Microbiological and physico-chemical characterization of khadi, a traditional

non-cereal based alcoholic beverage of Botswana

By

Koketso Motlhanka

Student ID: 16100233

Department of Biological Sciences and Biotechnology

Faculty of Science

Botswana International University of Science and Technology

A Dissertation/Thesis Submitted to the Faculty of Science in Partial Fulfilment of the

Requirements for the Award of the Degree of Master of Science in Biological Sciences and

Biotechnology at BIUST

Supervisor(s): Dr Kebaneilwe Lebani and Dr Nerve Zhou

Signature: __ ________ _____ Date: 10 December 2020

i
 Declaration and Copyright

I, _Koketso Motlhanka_, declare that this thesis is my own original work and that it has not

been presented and will not be presented to any other university for a similar or any other degree

award.

Signature: ___________ Date: 10 December 2020

This dissertation/thesis is copyright material protected under the Berne Convention, the

Copyright Act of 1999 and other international and national enactments, in that behalf, on

intellectual property. It must not be reproduced by any means, in full or in part, except for short

extracts in fair dealing; for researcher private study, critical scholarly review or discourse with an

acknowledgement, on behalf of both the author and the BIUST.

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 Certification

The undersigned certifies that he has read and hereby recommends for acceptance by the College

of Science a thesis titled: Microbiological and physico-chemical characterization of khadi, a

traditional non-cereal based alcoholic beverage of Botswana, in fulfilment of the requirements

for the degree of Master of Science in Biological Sciences and Biotechnology at BIUST.

Dr. Kebaneilwe Lebani Dr. Nerve Zhou

(Supervisor) (Co-Supervisor)

Date: 10 December 2020 Date: 10 December 2020

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Abstract

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

iv
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.

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 Acknowledgements

First, I would like to express my sincere gratitude to Botswana International University of

Science and Technology for funding my studies. Secondly, my supervisor’s Dr Kebaneilwe

Lebani and Dr Nerve Zhou for their continuous support, patience, motivation, enthusiasm, and

immense knowledge. Their supervision was of utmost importance throughout the time of my

research, including writing of this thesis. I could not have imagined having better advisors and

mentors for my study. I would like to thank Monamodi Kesamang (a friend and a colleague) for

helping me with molecular based techniques on this project, Kesego Brooks (MSc student and a

friend) for helping me with sample preparations, Eric Motsamai (Technician) for helping with

media preparation, Matthews Mokoba (Senior Technician) for helping me operate most of the

equipment in the laboratory, Hawa Mukami (MSc student) for her assistance in the chemistry

analysis and Gakeonyatse Modirwagale (Technician) for her assistance during sample

collection. My sincere thanks also go to the rest of the technical staff of the Department of

Biological Sciences and Biotechnology for the help they gave during the course of my studies. I

am also grateful to my other colleagues for their advice and the stimulating discussions we had.

I would like to extend my gratitude and appreciation to the National Food Technology Research

Centre (NFTRC) for allowing me to run some of my experiments in their laboratory.

Above all, I would like to thank God for the success of this project.

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 Dedication

I would like to dedicate this thesis to my daughter Gofiwa Segwati and my family who stood by

me every day and made me who I am today.

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Contents

Declaration and Copyright........................................................................................................... ii

Certification .................................................................................................................................. iii
Abstract......................................................................................................................................... iv
Acknowledgements ...................................................................................................................... vi
Dedication .................................................................................................................................... vii
List of Figures .............................................................................................................................. xii
Chapter 1: Introduction ............................................................................................................... 1
1.1 Introduction ........................................................................................................................... 1
1.2 Rationale of the study ........................................................................................................... 3
1.3 Hypothesis and study objectives ........................................................................................... 3
1.3.1 Hypothesis.............................................................................................................................. 3
1.3.2 Objectives .............................................................................................................................. 3
Chapter 2: Literature Review ...................................................................................................... 4
2.1 Fermentation: an overview ................................................................................................... 4
2.2 Fermentation substrates and fermenting yeasts .................................................................... 6
2.3 Cereal-based and non-cereal-based alcoholic beverages ...................................................... 9
2.4 Metabolites produced during alcoholic fermentation ......................................................... 20
2.4.1 Aroma compounds ............................................................................................................... 20
2.4.2 Mycotoxins .......................................................................................................................... 25
2.5 Khadi – a traditional alcoholic beverage of Botswana ....................................................... 29
2.5.1 Khadi .................................................................................................................................... 29
Chapter 3: Materials and Methods ........................................................................................... 31
3.0 Diversity of microbiota from G. flava fruits and khadi ...................................................... 31
3.1 Survey on khadi brewing process ....................................................................................... 31
3.2 Sample collection ................................................................................................................ 31
3.3 Physico-chemical characterization of khadi ....................................................................... 32
3.3.1 Ethanol assays ...................................................................................................................... 32
3.3.2 pH measurement .................................................................................................................. 33

viii
 3.4 Enumeration and isolation of yeasts and bacteria G. flava fruits and khadi ....................... 33
3.4.1 Abundance of microbiota isolated from G. flava fruits ....................................................... 33
3.4.2 Abundance of fermentative microbiota isolated from khadi ............................................... 34
3.4.3 Isolation of molds from khadi samples ................................................................................ 34
3.5 Identification and characterization of bacterial isolates from G. flava fruits ..................... 35
3.5.1 DNA extraction and PCR from bacterial isolates ................................................................ 35
3.5.2 Characterization of bacteria from G. flava fruits ................................................................. 35
3.6 Identification of yeast isolates from G. flava fruits ............................................................ 37
3.7 Identification and characterization of yeast isolates from khadi ........................................ 38
3.7.1 Yeast colony PCR ................................................................................................................ 38
3.7.2 Phylogenetic analyses .......................................................................................................... 38
3.7.3 In-silico PCR-RFLP of Saccharomyces and non-Saccharomyces yeasts ............................ 38
3.8 Bioprocess development ..................................................................................................... 39
3.8.1 Carbon assimilation in yeasts............................................................................................... 39
3.8.2 Yeast characterization: Determination of fermentative capacity. ........................................ 40
3.8.3 Yeast stress tolerance ........................................................................................................... 40
3.9 Safety of khadi .................................................................................................................... 41
3.9.1 DNA extraction and identification of molds isolates ........................................................... 42
3.9.2 Mycotoxin quantification ..................................................................................................... 42
3.11 Statistical analyses ............................................................................................................ 44
Chapter 4: Results and Discussion ............................................................................................ 45
4.1 Khadi brewing process ........................................................................................................ 45
4.1.1 Survey responses .................................................................................................................. 45
4.2 Physico-chemical properties of khadi ................................................................................. 49
4.2.1 Ethanol content of khadi ...................................................................................................... 49
4.2.2 pH of khadi, an important factor in preservation of the brew .............................................. 50
4.3 Fermentative microbiota from G. flava fruits and khadi .................................................... 52
4.3.1 Abundance of microbiota isolated from G. flava fruits ....................................................... 52
4.3.2 Abundance of microbiota isolated from khadi..................................................................... 53

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 4.4 Investigation of bacterial isolates from G. flava fruits ....................................................... 54
4.4.1 Diversity of bacterial isolates............................................................................................... 54
4.4.2 Biochemical characterization of the bacteria isolates .......................................................... 56
4.5 Investigation of yeast isolates from G. flava fruits ............................................................. 58
4.5.1 Diversity of yeast isolates .................................................................................................... 58
4.6 Yeasts as major contributors in fermentation of khadi ....................................................... 59
4.6.1 Diversity of yeasts................................................................................................................ 59
4.6.2 The fermenting microorganism behind khadi ...................................................................... 65
4.6.3 Bioprocess development initiative ....................................................................................... 71
4.7 Safety in bioprocessing of khadi ......................................................................................... 84
4.7.1 Identification of molds contaminants of khadi .................................................................... 84
4.7.2 Mycotoxins quantification: Evidence Investigator calibration ............................................ 88
4.7.3 Mycotoxins from khadi ........................................................................................................ 90
Chapter 5: General discussion and conclusion ........................................................................ 93
5.1 Summary of study investigations and findings ................................................................... 93
5.2 The significance of the findings to food hygiene and safety .............................................. 94
5.3 The major strengths and weaknesses of study methodology .............................................. 95
5.4 Conclusion .......................................................................................................................... 96
5.5 Recommendations ............................................................................................................... 97
References .................................................................................................................................... 98
Appendix A ................................................................................................................................ 117
Appendix B ................................................................................................................................ 119
Appendix C ................................................................................................................................ 120
Appendix D ................................................................................................................................ 128
Appendix E ................................................................................................................................ 129

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List of Tables

Table 1: The fermentative microbes from substrates of non-cereal-based alcoholic beverages. ... 8
Table 2: Microbial diversity and physico-chemical properties of cereal-based alcoholic
beverages....................................................................................................................................... 11
Table 3: Microbial diversity and physico-chemical properties of non-cereal-based alcoholic
beverages....................................................................................................................................... 15
Table 4: Contribution of secondary fermentation metabolites to flavor and aroma of alcoholic
beverages....................................................................................................................................... 24
Table 5: Common mycotoxins, their source and their effects on mammalian cells. .................. 27
Table 6: Abundance of yeasts and bacteria isolated from the ripened sun-dried G. flava fruits
samples expressed in Log 10CFU/mL (colony forming units per millimeter). ............................. 52
Table 7: Identity of bacteria isolated from Grewia flava fruits based on D1/D2 region of the 16S
rDNA............................................................................................................................................. 55
Table 8: Biochemical characterization and colony pigmentation of bacterial isolates from G.
flava fruits. .................................................................................................................................... 57
Table 9: Identity of yeast isolated from G. flava fruits ................................................................ 59
Table 10: Identity of yeast isolates from the khadi samples based on D1/D2 domains of 26S
rDNA............................................................................................................................................. 64
Table 11: The number and percentage of yeast isolates from khadi samples. ............................. 65
Table 12: Carbon assimilation of the yeast isolates from G. flava fruits and khadi samples. ..... 73
Table 13: Identity of molds isolates from the khadi samples based on D1/D2 domains of 26S
rDNA............................................................................................................................................. 86
Table 14: The calibration results from the Randox Evidence Investigator.................................. 89
Table 15: Comparison of the product recovery from the spiked samples using the acetonitrile
method and the Myco-10 method. ................................................................................................ 90
Table 16: The quantification of mycotoxins from khadi samples in ppb. ................................... 92

xi
List of Figures
Figure 1: The yeast alcoholic fermentation pathway showing pathways involved in glucose
and ethanol assimilation under aerobic conditions ................................................................... 5
Figure 2: Phylogenetic schematic of conventional and non-conventional yeasts with their
fermentation capabilities ............................................................................................................ 19
Figure 3: Basic yeast metabolism .............................................................................................. 22
Figure 4: Unripe and Ripe sun-dried Grewia flava fruits, a khadi fermentation substrate
([Link] ........................................................................................... 30
Figure 5: The map of Botswana showing the sampling locations. .......................................... 32
Figure 6: Summary of the khadi fermentation process for a fermentation of 100L. ............ 45
Figure 7: Results of the questionnaire on the khadi brewing process. ................................... 48
Figure 8: The physico-chemical properties of the khadi samples ........................................... 51
Figure 9: Variability of concentrations of yeast (log10 CFU/mL) isolated from khadi samples
from different parts of the Central and Northern Botswana. ................................................ 54
Figure 10: Maximum likelihood analysis of the D1/D2 domains of 26S rDNA of
Saccharomyces yeasts isolated from khadi samples................................................................. 63
Figure 11: PCR-RFLP for yeast isolates from khadi. .............................................................. 68
Figure 12: Ability of the isolated yeast strains to utilize different carbon sources ............... 75
Figure 13: Fermentative capacity of yeast isolates .................................................................. 77
Figure 14: Carbon dioxide production rate of yeast isolates. ................................................. 78
Figure 15: Growth inhibition of the yeast strains due to different ethanol concentrations..80
Figure 16: Osmo-tolerance of the yeast isolates due to supplementation with various NaCl
concentrations. ............................................................................................................................ 81
Figure 17: The pH stress tolerance of the yeast isolates. ......................................................... 82
Figure 18: The thermo-tolerance of the isolates....................................................................... 84
Figure 19: A picture of the molds isolates from khadi samples namely: (a) Cladosporium
sphaerospermum, (b) Periconia thailandica, (c) Cladosporium cladosporioides, (d)
Setosphaeria turcica (e) Flavodon ambrosius (f) Aspergillus ochraceus, (g) Phoma eupyrena
and (h) Chaetomium longiciliata. ............................................................................................... 87

xii
 Chapter 1: Introduction

1.1 Introduction

Fermentation is accentuated by the wide spectrum of foods marketed both in developing and

industrialized countries. Its benefits to foods range from preservation, safety to improvement of

sensory characteristics (Holzapfel, 2002). Traditionally fermented foods are cherished for their

contribution to food security, value addition, enhancement of nutritional quality, digestibility and

improvement of food safety. These foods are traditionally acceptable and accessible as major

nutritional constituents in many developing countries (Holzapfel, 2002; Rolle and Satin, 2002).

