristic of khadi. The aroma components of 18 khadi samples were extracted using headspace solid phase micro-extraction (HS-SPME) and identified using a GC-MS. We detected esters as the majority of volatile compounds in khadi, typical of the aromatic signature of both khadi and L. fermentati associated fermentations. This work shows that L. fermentati has potential for commercial production of khadi.