Preservation and safeguarding of foods and beverages remain the principal objectives of

fermentation, with wholesomeness, acceptability and overall quality, having become increasingly

valued features to consumers, especially in rural areas where old traditions and cultural

particularities in food fermentations are generally well maintained (Marshall and Mejia, 2011).

A large variety of fermented non-cereal-based alcoholic beverages are consumed worldwide.

Most fermented alcoholic beverages that are produced commercially in large quantities are from

cereals. For instance, in Botswana, chibuku, produced from sorghum, has been commercialized

(Motlhanka et al., 2018). Standardized and well-controlled production processes have been

developed. However, fermented beverages from non-cereal-based substrates in Botswana and

beyond are produced from spontaneous fermentation whereby the desirable microbes are

naturally present in the raw material. Spontaneous fermentation is unpredictable due to the

presence of undesirable microorganisms on the raw materials or fermentation equipment and

vessels. Henceforth, there is a need for standardization of processes and development of local

food processing capabilities to alleviate food insecurity in Africa (Dendy and Dobraszczyk,

1
2001). Documentation of traditional food fermentation processes of alcoholic beverages remain a

major stumbling block to unlocking the potential of non-cereal-based fermented beverages.

Studies on indigenous processes of food fermentation aimed at upgrading or modernizing the

methods involved in their preparations are therefore necessary. In Botswana, our fermented non-

cereal-based alcoholic beverages include setopoti, bojalwa-jwa-morula, muchema and khadi, to

list a few (Motlhanka et al., 2018; Sekwati-Monang and Gänzle, 2011).

Khadi is a popular non-cereal-based alcoholic beverage produced from Grewia flava fruits in

Botswana. However, there is little or no documentation on its production processes and

associated safety for consumption despite its popularity and serving as a source of income for

many households. Poor documentation and non-standardized production processes could be

partly blamed for reported khadi poisoning cases recorded in 2010 that led to a ban of khadi sales

in the Kgatleng region

([Link] and 2017

([Link] where five people allegedly died from alcohol

poisoning in Ghanzi. Previous work on the characterization of khadi only focused on the

quantification of the volatile compounds (Mapitse, Okatch, & Moshoeshoe, 2014), leaving

leaving the most important factors that affect the overall quality of the product such as a full

documentation of the production process, the fermenting microbiota and the safety of the final

product uninvestigated. The aim of this work was to document and characterize the brewing

process as well as to investigate the microbiota responsible for fermentation of khadi. Such

information is crucial to ensure batch-to-batch consistency in quality and safety of the beverage.

Standardized methods for a safe and hygienic production of khadi is of utmost importance for

entrance into the commercial markets.

2
1.2 Rationale of the study
The main aim of this study is to elucidate the microorganisms that are involved khadi

fermentation processes. Currently, fermentation is carried out under non-standard conditions due

to absence of documentation of the processes. Such information is relevant for consistency in

microbiological and/or chemical characteristics of the alcoholic beverage. It is noteworthy that

discrepancies in processing affect taste, primary and secondary metabolites, result in spoilage

and even affect human health.

1.3 Hypothesis and study objectives

1.3.1 Hypothesis
Khadi is a product of spontaneous fermentation of fruits by diverse microbiota and therefore the

overall aim of this study was to better understand this fermentation process to improve the

fermentation protocol and ultimately the safety of khadi.

1.3.2 Objectives
 To document the production process of khadi in Botswana.

 To determine the physico-chemical properties of khadi.

 To determine the fermentative microbiota on the fruits and khadi.

 To evaluate yeast isolates from G. flava fruits and khadi as potential starter cultures.

 To investigate fungal mycotoxins as a measure of safety for consumption.

3
 Chapter 2: Literature Review
2.1 Fermentation: an overview

Fermentation involves the biochemical modification of foods and food products brought about

by enzymes secreted by microorganisms. Fermentation is an energy-yielding catabolic pathway

that proceeds with no net change in the oxidative state of the products compared to that of the

substrate. This process basically entails the extraction of energy from carbohydrates and other

organic substrates without using oxygen as an electron acceptor. Exceptions exist with Crabtree

positive yeasts, which tend to ferment even in the presence of excess oxygen and a fermentable

carbon source (De Deken, 1966). In the presence of excess sugar and oxygen, a few

microorganisms, for example, some yeasts from the Saccharomycetaceae family, generate ATP

through a respiratory or fermentative metabolism or both pathways concurrently, thus known as

respiro-fermentative yeasts (De Deken, 1966).

Crabtree positive yeasts undertake an alternatively exclusive pyruvate dissimilatory route for

redox balancing purposes involving the conversion of pyruvate to ethanol. The process recycles

NADH by regeneration of NAD+ essential for ATP production during glycolysis (Navarrete et

al., 2014). In comparison to the respiration metabolic strategy, no additional ATP is formed, but

fermentation forms a faster oxygen independent ATP production alternative, which is of utmost

importance in the utilization of anaerobic niches (Hagman et al., 2013; Piškur et al., 2006; Pronk

et al., 1996). In general the absence of a final electron receptor, oxygen, fermentation becomes

the cell's primary means of ATP production (Ban and Yang, 1998). A series of glycolytic steps

have long been elucidated and their mechanisms well described as shown in Figure 1. In most

microorganisms, in the presence of O2, NADH and pyruvate are used to generate ATP through

the respiratory pathway involving the electron transport chain. This is referred to as oxidative

4
phosphorylation, and it generates much more energy than glycolysis would alone. As such, cells

benefit from avoiding fermentation when oxygen is available with the exception being obligate

anaerobes which cannot tolerate oxygen.

Figure 1: The yeast alcoholic fermentation pathway showing pathways involved in glucose

and ethanol assimilation under aerobic conditions (Patrascu et al., 2009).

5
Glucose is broken down in a series of steps illustrated above (Figure 1) to produce ethanol and

CO2. Based on the fate of pyruvate, at the dissimilatory pyruvate branching point dependent on

the oxygen tension and metabolic regulation (Gancedo, 1998; Pronk et al., 1996). Glucose is

broken down into pyruvate in cellular cytoplasm in a series of steps, a process called glycolysis.

The whole pathway of glycolysis, contains 10 steps of chemical reactions each catalyzed by

enzymes namely hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase,

triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase, phosphoglycerate

kinase, phosphoglycerate mutase, enolase, and pyruvate kinase (Pelicano et al., 2006). In

glycolysis, one molecule of glucose (a six-carbon compound) is converted to fructose-1,6-

bisphosphate (also a six-carbon compound), which eventually gives rise to two molecules of

pyruvate (a three-carbon compound) as seen in Figure 1. Depending on the presence of oxygen,

the pyruvate can be broken down either aerobically or anaerobically. In aerobic metabolism,

pyruvate loses carbon dioxide and the remaining two carbon atoms become linked to coenzyme

A as an acetyl group to form acetyl-CoA, which then enters the citric acid cycle (Pronk et al.,

1996). There are two fates for pyruvate in anaerobic metabolism; ethanol production or lactic

acid production. In organisms capable of alcoholic fermentation such as yeasts, pyruvate loses

carbon dioxide, this time producing acetaldehyde, which, in turn, is reduced to produce ethanol

(Jansen et al., 2017).

2.2 Fermentation substrates and fermenting yeasts

Fermentation substrates are the sources of carbon and sometimes they are the source of the

fermentative microbes responsible for the fermentation process. One of the most well studied

substrates in the production of alcoholic beverages are grapes. Several studies have reported that

6
grape skins harbor a number of fermenting yeasts. Examples of yeasts isolated from grapes are as

follows; Curvibasidium pallidicorallinum, Metschnikowia viticola, Rhodosporidium babjevae,

Sporidiobolus pararoseus, Filobasidium floriform, Kloeckera spp., Hanseniaspora spp.,

Candida spp., Pichia spp., Kluyveromyces spp., Metschnikowia spp., and Cryptococcus spp.

(Barata et al., 2012; Bouki et al., 2020; Brysch-Herzberg and Seidel, 2015; Ponzzes-Gomes et

al., 2014; Walker and Stewart, 2016). Hanseniaspora uvarum, Pichia terricola, and

Metschnikowia pulcherrima have been reported to be the most representative species isolated

from grapes (Drumonde-Neves et al., 2017). However, the most well-known fermenting yeast, S.

cerevisiae, is found in very low frequencies on some grape skins, and in vineyards soils

(Swiegers et al., 2009). Some of the non-conventional yeast strains isolated from fermented

foods and alcoholic beverages elsewhere in the world include Brettanomyces spp., Candida spp.,

Cryptococcus spp., Debaryomyces spp., Dekkera spp., Galactomyces spp., Geotrichum spp.,

Hansenula spp., Hanseniaspora spp., Hyphopichia spp., Issatchenkia spp., Kazachstania spp.,

Kluyveromyces spp., Metschnikowia spp., Pichia spp., Trichosporon spp., Rhodotorula spp.,

Rhodosporidium spp., Saccharomyces spp., Saccharomycopsis spp., Schizosaccharomyces spp.,

Saccharomycodes spp., Sporobolomyces spp., Torulopsis spp., Torulaspora spp.,

Zygosaccharomyces spp., and Yarrowia spp (Bouki et al., 2020; Tamang et al., 2016a). Non-

conventional yeasts are a source of novel traits imparting unique flavors (Capece et al., 2018;

Dashko et al., 2015a; Ravasio et al., 2018) in both commercialized and traditional alcoholic

beverages like khadi. Table 1 summarizes the fermentative microbes isolated from different

substrates used to produce non-cereal-based alcoholic beverages.

7
 Table 1: The fermentative microbes from substrates of non-cereal-based alcoholic beverages.

Substrate Beverage Country Microbes References

Honey Tej Ethiopia Saccharomyces spp., Rhodotorula spp., Debaryomyces spp., Hansenula spp., Lipomyces spp., Oosporidium (Pereira et al., 2016;

spp., Pichia spp., Torulopsis spp., Trichosporon spp., Nematospora spp., Schizosaccharomyces spp., Snowdon and Cliver,

Schwanniomyces spp., Torula spp., and Zygosaccharomyces spp. 1996)

Masau (Ziziphus Kachasu Zimbabwe S. cerevisiae, Issatchenkia orientalis, P. fabianii and Aureobasidium pullulans (Nyanga et al., 2013a,

mauritiana) 2008, 2007)

Marula Mukumbi Zimbabwe Aureobasidium pullulans, Geotrichum capitatum, Trichosporon brassicae, Rhodotorula mucilaginosa, H. (Okagbue and Siwela,

(Sclerocarya anomala, H. jadinii and Hansenula spp. 2002)

birrea)

Palm sap (Palmae Palm wine Leuconostoc mesenteroides, L. delbrueckii, Zymomonas spp., Gluconobacter spp., Acetobacter spp., S. (Manel et al., 2011;

family) cerevisiae and S. chevalieris Santiago-Urbina and

Rui´z-Tera´n, 2014;

Tapsoba et al., 2015)

Grapes Wine China, France, USA, S. cerevisiae, Rhodotorula mucilaginosa, P. kudriavzevii, C. parapsilosis, Meyerozyma guilliermondii, (Bouki et al., 2020;

Italy, England, India, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis, C. zemplinina, H. uvarum, Chen et al., 2020; Gao

etc Cryptococcus flavescens, P. fermentans, C. zemplinina, Cryptococcus carnescens, C. inconpicua, Z. et al., 2019; König et

fermentati, Issatchenkia terricola, C. quercitrusa, Hanseniaspora guillier- mondii, C. bombi, Z. bailii, al., 2017; Li et al.,

Sporidiobolus pararoseus, Cryptococcus magnus, Metschnikowia pulcherrima, Issatchenkia orientalis and 2010; Ponzzes-Gomes

P. guilliermondii. H. uvarum and C. flavescen et al., 2014)

8
The fermentation of alcoholic beverages in Africa relies on cereals and non-cereal-based

substrates. The non-cereal-based substrates include sugarcane (Saccharum officinarum),

Berchemia discolor, palm tree fruits (Arecaceae family) and exudates, banana pulp (Musa

acuminata), watermelons (Citrullus lanatus), Hyphaene petersiana (mokolwane/moxao), marula

fruit (Sclerocarya birrea), Grewia flava (mogwana), Grewia occidentalis (moretlwa), Grewia

flavascens (mokgomphatha), Popowia obovata, Balanites aegyptiaca, Ziziphus mauritiana

(masau), Kedrostis hirtella (mogakangwaga), Khadia acutipetala, cassava (Manihot esculenta),

and honey (Gadaga et al., 1999; Kubo et al., 2014; Motlhanka et al., 2018; Nyanga et al., 2007;

Van Wyk, B.E. and Gericke, 2000) while the cereal-based substrates include maize (Zea mays),

millet (Panicum miliaceum, Eleusine coracana and Pennisetum glaucum), sorghum (Sorghum

bicolor), barley (Hordeum vulgare), teff (Eragrostis tef) and wheat (Triticum aestivum).

2.3 Cereal-based and non-cereal-based alcoholic beverages

The abundance of a variety of cereals across the African continent could be responsible for

popularity of cereal-based alcoholic beverages as compared to non-cereal-based alcoholic

beverages. The variety of cereals means that a lot of alcoholic beverages can be produced using

these cereals either individually or as mixture. These cereal-based alcoholic beverages are

popularly produced using maize, millet, sorghum, barley, teff and wheat. The utilization of these

different cereals gives the beverages their distinct flavor and taste. The production of cereal-

based alcoholic beverages is characterized by malting or germination process prior to

fermentation. The common malted beer in Africa are known with different names, for example,

bushera or muramba in Uganda, chibuku in Zimbabwe, dolo, burukutu, and pito in West Africa

and ikigage in Rwanda (Iwuoha and Eke, 1996; Lyumugabe et al., 2010; Muyanja et al., 2003;

9
Togo et al., 2002). The fermentation process of cereal-based alcoholic beverages is characterized

by a two-stage process namely lactic acid fermentation followed by alcoholic fermentation

usually carried out by lactic acid bacteria (LAB) and yeast species, respectively (Lyumugabe et

al., 2012). The symbiotic relationship between LAB and yeasts is reported to be responsible for

production of different secondary metabolites hence distinct aroma complexities among the

cereal-based alcoholic beverages (Tamang et al., 2016b). Table 2 below summarizes the

fermentative microbiota, physico-chemical properties and secondary metabolites of cereal-based

alcoholic beverages of sub-Saharan Africa.

10
 Table 2: Microbial diversity and physico-chemical properties of cereal-based alcoholic beverages.

Alcoholic Substrate Sensory property Alcohol content Fermentative microbes Secondary metabolites Country Reference

beverage Nature and pH

Tella Barley, Sour, cider taste and 2.5 – 5% (v/v) S. cerevisiae, Lactobacillus Ethanol Ethiopia (Lee et al., 2015; Tafere,

wheat, light yellow to dark pastorianumi and 2015; Yohannes et al.,

and 4.5 – 4.8
millet, brown Acetobacter spp. 2013)

sorghum

and teff

Amgba Sorghum Sour and cloudy 4.5 – 7% (v/v) S. cerevisiae, Kluyveromyces Methanol, butanol-1, 2-methyl 1- Cameron and Chad (Darman Roger et al.,

and maize marxianus, Cryptococcus propanol, 2-methyl 1-butanol, 3- 2013; Lyumugabe et al.,
and 2.5 – 6.2
albidius, Debaryomyces methyl 1-butanol, 1-hexanol, cis-3- 2012; Maoura et al.,

hanseni and LAB hexen-1-ol, trans-3-hexen-1-ol, 2005)

trans-2-hexen-1-ol, 2-phenyl-

ethanol, higher alcohols and

acetaldehydes

Doro Sorghum, Cloudy and light 4% (v/v) S. cerevisiae, L. plantarum, Ethanol Zimbabwe (Gadaga et al., 1999;

maize, brown L. delbrueckii, L. lactis and Jespersen, 2003;

millet L. raffinolactis Lyumugabe et al., 2012;

Togo et al., 2002)

11
Pito Sorghum Golden yellow to 0 – 1.8% (v/v) S. cerevisiae, C. tropicalis, Alkanols, phenols Nigeria, Ghana, and (Kolawole et al., 2007;

dark brown in Kloeckera apiculata, H. Aldehydes, esters, acids and Togo Onyenekwe et al., 2016;
and 3.9 – 5.3
colour and slightly anomala, Torulaspora alkanone/ketones Sefa-Dedeh et al., 1999;

sweet to very sour delbrueckii, van der Aa Kühle et al.,

Schizosaccharomyces 2001)

pombe, Kluyveromyces

africanus, Lactobacillus spp.

and Leuconostoc spp.

Ikigage Sorghum Light yellow to dark 2.2% (v/v) and S. cerevisiae, Issatchenkia Ethanol and organic acids Rwanda (Lyumugabe et al., 2012,

brown in colour and 3.9 orientalis, L. fermentum, L. 2010; Shanel and

sour buchneri and Lactobacillus Matemu, 2015)

spp.

Umcombotsi Sorghum, Light brown with Lactotobacillus spp., Ethanol Swaziland (Kumari et al., 2015;

maize, sour taste Leuconostoc spp., Masarirambi et al., 2009;

bulrush Pediococcus spp., Simatende et al., 2015;

millet or Enterococcus spp., S. Solange et al., 2014)

finger millet cerevisiae, S. capsularis, C.

ethanolica, C. haemuloni, C.

sorbophila, Dekkera

anomala and D. bruxellensis

12
Tchoukoutou Sorghum Sour taste 2 – 3% (v/v) and S. cerevisiae, Torulaspora Ethanol Benin (Greppi et al., 2013;

delbrueckii, S. pastorianus, Kayodé et al., 2011;

3.61 – 3.98
L. divergens L. fermentum, Lyumugabe et al., 2012)

L. fructivorans and

Lactobacillus spp.

13
Non-cereal-based beverages are less generally consumed in comparison to the cereal-based

beverages in the sub-Saharan region. The discrepancy could be explained by the abundance of a

variety of cereals across the continent. The mass production of non-cereal-based alcoholic

beverages of Sub-Saharan Africa relies on uncontrolled spontaneous fermentation and exploits

inexpensive substrates that are available in the locality. Some of the beverages made from non-

cereal substrate include muchema, palm wine, setopoti, bojalwa-ja-morula, khadi, mukumbi,

urwagwa, pineapple wine, and tej. The fermentation of non-cereal-based beverages, although not

all fully characterized, is reported to be carried out by yeasts, LAB and acetic acid bacteria

during a spontaneous fermentation production procedure (Ouoba et al., 2012). Mixed culture

fermentation, as shown in Table 3, could influence the distinctive flavor quality of these

traditional non-cereal-based alcoholic beverages. Table 3 summarizes the microbial diversity and

physico-chemical properties of non-cereal-based alcoholic beverages of sub-Saharan Africa

region produced from some of the substrates in Table 1 as well as their secondary metabolites.

14
 Table 3: Microbial diversity and physico-chemical properties of non-cereal-based alcoholic beverages.

Alcoholic Beverage Substrate Sensory Properties and Alcohol Fermentative microbes Secondary Metabolites Country References

Nature content (v/v)

and pH

Tej Honey Sweet, Effervescent, yellow and 8.94 – 13.16% S. cerevisiae, Kluyveromyces Ethanol, methanol, esters and Ethiopia (Bahiru et al., 2006;

cloudy bulgaricus, Debaromyces phaffi, K. organic acids Tamang et al., 2016b;

(v/v) and pH
veronae, Zygosaccharomyces rouxii, Teshome et al., 2017)
3.56 – 4.45
Hanensula subpeliculosa, S. norbensis,

K. vanudenii, Endomycopsis burtonii,

Lactobacillus spp., Streptococcus spp.,

Leuconostoc spp., and

Pediococcus spp.

Urwagwa Banana Pungent, fruity, herbaceous, 7 – 18.1% (v/v) S. cerevisiae Ethanol, 1-propanol, 2-hexanol, Rwanda, (Shale et al., 2013)

pulp astringent/acidic taste and acetic acid, 5-hexanoic acid, Burundi,

and no pH
Cloudy benzoic acid, propanoic acid, Uganda,
information
formic acid, ethyl acetate, Tanzania

butanoic acid, ethyl ester, 1- and

Butanol 3-methyl-ethyl ester and Kenya

hexanoic acid ethyl ester

Palm wine Palm tree Sour, heavy, astringent taste, 1.5 – 7.1% Staphylococcus spp., Micrococcus Lactic acid, citric acid, tartaric Nigeria, (Nwachukwu et al.,

sap whitish and milk-white spp., Serratia spp., Bacillus spp., acids, ethanol and acetic acid, Bukina 2016; Sampson et al.,
(v/v) and pH 4.0
opalescent liquid Streptococcus spp., S. cerevisiae, esters, carbonyls, higher Faso and 2007; Santiago-Urbina
– 5.5 Schizosaccharomyces pombe, C. alcohols, phenols, sulphur Cameroo and Rui´z-Tera´n, 2014;

tropicalis, C. krusei, Candida spp., compounds, terpenes, n Steinkraus, 2002)

15
 Pichia spp., S. chevalieri, Zymomonas hydrocarbons, acetals, nitrogen

mobilis, Hanseniaspora compounds and lactone

guilliermondii, H. uvarum, L.

plantarum, Leuconostoc

mesenteroides, L. nagelii, L. sucicola,

Acetobacter pasteurianus, A.

indonesiensis and

Gluconobacter spp.

Mukumbi Marula Thick creamy liquor and 15% (v/v) and S. cerevisiae, P. anomala, P. Ethanol Namibia, (Misihairabgwi and

fruits yellowish-brown no pH guilliermondii, C. tropicalis, and C. Botswan Cheikhyoussef, 2017;

information intermedia a, Nyanga et al., 2007)

Swazilan

d,

Zimbab

we and

Zambia

Pineapple wine Pineapples Fruity, sweet, cream/fatty notes 6.0 – 7.0% H. guilliermondii, Acetic acid, ethyl acetate, Angola (Ayogu, 1999;

and golden yellowish P. anomala, Meyerozyma isobutanol, 3-methyl-1-butanol, Dellacassa et al., 2017;
(v/v) and pH
guilliermondii, H. uvarum, 2-methyl-1-butanol, ethyl 2- Pino and Queris, 2010;
3.78
Wickerhamomyces anomalus and methylpropanoate, 3-methyl-1- Roda et al., 2017)

H. opuntiae butyl acetate, ethyl hexanoate,

methyl benzoate, 2-

phenylethanol, methyl 2-

methylheptanoate, ethyl

benzoate, diethyl succinate, ethyl

16
 octanoate, ethyl phenylacetate, 2-

phenylethyl acetate, ethyl 9-

decenoate and ethyl decanoate

Kachasu Masau Sour, acidic and clear distillate 9 – 41% (v/v) S. cerevisiae, Isoamyl alcohol, isobutanol, Zimbab (Gadaga et al., 1999;

fruits Issatchenkia orientalis, P. fabianii, methanol, acetaldehyde, acetone, we Nyanga et al., 2007)
and pH
(Ziziphus Aureobasidium pullulans, L. agilis, L. ethyl acetate, and furfurals
information
mauritiana minor,

), L. confusus, L. fructosus,

Adansonia L. bifermentans, L. divergens,

digitate L. fermentum, L. hilgardii and

and Streptococcus spp.

Tamarindu

s indica

17
Mixed culture fermentation has always been practiced inadvertently during spontaneous

fermentation of traditional alcoholic beverages. Substrates are one of the possible sources of

fermentation microbes thus the reason for mixed culture fermentation of alcoholic beverages.

Mixed culture fermentation characterized by a diversity in carbon metabolism pathways among

the different microbes in the consortia, is responsible for production of a variety of primary and

secondary metabolites that influence the overall aromatic complexity of the alcoholic beverage

(Dashko et al., 2014; Lencioni et al., 2016; Walker and Stewart, 2016). The technique of mixed

culture fermentation has become common in the wine and beer industries in an effort to improve

the distinctive flavors of the alcoholic beverages. For example, the use of S. cerevisiae and T.

delbrueckii lead to the production of beverages with elevated levels of higher alcohols, ethyl

acetate and isoamyl acetate, and lower levels of ethyl hexanoate and ethyl octanoate when

compared to the monocultures of the two yeasts (Canonico et al., 2017) thus enhancing the taste

and flavor. In the wine industry starter cultures are dominated by Saccharomyces species, but the

need for new tastes to address diverse customer demands have presented great potential for non-

Saccharomyces species to be used for mixed culture fermentation (Dashko et al., 2015b;

Garofalo et al., 2016).

Mixed culture fermentation utilizing species from both conventional (Saccharomyces and

Schizosaccharomyces spp) and the recently documented fermentative non-conventional genera

such as Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma,

Zygosaccharomyces, Zygotorulaspora, Lachancea, Kluyveromyces, Eremothecium, Dekkera and

others (Hagman et al., 2013) could enhance flavor complexities as well as novel traits (Figure 2).

Although some of these species are characterized by poor fermentation, their contribution in

mixed cultures is becoming clear.

18
Figure 2: Phylogenetic schematic of conventional and non-conventional yeasts with their

fermentation capabilities. This figure was reproduced from (Zhou, 2015). The figure also

shows the ethanol production capabilities of respective yeasts.

A consortium in an alcoholic beverage can be made up of yeasts, acetic acid bacteria and (LAB)

– which are responsible for the fermentation process of a product. The commonly isolated yeasts

include S. cerevisiae, Saccharomycodes ludwigii (Stringini et al., 2009), C. tropicalis (Lee et al.,

2011), T. delbrueckii, Z. rouxii (Lleixà et al., 2016), and P. kluyveri (Mu et al., 2012). Other than

yeasts, some bacteria are known to ferment simple sugars but the end product is usually non-

19
alcoholic if allowed to ferment exclusively, exceptions being Zymomonas mobilis, Clostridium

acetobutylicum, and Klebsiella pneumonia existing (Motlhanka et al., 2018). Major genera of the

LAB species isolated from various fermented beverages include Alkalibacterium spp.,

Carnobacterium spp., Enterococcus spp., Lactobacillus spp., Lactococcus spp., Leuconostoc

spp., Oneococcus spp., Pediococcus spp., Streptococcus spp., Tetragenococcus spp., Vagococcus

spp., and Weissella spp. and these are known for the sour taste and low pH they confer to the

product due to lactic acid production.

2.4 Metabolites produced during alcoholic fermentation

2.4.1 Aroma compounds

During alcoholic fermentation, a multitude of primary and secondary metabolites are produced

(Styger et al., 2011). Primary metabolites are products of metabolism that are produced during

the growth phase of an organism. Primary metabolites perform the physiological functions and

support in the overall development of the cell and primary metabolites include hormones,

glycerol and enzymes (Dussap and Poughon, 2016; Kong et al., 2007; Navarrete et al., 2014).

Secondary metabolites (such as esters, alkanols, organic acids, phenols, aldehydes and ketones),

on the other hand, are the end products of primary metabolism that are synthesized after the

growth phase has been completed (Figure 3). Such metabolites are vital in the ecological

function of the cell. The combination, variety and abundance of aromatic molecules are relevant

to sensory properties of alcoholic beverages and thus their unique flavor profile.

Different substrates have different types and amounts of fermentable sugars and amino acids.

The presence of different microorganisms in different concentrations during fermentation, each

with their varying fermentable sugar preference and primary metabolic requirements leads to

20
various species-specific pathways being turned on and resulting in production of different

secondary metabolites (Tables 2, 3 and 4). These characteristics affect the overall taste and flavor

of alcoholic beverages. Differential production of higher alcohols among microbes contributes to

the distinctive flavors. Examples of higher alcohols that intensify flavor characteristics of beers

include n-propanol, iso-butanol, 2-methylbutanol and 3-methlybutanol (Carrau et al., 2008).

Some of the secondary metabolites such as 2-methyl-1-propanol, 2/3-methyl-1-butanol, ethyl

lactate and ethyl acetate, which are typical aromatic volatiles reported in beer, wines and spirits,

have been isolated in non-cereal-based alcoholic beverages such as khadi (Mapitse et al., 2014).

Figure 3 summarizes secondary metabolites produced by yeasts.

21
Figure 3: Basic yeast metabolism. Secondary metabolites are shown in (pink) and

metabolic intermediates (lime, orange, purple and blue) are also shown (Motlhanka et al.,

2018).

Some secondary metabolites act as preservatives, some of which include organic acids such as

oxalic, citric, tartaric, malic, ascorbic, fumaric, lactic, and acetic acids. Organic acids are

responsible for the rapid acidification of alcoholic beverages (Karamoko et al., 2012). They

lower pH to below 4.6, which inhibits the growth of unwanted spoilage microorganisms (Chelule

et al., 2010). Not all secondary metabolites contribute positive characteristics to the alcoholic

beverages. For example, acetaldehyde has an unpleasant pungent odor at higher concentrations,

imparting a freshly cut green leaves flavor to the beverage (Magalhães et al., 2011). Methanol

22
and its metabolites (such as formaldehyde and formic acid) are produced during fermentation of

alcoholic beverages like kachasu (Nyanga et al., 2008) and pose health challenges due to their

toxicity effects (Arslan et al., 2015). The toxicity of kachasu due to the presence of isoamyl

alcohol, iso-butanol, acetone, methanol, ethyl-acetate, and furfurals has led to the prohibition of

kachasu in Zimbabwe since 1971 (Gadaga et al., 1999). Secondary metabolites of alcoholic

fermentation are summarized in Table 4 and their contribution to the flavor of the alcoholic

beverage is also briefly noted.

23
 Table 4: Contribution of secondary fermentation metabolites to flavor and aroma of alcoholic beverages.

Metabolite class Examples Contribution to Flavor and Aroma References

Organic acids Succinic acid, acetic acid, lactic acid, citric acid contribute to the astringency of fermented beverages. The presence of (Manel et al., 2011; Schifferdecker et al.,

some acids, notably lactic acid, can indicate undesirable bacterial 2014; Swiegers et al., 2009)

spoilage.

Higher alcohols Isoamyl alcohol, phenylethanol, isopropanol impart desirable flavor and aromas to fermented beverages but only (Magalhães et al., 2011)

within certain limits

Carbonyl compounds Acetaldehyde, aldehydes, ketones Above its flavor threshold in beverages, this compound can impart a (Hansen et al., 2001; Swiegers et al., 2009)

“grassy” or “green apple” (related to acetaldehyde) flavor but this can

be removed by secondary yeast fermentation during conditioning.

Vicinal diketones Diacetyl, pentane-2,3-dione Diacetyl in most beverages is undesirable, imparting a rancid-butter or (Naydenova et al., 2014; Pereira et al.,

“butterscotch” flavor. 2016; Regodón Mateos et al., 2006)

Polyols Butandiol, propandiol These compounds make no direct contribution to the aroma, some of (Cao et al., 2010; Hansen et al., 2001;

them are reactive and can break down with great ease to give pleasant Rosa et al., 2017)

and potent down with great ease to give pleasant and potent volatiles

e.g. diendiol.

Sulfur compounds Hydrogen sulphide, dimethyl sulphide, sulphur Dimethyl sulphide (DMS) in low concentration is a desirable attribute (Escudero et al., 2007; Walker and

dioxide, Thiols of lagers, but higher concentration imparts off-flavors. Stewart, 2016)

Esters Ethyl acetate, ethyl caprylate, ethyl butyrate, ethyl Associated with a fruity aroma and floral flavors and aromas to (De Deken, 1966; Zhou et al., 2017c)

hexanoate, 2-phenethyl acetate, ethyl laurate, ethyl fermented beverages.

decanoate, hexyl acetate

24
2.4.2 Mycotoxins

Growth of molds on foods and beverages is usually followed by the production of mycotoxins.

These secondary metabolites, which are toxic to humans and animals even in small

concentrations has been extensively reviewed (Nguegwouo et al., 2018; Roseanu et al., 2010).

Mycotoxins production is dependent on many factors such as conditions of storage, ecological

and environmental conditions. Poor storage of commodities leads to molds growth which can

subsequently lead to spoilage and mycotoxins production (Gori, 2012; Zachariášová et al., 2010).

The introduction of mycotoxins in alcoholic beverages could likely be attributed to

contamination of raw materials used in the brewing process (Nkwe et al., 2005) and favourable

environmental factors such as water, temperature, light, nutrients, and pH (Adekoya et al.,

2018b; Sokefun et al., 2018; Zhang et al., 2016). These aspects support the nutritive and

proliferative aspects of molds. The mycotoxigenic potential due to mold growth is species or

strain dependent (Bennett et al., 2003). Although measures to reduce growth of molds are

possible, in most cases not all secondary metabolites are toxic.

Mycotoxins are usually produced by mycotoxigenic genera such as Aspergillus, Alternaria,

Fusarium, Stachybotrys and Penicillium (Kharayat and Singh, 2018; Snowdon and Cliver, 1996).

The most common mycotoxins are aflatoxins, deoxynivalenol (DON), fumonisin, ochratoxins,

citrinin, ergot alkaloids, patulin and zearalenone (ZEN) which have previously been reported in

beers from Africa and Europe (Ezekiel et al., 2015; Fernández-Cruz et al., 2010; Galvis-Sánchez

et al., 2008; Inoue et al., 2013; Kirui et al., 2014; Richard, 2007; Zain, 2011). The significance of

the presence of mycotoxins in beverages could be used as an indicator for molds growth on the

beverage or the raw materials used. The mycotoxins that have been detected from wines include

aflatoxins, ochratoxin A, patulin and Alternaria toxins (Matei and Kosseva, 2017).

25
Some previous work have shown that yeast cells are able to absorb and degrade mycotoxins

(Jespersen, 2003; Querol and Fleet, 2012) but alcoholic fermentation does not reduce mycotoxins

concentration to non-significant levels thus they are present even after the fermentation process

(Inoue et al., 2013). This is made possible by the presence of β-(1→3) and β-(1→6)-glucans and

mannoprotein, which represent about 20–30 % of the cell dry weight. Table 5 summarizes some

mycotoxins that are pertinent to the context of fermentation.

26
 Table 5: Common mycotoxins, their source and their effects on mammalian cells.

Toxin Source Effect on mammalian cells References

Aflatoxin A. flavus, A. parasiticus, A. bombycis, A. ochraceus, A. Carcinogenic (Bircan, 2009; Falegan and Akoja, 2014; Kharayat

nomius, and A. pseudotamar and Singh, 2018; Kumari et al., 2015; Lyumugabe et

al., 2012; Roseanu et al., 2010; Zain, 2011)

Ergot alkaloids Sclerotia of Claviceps spp. Gangrene, central nervous system, and (Kharayat and Singh, 2018; Lepp, 2010; Morcia et al.,

gastrointestinal effects 2013; Richard, 2007)

Fumonisins F. verticillioides, F. moniliforme, F. proliferatum, F. Carcinogenic and hepatotoxic (Bertuzzi et al., 2011; Morcia et al., 2013; Richard,

nygamai, and Alternaria alternata f. sp. Lycopersici 2007; Roseanu et al., 2010; Rubert et al., 2013)

Ochratoxin (OTA) A. ochraceus, A. alliaceus, A. auricomus, A. carbonarius, A. Carcinogenic, nephourotoxic, hepatotoxic and (Barata et al., 2012; Bertuzzi et al., 2011; Fernández-

glaucus, A. melleus, and A. niger teratogenic Cruz et al., 2010; Galvis-Sánchez et al., 2008;

Karbancioǧlu-Güler and Heperkan, 2008; Roseanu et

al., 2010; Rubert et al., 2013)

T-2 toxin F. sporotrichioides, F. poae, F. equiseti, and F. acuminatum Cytotoxic and immunosuppressive (Richard, 2007; Roseanu et al., 2010; Rubert et al.,

2013)

Deoxynivalenol F. graminearum and F. culmorum Inhibition of protein synthesis (Kharayat and Singh, 2018; Nathanail et al., 2015;

Rahmani et al., 2009; Richard, 2007; Rubert et al.,

2013)

27
Diacetoxyscirpenol F. poae, F. semitectum, F. verticillioides, F. sporotrichioides, Cytotoxic and immunosuppressive (Roseanu et al., 2010; Rubert et al., 2013; Zain, 2011)

F. acuminatum, F. culmorum, F. crookwellense, F.

venenatum, F. sambucinum, F. equiseti, F. graminearum, F.

avenaceum, and F. langsethiae

Zearalenone F. roseum and F. tricinctum Estrogenic activity, potential carcinogenic and (Bertuzzi et al., 2011; Ezekiel et al., 2015; Hill, 2015;

teratogenic Kumari et al., 2015; Roseanu et al., 2010; Rubert et

al., 2013)

28
2.5 Khadi – a traditional alcoholic beverage of Botswana

Botswana has a variety of unrecorded traditional alcoholic beverages, which are produced using

either cereals, or non-cereal-based substrates. The cereal-based alcoholic beverages include

bojalwa-jwa-setswana (produced from sorghum) and sekhokho (sorghum-based) while the non-

cereal-based include khadi (produced from G. flava fruits) and setopoti (produced from

watermelon juice) (Motlhanka et al., 2018; Rudloff et al., 2012). This project is focused on the

khadi, an alcoholic beverage of Botswana produced from G. flava fruits and the identification of

its fermentative microbiota.

2.5.1 Khadi

Khadi is a reddish-brown traditional alcoholic beverage made from the fermentation of ripe sun-

dried G. flava (mogwana) fruits (Figure 4). Khadi has been previously reported to contain an

alcohol content of between 1.66 % and 5.71 % (v/v), with a pH from 2.87 to 3.16. The beverage

has been compared favorably to commercial wine (Mapitse et al., 2014). Brown table sugar is

added to the fruit juice before fermentation. One would posit that the sugar is required to aid

fermentation of the juice or increase alcohol content. However, there is no available literature

documenting the sugar content in the G. flava fruits. The other fruits used in khadi fermentation

include G. occidentalis (moretlwa), G. flavascens (mokgomphatha), Kedrostis hirtella

(mogakangwaga), and Khadia acutipetala (Van Wyk, B.E. and Gericke, 2000). The most well-

known technique used in brewing khadi is the back-slopping fermentation technique where an

already fermented khadi beverage is used as a source of starter culture for the brewing. A Global

Status Report on Alcohol consumption by the World Health Organization (WHO) reported that

29
the mixing in of mashed wild pumpkins, wild fruits, wild tuberous roots, can occur in the

production of khadi thus making khadi to be termed a ‘designer beer’ (WHO, 2004).

Unorthodox methods of production of this beverage have been known amongst the producers.

Use of marijuana, car battery acid, tobacco, dagga, during preparation are some well-known

practices (Rudloff et al., 2012). The reasons why such harmful additives are used are not

documented although some villagers claim that the battery acid increases intoxication (Rudloff et

al., 2012). Khadi is a unique traditional alcoholic beverage with no similarities to any traditional

alcoholic beverage. Mapitse et al., 2014 however detected some volatile compounds from khadi,

which are present in wine. These volatile compounds are 2-methyl-1-propanol, 2/3-methyl-1-

butanol, ethyl lactate, and ethyl acetate. In some respect, the aromatic volatile compounds of

khadi show comparability to wine. Although the beverage is seemingly popular amongst

Batswana, there is no literature available documenting the microbiological characteristics of

khadi. The microbiological characterization and consequent chemical profiling of khadi will be

important to ascertain the resultant variations of the beverage.

Figure 4: Unripe and Ripe sun-dried Grewia flava fruits, a khadi fermentation substrate
([Link]

30
 Chapter 3: Materials and Methods

3.0 Diversity of microbiota from G. flava fruits and khadi

3.1 Survey on khadi brewing process

A questionnaire was administered to the khadi brewers in 1 town and 5 villages in Central and

Northern Botswana (Appendix A). The questionnaire survey was carried out to document the

process of khadi fermentation, the ingredients and their quantities, storage of the ingredients, the

fermentation conditions, fermentation duration and the factors to determine completion of

fermentation. The questionnaire was completed by a total of 18 respondents, 3 from each

location where samples were collected as well as 6 respondents who drink khadi.

3.2 Sample collection

During the sampling process, 250 g of ripened sun-dried G. flava fruits and 500 mL khadi were

randomly collected from local khadi brewers from Botswana. Collection of samples was only

done in central and northern towns and villages (Tonota, Palapye, Serowe, Letlhakane,

Mmashoro and Maun) (Figure 5) because khadi brewers in the southern part of Botswana use

tubers instead of G. flava fruits for fermentation of khadi. A total of 6 batches of ripened sun-

dried G. flava fruits and 18 khadi samples were collected. G. flava fruit samples were collected

aseptically using sterile zip-lock bags whereas khadi samples were collected in sterile 500 mL

bottles. Samples were transported to the laboratory at a temperature of 0 – 4 °C in a cooler box

with frozen ice packs before analyses. All samples were analyzed at most 6 hours after

collection. All the khadi samples were collected at a stage when the khadi was ready for

consumption i.e. after completion of fermentation.

31
Figure 5: The map of Botswana showing the sampling locations. The map shows the
locations in the central and northern parts of Botswana (black dots) from which ripened sun-
dried G. flava fruits and khadi were collected. The green triangles show the number of
replicates per location.

3.3 Physico-chemical characterization of khadi

3.3.1 Ethanol assays

Khadi samples were centrifuged and the resultant cell-free supernatant was used to determine the

ethanol content using an enzymatic ethanol assay kit (K-ETOH 08-18; Megazyme, Ireland

according to the manufacturer’s instructions. The samples were analyzed in duplicates.

32
3.3.2 pH measurement

Within 6 hours of collection and storage at 4 °C, khadi samples were tested in triplicate to

determine their pH. A pH electrode (HANNA H1208, UK) was directly placed into each sample

and the results were recorded. The pH meter was first calibrated using a 3-point calibration steps

according to manufacturer´s instructions, using buffers standards of pH 2, 7 and 9.0 before use.

3.4 Enumeration and isolation of yeasts and bacteria G. flava fruits and khadi

3.4.1 Abundance of microbiota isolated from G. flava fruits

A modified protocol for isolating microorganism fruits was used from Lee et al., (2011). A

maximum of 5 fruits of each of the fruit samples were crushed and re-suspended in 2 mL × PBS

(phosphate buffered saline). The mixture was then vortexed vigorously for 5 mins and stored on

ice. To isolate yeast, 1 mL from each sample was pelleted in a micro-centrifuge for 1 min at 14

000 x g. The pellets were then re-suspended in 200 µL lysozyme solution (10 mg/mL lysozyme;

20 mM Tris-HCl; 2mMEDTA; 1 % Triton X-100 (pH 8)) and maintained at room temperature

for 30 minutes to lyse the bacteria. 100 µL of tenfold serially diluted samples (in 1 × PBS) was

then spread plated on yeast peptone dextrose agar (YPD) (2 % glucose; 1 % yeast extract; 2 %

peptone; and 1.5 % agar, at a pH of 6.2) supplemented with a cocktail of antibiotics (100 µg/mL

streptomycin, 100 µg/mL penicillin and 100 µg/mL ampicillin) for selective growth of yeast

before enumeration. The plates were incubated at 30 °C for 48 hours to 72 hours. After

incubation, colony-forming units (CFU) were counted and recorded. For further studies,

representatives of morphologically distinct colonies were picked, observed under a microscope

(DE/Axioplan 2; Carl Zeiss, Oberkochen, Germany) and purified by sub-culturing twice on YPD

plates. The pure cultures were then cryopreserved at −80 °C in 25 % glycerol. On the other hand,

33
isolation of bacteria was done by using the remaining 2 mL of the sample. The samples were

serially diluted and spread plated on lysogeny broth agar (1 % NaCl; 1 % peptone; 0.5 % yeast

extract; and 1.5 % agar at a pH of 7.4) supplemented with 10 μg/mL of cycloheximide to select

against yeast and other fungi. After incubation at 37 °C for 24 hours to 72 hours and

representative of different colonies were purified and stored as mentioned above. The

experiments were done in triplicates.

3.4.2 Abundance of fermentative microbiota isolated from khadi

100 µL aliquots from all khadi samples were 10-fold serially diluted in 1 × PBS, kept on ice, and

plated out on either selective YPD or LB agar media to enumerate yeast or bacteria, respectively,

as in the section above (section 2.3.1). De Man, Rogosa and Sharpe agar (MRS agar) plates

(CONDA Laboratory, Spain) supplemented with 10 μg/mL of cycloheximide were used to

determine and enumerate lactic acid bacteria. Plates were incubated at 37 °C (modified method

by (Wilson, 2012)) at anaerobic conditions (10 % of CO2) for 72 hours to promote the growth of

LAB. Colonies were counted and expressed as log colony-forming units per milliliter (log

CFU/mL) and recorded. The experiments were done in triplicates of triplicates meaning each

sample was plated onto 27 plates.

3.4.3 Isolation of molds from khadi samples

Isolation of molds was done as in section 3.4.2. The mold colonies on the YPD plates were then

cut and transferred to malt extract agar (MEA; 3 % malt extract, 0.5 % mycological peptone, 1.5

% agar and pH 5.4) (Oxoid, England) and potato dextrose agar plates (PDA; 0.4 % potato

infusion, 2 % dextrose, 2 % agar and pH 3.5) (Difco, Detroit, USA) to get pure cultures which

were then incubated at 30 °C for 5 days. Purified cultures were stored in 25 % glycerol at −80 °C

for further analysis.

34
3.5 Identification and characterization of bacterial isolates from G. flava fruits

3.5.1 DNA extraction and PCR from bacterial isolates

DNA was extracted from a single colony using GenElute ™ Bacterial Genomic DNA Kit

(Sigma-Aldrich, USA) according to manufacturer’s instructions. The extracted genomic DNA

was used for PCR amplification using 16S rDNA universal primers, pAF (5'-

AGAGTTTGATCCTGGCTCAG-3') and pER (5'CCGTCAATTCCTTTGAGTTT-3') (Ouoba et

al., 2004) and the following cycling conditions: initial denaturation at 98 °C for 30 s; 35 cycles

of denaturation (98 °C for 30 s), annealing (47 °C for 30 s), and extension (72 °C for 30 s), a

final extension step at 72 °C for 7 min and held at 4 °C ∞. Negative controls in which the

template DNA in the PCR mixture was replaced with sterile distilled water were also included.

All amplicons were purified using a QIAquick PCR product purification kit (Qiagen, GmBH,

Germany) according to manufacturer´s instructions. The amplicons were sequenced by Inqaba

Biotech (Pretoria, South Africa). SnapGene® Viewer software ver. 4.2.11 (GSL Biotech)

sequence editing tool was used to generate contiguous sequences ([Link]

Species identification was done by using a BLAST nucleotide sequence analysis tool

([Link]

K_LOC=blasthome).

3.5.2 Characterization of bacteria from G. flava fruits

Bacterial isolates were subjected to biochemical tests which included Gram staining, lactose test

(LAC), indole test (IND), methyl red test (MR), Voges-Proskauer test (VP), urease test (URE)

35
and catalase test (CAT). Gram staining was performed by staining a single with crystal violet and

counterstaining it again with safranin and observing the Gram type under a microscope. Lactose

test was performed by inoculating the lactose broth with the bacterial cultures and the broth was

incubated at 37 0C for 24 hours. The positive results were evident when there was a color change

from yellow to a pink or red color at the surface of the medium while negative results had no

color formation. The IND test was performed by inoculating a loopful of bacterial culture into 2

mL tryptone water and incubating the vials at 37 °C for 24 hours. After incubation, the Kovac’s

reagent was added to the broth. The positive results were characterized by a pink layer while

negative results had no pink layer formation. The MR test was performed by inoculating cultures

into methyl red-Voges-Proskauer medium (MR-VP medium) and incubation at 30 °C for 3 days.

After 3 days, 5 drops of methyl red solution were added and further incubated for 48 hours at the

same temperature as above. The positive results were a red color formation and the negative

results were a yellow color. Under the VP test, the MR-VP medium was inoculated with the

cultures and incubated at 37 °C for 48 hours. After incubation, 0.6 mL of alpha-naphthol was

added to the broth and 0.2 mL of potassium hydroxide. The tubes were then shaken by hand for 1

minute to expose the medium to atmospheric oxygen in order to oxidize the acetoin and allowed

to stand for 10 minutes. The positive results were a red color formation while the negative results

were no color formation. The URE test was performed by inoculating a loop full of the culture to

the urease broth with the cultures and the broth was incubated at 37 °C for 48 hours. The positive

results were a color change from yellow-orange to bright-pink and the negative results were no

color change. The CAT test was performed by adding a small amount of the bacterial culture into

a 100 µL freshly prepared 1 % hydrogen peroxide. The positive results were production of

bubbles while the negative results were no bubble production.

36
3.6 Identification of yeast isolates from G. flava fruits

A single colony from each of the yeast isolates with distinct differences in morphology was

picked and purified by re-streaking on YPD. These colonies were then re-suspended in 50 µL

sterile deionized water. The mixture was then boiled at 98 °C for 15 mins and then centrifuged

for 1 min at 16 000 × g using a micro-centrifuge. 2 µL was then used as a template for PCR

amplification of a 560 bp DNA fragment of the 26S rDNA using universal PCR primers, NL1

(5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-

3') as reported elsewhere (Kurtzman and Robnett, 1997). PCR amplification was performed

using Applied Biosystems ProFlex PCR systems in a 20 µL (containing 2.5 µL 10× PCR buffer,

2.0 µM of each primer, 0.2 mM dNTPs, and 1.25 U rTaq DNA polymerase (Takara Bio Inc.,

Japan) using the following cycling conditions: initial denaturation at 98 °C for 3 min; 38 cycles

of denaturation (98 °C for 15 s), annealing (54 °C for 1 min), and extension (72 °C for 1 min), a

final extension step at 72 °C for 7 min and held at 4 °C ∞. Negative controls were as in the above

section (section 2.4.1). PCR product purification, sequencing and identification of yeast were

done as in the previous section above (section 2.4.1) as well as using a/the pairwise identification

tool available on the Westerdijk Fungal Biodiversity Institute (CBS-KNAW) strain database,

([Link] The control yeast used in this study is a commercial baker’s

yeast obtained from local supermarkets. It should be noted that there are no retail outlets in

Botswana that sell commercial brewer’s yeast that could have been used as a control in this

study.

37
3.7 Identification and characterization of yeast isolates from khadi

3.7.1 Yeast colony PCR

Yeast colony PCR and identification were carried out as outlined in section 3.6.

3.7.2 Phylogenetic analyses

To determine the phylogenetic relationship of the isolates from the khadi samples, we used

MEGA 7.0.26 software (Kumar et al., 2016). The sequences were aligned using MUSCLE

within the MEGA software and then the aligned sequences were used to calculate the best

maximum likelihood model (Jukes-Cantor, Kimura 2-parameter, Tamura 3-parameter,

Hasegawa-Kishino-Yano, Tamura-Nei and General time reversible models) to be used to

construct the phylogenetic tree.

3.7.3 In-silico PCR-RFLP of Saccharomyces and non-Saccharomyces yeasts

We sought to investigate all S. cerevisiae isolates that showed a significantly high percentage of

global similarity to the readily available baker’s yeasts to ascertain whether the wine makers

used it as starter cultures or depended on the naturally occurring yeasts isolated from the dried

fruits. To do so, we performed an in-silico PCR-RFLP (restriction fragment length

polymorphism) using 26S rDNA amplicons with a SnapGene® Viewer software ver.4.2.11 (GSL

Biotech) ([Link] The simulation of gel electrophoresis with a set parameter

of 4 % agarose gel option was chosen for visualization after an in-silico digestion with the

following restriction enzymes GluI, HaeIII, HinfI and RsaI. pUC19 – Sau3AI digest was selected

as a molecular weight ladder to compare the mobility of the fragments.

We also used the same technique to investigate whether the same non-Saccharomyces yeasts

found among wines from across Botswana were similar. The 26S rDNA sequences of the ale

38
yeasts (S. cerevisiae, SafAle T-58, Fermentis, France), lager yeasts (S. pastorianus, Lallemand

Brewing, Austria) and wine yeasts (S. cerevisiae, Lalvin EC-1118, Lallemand Brewing, Austria)

where downloaded from NCBI ([Link] These where ran in parallel

with readily available commercial baker’s yeast.

3.8 Bioprocess development

To select potential starter culture yeasts to be used for a standardized production of a commercial

grade of khadi, the yeasts isolated from both the G. flava fruits and khadi were tested. The tests

were aimed at identifying the best yeast isolates with the following attributes; higher

fermentative capacity, ability to utilize different carbon sources as well as those yeasts with

higher stress tolerance to brewing-associated stresses. Such attributes are important for an

effective fermentation process.

3.8.1 Carbon assimilation in yeasts

The utilization of a wide substrate range is one of the important attributes for potential brewing

starter cultures. The attributes determine the economical production of beer and determine the

attenuation ability responsible for either low alcohol or high alcohol beverage. G. flava fruits like

any other fruits contain multiple fruit sugars (not determined in this study) and thus a yeast

capable of converting these sugars into ethanol will be an ideal starter culture. Therefore, the

ability of the yeast isolates to assimilate selected carbon compounds was tested using the API

20C AUX kit (Bio-Mérieux, Marcy l'Etoile, France) according to the manufacturer's instructions.

The yeast isolates were also grown in different locally available sugars [maltose (2 %), fructose

(2 %), sucrose (10 % and 20 %) and lactose (2 %)] to confirm their utilization of the sugars.

39
3.8.2 Yeast characterization: Determination of fermentative capacity.

The ability to ferment reducing sugars and other carbon sources found in brewing substrates into

ethanol is one indispensable attribute of a brewing yeast starter culture. The isolates were

therefore tested for their fermentative capacity as reported by (Zhou et al., 2017b) with minor

adjustments. In brief, a single colony of each of the isolated strains and the controls (ale yeast,

baker’s yeast and wine yeast) were grown overnight in media containing G. flava fruits (200 g/L)

and brown sugar (GFS media) (100 g/L) in 5 mL test tubes. The samples were incubated in an

incubating shaker at 180 rpm at 30 °C. Overnight cultures were pelleted, washed with sterile

deionized water and used for inoculation in GFS media. The GFS media (3 mL) was inoculated

with a yeast suspension culture to give an initial OD600nm of 1.0 and introduced into a 30 mL

marked syringes in triplicates. The syringes were incubated in a shaker at 30 °C at 180 rpm for

24 hours. Fermentative capacity was determined by measuring carbon dioxide evolved during

fermentation, which corresponded to the movement of the syringe plunger. The results were

recorded at hourly intervals until fermentation was complete. The experiments were done in

duplicates and repeated twice.

3.8.3 Yeast stress tolerance

Yeasts are subjected to a variety of stresses during brewing. These stresses impact their

fermentative performance, which subsequently affect the overall quality of a beverage.

Investigating the capacity of the potential starter cultures to withstand osmotic, ethanol, pH, and

high temperature fermentation stresses associated with brewing is essential. Specific isolates

from G. flava fruits and khadi used in stress tolerance tests were grown overnight in liquid YPD

in 5 mL tubes at 30 °C and 180 rpm on a shaker as described elsewhere (Zhou et al., 2017b). The

40
cells were harvested by spinning down at 13 000 rpm for 2 minutes and washed with deionized

sterile water. The cells were then adjusted to OD600 nm of 0.5 in phosphate buffered saline. The

cultures were further diluted in 96-well plates to give working OD600 nm of 0.5, 0.25 and 0.125.

Cells in these dilution ranges were then spotted using a spot test stamp (replicator) on different

solid media plates containing YPD supplemented with different reagents to test for different

stresses. For osmotolerance, YPD was supplemented with different NaCl concentrations to

working concentrations of 0.5M, 1M, 1.5M, 2M and a control with a working concentration of

0M NaCl. To test for thermotolerance, isolates were spotted on YPD plates and incubated at

different temperatures (37, 40, 41, 42, 43 and a control temperature of 30 °C). pH stress

tolerance was tested by growing the yeasts in liquid media set at different pH (1, 2, 3, 5 and a

control pH of 6.2). The ability of isolates to tolerate different ethanol concentrations was also

tested by spotting isolates on media with different ethanol concentrations (3, 5, 7, 9 and a control

with 0 % ethanol). In all other tests for stress, incubation was done at 30 °C. The ability to

tolerate different types of stressors was evaluated semi-quantitatively by estimating the

abundance of colonies that grew on a control plate in comparison to those that grew on a

selective media plate. The plates were photographed every 24 hours and the plate photos with the

best differences were recorded.

3.9 Safety of khadi

Isolation of molds prompted further investigation to ascertain the presence of mycotoxigenic

fungi. Since, no bacteria isolates or pathogenic yeasts were isolated these microorganisms were

not considered for investigation of safety of khadi.

41
3.9.1 DNA extraction and identification of molds isolates

The molds were collected and cultured as described above (Section 3.4.2). Genomic DNA was

purified by the microwave method (Bollet et al., 1991). The fungi mat was rinsed with 1 mL TE

buffer, centrifuged, and lysed with 100 µL TE buffer and 50 µL 10 % SDS. The molds mat was

then incubated at 65 °C for 30 min and centrifuged to remove the supernatant. The cell pellet was

placed in a microwave oven, heated twice for 1 min at 900 W. The pellet was then resuspended

in 200 µL TE buffer with one volume of phenol: chloroform: isoamyl alcohol (25:24:1) for 15

min. The aqueous phase was recovered by centrifugation; the DNA was precipitated with 95 %

ethanol and centrifuged at 12 000 rpm for 20 mins. Then, the DNA was rinsed with 1 mL 70 %

ethanol and resuspended in 200 µL deionized water for PCR assays. PCR conditions and

identification were carried out as outlined in Section 3.6.

3.9.2 Mycotoxin quantification

[Link] Randox Evidence Investigator Myco-10 calibration

The Randox Evidence Investigator was calibrated based on the nine-point calibration using

Randox Evidence Investigator Myco-10 calibrator, which covered the calibration range of all

assays. The calibration data was used to calculate the linearity, LOD and LOQ of the calibration

process. The LOD is the detection limit of an analytical procedure is the lowest concentration of

analyte in a sample that can be detected while the LOQ is the smallest concentration of a

measurand that can be reliably measured by an analytical procedure.

42
[Link] Extraction of mycotoxins from khadi samples

After calibration, a total of 2.5 mL of each khadi sample was mixed with 20 mL pure

Acetonitrile (Sigma–Aldrich, USA) in a 50 mL centrifuge tube. Sodium chloride (2 g) and

magnesium sulphate (0.25 g) were added to the mixture. The resultant mixture was vortexed for

3 minutes followed by centrifugation at 4000 rpm for 10 minutes at 4 °C.

[Link] Mounting of samples onto a Myco-10 biochip

150 µL of diluted wash buffer was pipetted into each of the 9 biochip wells (Myco-10 biochip,

Randox Diagnostics, UK). 50 µL of the extracted sample was added to the appropriate biochip

well. After gently tapping the edges of the handling tray to mix the samples and reagents, the

trays were placed in the thermo-shaker and incubated at 25 °C for 30 minutes at 370 rpm. A

working strength solution conjugate of 100 µL was pipetted in each biochip well. The biochips

were then incubated at 25 °C for 60 minutes at 370 rpm. The biochips were then immediately

washed twice by pouring 375 µL of wash buffer to each biochip well. Another wash cycle was

carried 4 times and the biochips were finally soaked in wash buffer for 2 minutes. After the final

wash, the biochips were tapped onto a lint free paper towel to remove any residual wash buffer.

After tapping, the biochip wells were filled with wash buffer and left to soak until directly prior

to imaging.

[Link] Myco-10 biochip imaging and mycotoxins quantification

Each biochip carrier was imaged individually and biochips awaiting imaging were protected

from light by covering them with a lint free paper towel. Each biochip well was tapped onto a

lint free paper towel to remove the wash buffer. A signal reagent of 250 µL was added to each

biochip well and covered to protect from light for 2 minutes. The biochip carrier was placed into

43
the Evidence Investigator (Randox Diagnostics, UK), images from each well were captured and

mycotoxins per well quantified. In Evidence Investigator, degree of binding is determined using

a chemiluminescent light source, quantified using a Charge Coupled Device (CCD) camera and

imaging system. The Randox Evidence Investigator detects and quantifies a total of 10

mycotoxins per sample (paxilline, fumonisin, ochratoxin A, aflatoxin G1/G2, ergot alkaloids,

diacetoxyscirpenol, deoxynivalenol, aflatoxin B1/B2 and zearalenone). The control provided

with the kit of a known concentration was ran along with the samples to monitor the efficiency

and accuracy of the kit.

The extraction method was confirmed for accuracy and efficiency by spiking 3 samples from 3

different sampling locations with known concentrations of AFB1 (0.1 ppb), FB1 (1 ppb) and

OTA (2 ppb). The Myco-10 extraction method was also used as instructed by the manufacturer

(Randox Diagnostics, UK) and compared with the above extraction method. The method

involved taking 5 mL of the khadi sample and adding 25 mL of acetonitrile: methanol: water

(50:40:10). The mixture was then vortexed for 60 seconds and hand rolled for 10 minutes. After

rolling, the mixture was centrifuged for 10 minutes at 3 000 rpm to complete the extraction. The

mycotoxin quantification and spiking were done as stated above.

3.11 Statistical analyses

One-way ANOVA was used to compare differences among the CFUs/mL of yeasts isolated from

the khadi samples (Microsoft Office 2019, USA). The baker’s yeast was used as a control isolate

in the gassing power experiment and the differences in the gassing power between each khadi

isolate and the control bakers’ yeast was analyzed using a t-test.

44
 Chapter 4: Results and Discussion
4.1 Khadi brewing process

4.1.1 Survey responses

Khadi brewing process employs a back-slopping technique in which the previously fermented

fruits (seretse, 30 L) is pitched as the raw material for the next fermentation step. To supplement

on the available fermentable sugars in the ripe sun-dried G. flava fruits (20 L), table brown sugar

at a concentration of 10 kg is added. Then fermentation is allowed to proceed for 18 to 24 hours

or 36 to 48 hours depending on the ambient temperature (Figure 6). Once the fermentation time

is reached, the product is sieved using old repurposed sacks before consumption. Repitching of

the previous fermented G. flava fruits was done for a maximum of 2 times. Figure 6 summarises

the khadi fermentation process.

Figure 6: Summary of the khadi fermentation process for a fermentation of 100L.

45
The responses from the questionnaires for khadi brewing process were analysed (Figure 7). The

results show that khadi is mostly produced by people who are not formally employed (80 %) and

the minority (20 %) are retired or in formal employment. The trend suggest that most brewers

probably depend on it as a source of income. In addition, the results suggests that khadi is

brewed using the following main ingredients; seretse (inoculum), G. flava fruits, brown table

sugar and warm water (Figure 7b).

The use of wild fruits in traditional beverages has been reported elsewhere for the production of

mukumbi of Zimbabwe produced from marula fruits (Dlamini and Dube, 2008; Mpofu et al.,

2008). Production of khadi is similar to mukumbi but sugar supplementation is not carried out in

the brewing process.

It should be noted that some brewers added grapes although it was observed among a few

brewers. In contrast to expectations that the brewers used some unique brewing pots, it was

found out that fermentation was done in tightly closed repurposed drums. Another observation is

that fermentation was done at room temperature which fluctuates seasonally and thus could be

one of the reasons for batch-batch variations. Surprisingly, some drums used in brewing did not

have lids but the brewers used large clear clean plastics and blankets to close the drums. Such a

setup creates differences in the final product quality as differences in amounts of oxygen in the

fermentation vessel affact the metabolism of yeasts which could oscillate between respiration

and fermentation. Therefore the amounts ethanol should not be extpected to be uniform in such

setups.

Khadi brewers determine completion of fermentation in 3 different ways that include taste,

bubbles and foam. Completion of fermentation includes aspects such as change in taste from

sweet to the khadi distinct taste, reduction in bubble production and disappearance of the foam.

46
Given that khadi is produced under non-standardised brewing conditions, it is noteworthy that

sugar and alcohol testing are not carried out

47
Figure 7: Results of the questionnaire on the khadi brewing process. (a) Livelihood of khadi brewers. (b) The ingredients used to

produce khadi. (c) Fermentation duration of khadi. (d) Fermentation vessels used during khadi production. (e) Attributes used by the

khadi brewers to indicate the end of fermentation. (f) Storage of G. flava fruits for khadi fermentation.
48
4.2 Physico-chemical properties of khadi

4.2.1 Ethanol content of khadi

The ethanol concentration among khadi ranged from 0.75 ± 0.02 to 8.73 ± 0.13 % (v/v)

(Figure 8). The highest ethanol content was recorded from Maun 1 whereas Maun 3 had the

lowest ethanol content. On average, ethanol content of khadi was 5.27 ± 2.02 % (v/v). This

value was higher than the average, 3.69 % (v/v), previously reported by Mapitse et al.,

(2014). Commercial beers range from 3 to 6 % (v/v) (Bamforth, 2002). The diversity of

fermenting yeasts among khadi samples could be one of the main reasons of the

discrepancies of amounts of ethanol accumulated. Species from the genus Saccharomyces

produce higher amounts of ethanol as compared to other genera and their non-

Saccharomyces counterparts (Hagman et al., 2013). In addition, the brewing of khadi,

which is carried out in non-standard conditions such as the use of uncontrolled and partially

aerobic fermenting drums could be another factor. This is because oxygen partial pressures

determine the alcoholic fermentation patterns (Merico et al., 2009). Lower oxygen partial

pressures increases the glycolytic flux towards a fermentative metabolism whereas the

opposite is true for environments with high oxygen concentrations (Piškur et al., 2006;

Pronk et al., 1996). It is also important to note that fermentation time was not standardized

in the brewing of khadi. Such discrepancies would lead to variations in ethanol

concentration in the brew at the end of fermentation. Yeasts accumulate and consume, a

characteristic of fermenting yeasts described by (Rozpedowska et al., 2011), suggest that if

fermentation is not stopped upon exhaustion of fermentable carbon sources and in the

presence of oxygen as the final electron acceptor, the yeasts will consume the accumulated

ethanol. Since the timing of end of fermentation was not prioritized or standardized this

could have revealed a batch-to- batch discrepancies in ethanol concentrations for brews. It

49
should also be noted that ethanol accumulates incrementally during brewing thus the

amount of ethanol during the perceived end of fermentation is actually stage-specific.

4.2.2 pH of khadi, an important factor in preservation of the brew

The pH of the khadi samples ranged from 2.64 to 3.62 (Table C3 and Figure 8). Letlhakane

2 sample was the least acidic (pH 3.62) as compared to all other samples whereas sample

Palapye 2 was the most acidic with a pH of 2.64. On average pH of khadi samples was 3.1

± 0.31. In comparison to other traditional beverages such as tej and palm wine, khadi was

found to be highly acidic. To be specific, tej was reported to have a pH of 3.56 to 4.45

(Yohannes et al., 2013) and palm wine with pH of 4.0 to 5.5 (Karamoko et al., 2012). These

results are in agreement with previous studies by Mapitse et al., (2014) who reported an

average pH of 3.08 ± 0.15. In comparison to most commercial beers, with a pH of 4 - 5,

khadi is acidic. pH influences the overall physiological characteristics of alcoholic

beverages such as taste, color and biological stability. Most importantly, pH also strongly

influences sanitation of the product, an attribute important for increasing shelf life of the

brew (Jukić et al., 2018). Other than the presence of ethanol, the acidic pH is likely the

other reason why they were no bacteria isolated from khadi because bacteria are known to

be sensitive to highly acidic conditions (Chelule et al., 2010; Hirshfield et al., 2003; Yu et

al., 2010). Although there are other factors such as mineral content and titratable acidity

among others that should be considered to make valid conclusions on the effects of acidic

brew, the information on the relative acidity of the brew would be important for

development of khadi commercialization.

50
Figure 8: The physico-chemical properties of the khadi samples. (a) Variable average ethanol content and (b) average pH of the khadi samples

used in the study. Color code: purple = Letlhakane, sky blue = Maun, navy blue = Mmashoro, red = Palapye, Black = Serowe and green = Tonota

51
4.3 Fermentative microbiota from G. flava fruits and khadi

4.3.1 Abundance of microbiota isolated from G. flava fruits

G. flava fruits play an important role in khadi fermentation as they are a possible source of

fermenting microorganisms, fermentable sugars and other growth factors. For this reason,

we enumerated the yeasts and bacteria found on G. flava fruits. It was noted that in each

area there was a single supplier of the G. flava fruits and hence only one batch of fruits per

town was used for investigation. The yeast colonies on the G. flava fruits ranged from 0 to

2x103 CFUs/mL with their average being 1x103 CFUs/mL while bacterial colonies ranged

from 2x103 to 4x103 CFUs/mL with an average of 3.5x103 CFUs/mL (Table 6). The

colonization of fruits by bacteria and yeasts is in agreement with studies described

elsewhere (Kurtzman et al., 2011) but the low number of colonies observed could be

attributed to the processing of fruits. The storage of fruits before fermentation could have

created a selective environment for proliferation of a few yeasts and bacteria with specific

attributes that allow them to survive in a new environment (Fleet, 2003). Drying of fruits

lowers the water activity needed for microbial survival thus reducing the number of

microbes (Beuchat et al., 2013). This means that grapes will have more microbes as

compared to raisins.

Table 6: Abundance of yeasts and bacteria isolated from the ripened sun-dried G. flava
fruits samples expressed in log 10CFU/mL (colony forming units per millimeter).
Sample Log10 CFU/mL
Yeasts Bacteria
Letlhakane 3.0 3.6
Maun 3.0 3.5
Mmashoro 0 3.6
Palapye 3.3 3.3
Serowe 3.0 3.6
Tonota 3.0 3.6
Average 2.6 3.5

52
4.3.2 Abundance of microbiota isolated from khadi

After identification of yeasts and bacteria on fruits we sought to investigate the diversity of

bacteria and yeasts after fermentation. Interestingly, there were no bacteria in all khadi

samples investigated. On the other hand, yeasts were isolated from all the brews. This

observation was not surprising, as yeasts are known to engineer the environment that favors

their proliferation through fermentation. Alcoholic fermentation is a niche reconstruction

lifestyle of the Crabtree positive yeasts which creates heat, CO2 and ethanol to inhibit

sensitive competitors such as bacteria (Dashko et al., 2014; Goddard, 2008; Rozpedowska

et al., 2011). Crabtree positive yeasts tend to ferment even in the presence of excess oxygen

and a fermentable carbon source (Dashko, Zhou, Compagno, & Pishkur, 2014). In the

presence of excess sugar and oxygen, a few microorganisms, for example, some yeasts

from the Saccharomycetaceae family, generate ATP through a respiratory or fermentative

metabolism or both pathways concurrently, thus known as respiro-fermentative yeasts (De

Deken, 1966).

The total number of yeasts on the khadi samples ranged from 5.12 ± 0.09 log10 CFUs/mL

(Mmashoro) to 6.41 ± 0.22 log10 CFUs/mL (Palapye) (Figure 7). On average, 5.6 ± 0.57

log10 CFUs/mL of yeasts were recorded. Using one-way ANOVA at a confidence interval

of 95 %, we noted that there was no significant difference in concentrations of yeasts at the

end of fermentation among the samples.

53
 8

6
Log10CFU/mL

Sample name

Figure 9: Variability of concentrations of yeast (log10 CFU/mL) isolated from khadi

samples from different parts of the Central and Northern Botswana. Six samples were

collected in triplicate and analyzed individually. Color code: purple = Letlhakane, sky blue

= Maun, navy blue = Mmashoro, red = Palapye, Black = Serowe and green = Tonota.

4.4 Investigation of bacterial isolates from G. flava fruits

4.4.1 Diversity of bacterial isolates

Bacteria are known to be sympatric with yeasts inhabiting sugar rich environments such as

ripening fruits (Barata et al., 2012; Zhou et al., 2017a). Investigation of bacteria found on

G. flava fruits yielded a total of 21 genetically diverse bacterial isolates (Table 7). Bacillus

species dominated the isolates accounting for 76.2 % of the identified isolates (16/21). The

dominance of Bacillus species was not surprising as they are ubiquitous microbes that are

common in soil, air, dust, water as well as dried and processed foods of plant origin

54
 (Stenfors Arnesen et al., 2008). Perhaps methods used to process fruits could have created

an environment that selected for Bacillus spp. In addition, the limited diversity of bacteria

skewed towards the Bacillus species suggests that these species could be responsible for

ecosystem modification. For example, B. velezensis is a well-known producer of

antibacterial secondary metabolites (Kim et al., 2017). The isolation of Bacillus spp.

known for food poisoning and causing illness in human beings such as B. cereus, B subtilis,

B. amyloliquefaciens and B. thuringiensis (André et al., 2017; de Blackburn and McClure,

2009) is a cause of concern but the concern can be overlooked as none of these were

isolated from the khadi samples. In addition, Acinetobacter lwoffii could also be another

bacteria of concern as the genus Acinetobacter comprises of multiple nosocomial

opportunistic pathogens that cause pneumonia (Margolin et al., 2016), but the bacterial

isolates did not survive the fermentation process.

Table 7: Identity of bacteria isolated from Grewia flava fruits based on D1/D2 region of

the 16S rDNA.

Species Name Isolate Collection Accession Number Identity (%)

Number Number
Acinetobacter lwoffii MMB4 Mmashoro KF818633.1 99
Acinetobacter lwoffii SB2 Serowe KF818633.1 99
Bacillus amyloliquefaciens PB1 Palapye MG892875.1 99
Bacillus cereus TB3 Tonota AB523744.1 99
Bacillus cereus LB4 Letlhakane MG021182.1 99
Bacillus methylotrophicus SB1 Serowe KM659219.1 99
Bacillus oleronius LB1 Letlhakane KY773585.1 98
Bacillus pichinotyi PB2 Palapye MG705701.1 98
Bacillus amyloliquefaciens subsp. plantarum MMB2 Mmashoro JN661699.1 99
Bacillus spp. MAB3 Maun KF646681.1 99
Bacillus spp. TB4 Tonota KT443870.1 99
Bacillus thuringiensis SB3 Serowe KY910253.1 99
Bacillus thuringiensis LB3 Letlhakane KY495218.1 99
Bacillus simplex LB2 Letlhakane AB547125.1 99
Bacillus subtilis MMB3 Mmashoro MH261154.1 99
Bacillus subtilis SB4 Serowe MH261154.1 98
Bacillus subtilis TB1 Tonota MH261154.1 99
Bacillus velezensis MMB1 Mmashoro MH000677.1 99
Desemzia incerta TB2 Tonota KF712891.1 99
Exiguobacterium indicum MAB1 Maun MH819520.1 93
Staphylococcus saprophyticus MAB2 Maun CP022093.2 99

55
4.4.2 Biochemical characterization of the bacteria isolates

The biochemical test results showed that 18 isolates were catalase positive, 17 isolates were

Gram positive, 11 isolates were oxidase positive, 9 isolates were methyl red positive, 9

isolates were lactose fermentation positive, 7 isolates were Voges-Proskauer positive, 6

isolates were urease positive and all the isolates were indole negative (Table 8). The Gram

staining results showed that the majority of the isolates were Gram positive in agreement to

the molecular identification results while TB4 and LB1 were Gram negative and were

identified as B. oleronius (Szkaradkiewicz et al., 2012). Even though staining Gram-

negative, B. oleronius has Gram-positive cell wall components shared amongst all Bacillus

species (Owusu-Darko et al., 2017). To identify those organisms that hydrolyze urea, the

urease test was performed and the results show that only 5 isolates can hydrolyze urea as

seen in Table 8. This means only these isolates have the urease enzyme thus allowing them

to hydrolyze urea producing carbon dioxide and ammonia (Yarrow, 1998). If these

organisms are allowed or capable of growing in an alcoholic beverage, they would spoil the

quality of the beverage due to the metabolites they produce such as ammonia and indole

(Comi, 2016). Table 8 summarizes the rest of the characterization results of the bacterial

isolates. Some bacteria have the ability to perform mixed acid fermentation of glucose

producing a complex mixture of acids, particularly lactate, acetate, succinate and formate as

well as ethanol and equal amounts of H2 and CO2 causing the medium to acquire an acidic

pH (Tassadaq et al., 2013). The results showed that 9 isolates were methyl red positive

meaning their presence in khadi could have possibly make the beverage acidic.

56
 Table 8: Biochemical characterization and colony pigmentation of bacterial isolates from

G. flava fruits.

Isolate name Isolate code Gram staining URE IND LAC VP CAT MR OXI Colony colour

Bacillus subtilis TB1 + - - + + + + - Opaque white

Desemzia incerta TB2 + + - + - + + + Opaque yellow

Bacillus cereus TB3 + - - - - - - - Pale yellow

Bacillus spp. TB4 - - - - - - - - Pale yellow

Bacillus methylotrophicus SB1 + - - + - + - + Opaque white

Acinetobacter lwoffii SB2 - - - - - + - + Opaque white

Bacillus thuringiensis SB3 + - - + + + + + Opaque white

Bacillus subtilis SB4 + + - + + + + + Opaque white

Bacillus amyloliquefaciens PB1 + - - + + + - + Cream white

Bacillus pichinotyi PB2 + - - - - + + + Cream white

Exiguobacterium indicum MAB1 + - - - + + + - Pale yellow

Staphylococcus saprophyticus MAB2 + + - + - + + - Opaque white

Bacillus spp. MAB3 + + - + + + + + Opaque white

Bacillus amyloliquefaciens subsp. MMB2 + - - + - + - - Dark yellow

plantarum

Bacillus velezensis MMB1 + - - - - + - - White

Bacillus oleronius LB1 - - - - - + - - Yellow

Bacillus simplex LB2 + - - - + + + + Opaque white

Bacillus thuringiensis LB3 + - - + + + + + Opaque white

Bacillus cereus LB4 + - - - - - - - Pale yellow

Bacillus subtilis MMB3 + - - + + + + + Opaque white

Acinetobacter lwoffii MMB4 - - - - - + - + Opaque white

57
*Note: URE – urease test, IND – indole test, LAC – lactose test, VP - Voges-Proskauer

test, CAT – catalase test, MR – methyl red test and OXI – oxidase test

4.5 Investigation of yeast isolates from G. flava fruits

4.5.1 Diversity of yeast isolates

After the observation that there were a number of yeasts with different morphologies, we

sought to identify yeasts by sequencing of the 26S rDNA. A total of 6 species dominated

the fruit samples namely; Aureobasidium leucospermi, A. melanogenum, Naganishia

diffluens, Aureobasidium spp., A. pullulans and S. cerevisiae (Table 9). Aureobasidium spp.

was the most predominant species in all samples. Different Aureobasidium spp. have been

reported to be among the non-Saccharomyces yeasts known to be involved in spontaneous

fermentations of wine, beer, tequila, mezcal and cachaça (Varela, 2016a) and hence they

could be contributors of alcoholic fermentation of khadi. In addition, the isolation of

Aureobasidium pullulans from other wild fruits and their fermented products reported

elsewhere (Nyanga et al., 2007), further suggest that this dominant genus could be involved

in the fementation of khadi. Some of the isolates from the fruits have an unknown

contribution to the fermentation of khadi such as Naganishia diffluens which has been

reportedly found to colonize flowers of edible fruit plants (such as pomegranate, mango,

guava and lemon) (Moubasher et al., 2018). It is not surprising that a well-known

fermenting species of yeast inhabiting sugar rich environments such as wild fruits, S.

cerevisiae, was only isolated from Letlhakane samples because S. cerevisiae is naturally

initially rare on fruit ecosystems, but it eventually dominates the niche after engineering the

ecosystem through fermentation (Goddard, 2008; Ponzzes-Gomes et al., 2014). Therefore,

58
it is likely that S. cerevisiae could have been present in other fruit samples but was not

isolated.

It should be noted that more microbial diversity could have been identified if more of the

G. flava fruits were used instead of only 2g. This could have limited the number and types

of microbes isolated as compared to if more fruits were used for the isolation of the

microbes thus the limitation of the selected method.

Table 9: Identity of yeast isolated from G. flava fruits

Species name Isolate Collection number Accession number Identity (%)

number

A. leucospermi D2 Palapye KX893326.1 99

A. melanogenum D3 Serowe MF370933.1 99

A. pullulans D6 Tonota MF979210.1 99

Aureobasidium spp. D5 Maun KT361586.1 99

Naganishia diffluens D4 Palapye KU316762.1 99

S. cerevisiae D7 Letlhakane HQ108377.1 99

4.6 Yeasts as major contributors in fermentation of khadi

4.6.1 Diversity of yeasts

Mainly Saccharomycotina and a few Basidomycota yeasts were isolated from all the brews.

This observation was not surprising, as Saccharomycotina yeasts are known to engineer the

environment that favors their proliferation through fermentation (Goddard, 2008).

Alcoholic fermentation is a niche reconstruction lifestyle of Crabtree positive yeasts which

creates heat, CO2 and ethanol to inhibit the growth of competitors (Dashko et al., 2014;

Goddard, 2008; Rozpedowska et al., 2011). The limited diversity of yeasts on the dried

fruits in comparison to the wide diversity of bacteria prompted further investigation to

59
establish which microorganisms could be responsible for the fermentation of khadi. A total

of 74 yeast isolates with a wide phylogenetic diversity were recorded from different khadi

samples (Table 10, Figure 10). The isolates belonged to 13 yeast species (Table 11).

Interestingly, most of those yeasts were not isolated from the ripened sun-dried fruits.

There are two possibilities to explain this; (i) there was a source of yeasts other than those

from the wild fruits such as fermentation vessels (Aouizerat et al., 2019) , (ii) there were

yeasts that were rare on the fruit surfaces and were not identified due to the limitation of

culture-based techniques (Baselga et al., 2017; Wade, 2002). Interestingly, no bacteria were

isolated from khadi and this might have been due to the possible temperature bias used for

the incubation of LAB.

S. cerevisiae dominated the isolates (51.4%) followed by Candida ethanolica (12.2%) and

Saccharomycodes ludwigii (10.8%) (Tables 10 and 11). The dominance of S. cerevisiae at

the end of the alcoholic fermentation, despite the dominance of non-conventional yeasts

and bacteria, during the early stages of fermentation is well documented (Goddard, 2008;

Ponzzes-Gomes et al., 2014; Varela, 2016b). These results suggest that khadi is a product

of mixed-culture fermentation with phylogenetically diverse yeasts dominated by S.

cerevisiae (Table 10).

Mixed-culture fermentations made up of conventional and non-conventional yeasts leads to

distinctive aromatic profiles of alcoholic beverages (Andorrà et al., 2012; Ciani et al., 2009;

Dashko et al., 2015a; Gamero et al., 2016; Lencioni et al., 2016). The production of aroma

compounds in alcoholic beverages also requires non-conventional yeasts. For example,

Lachancea spp. are well-known producers of fruity esters, which impart a characteristic

fruity flavor to alcoholic brews (Cordero-Bueso et al., 2012; Porter, 2017; Romano et al.,

1997). Fermentative species of the genus Candida, such as C. ethanolica and C. sake, also

known to generate unique flavors of a brew, were isolated (Maturano et al., 2015) from

60
brews from Letlhakane, Tonota and Maun. However, the isolation of spoilage yeasts, such

as Brettanomyces spp. and Zygosaccharomyces spp. (Alonso et al., 2015; James and

Stratford, 2003; Schifferdecker et al., 2016), in samples from Serowe and Tonota,

respectively, could negatively impact on the quality of a spontaneously fermented brew.

Brettanomyces bruxellensis is known to be both a spoilage yeast and good fermenting

yeast. This yeast has been isolated from wines that have characteristically developed

unpleasant and distinctive aromas due to the production of volatile phenolic compounds

and diacetyl (Chatonnet et al., 1995; Hittinger et al., 2015; Licker et al., 1998;

Schifferdecker et al., 2016). These unpleasant and distinctive aromas production is due to

the production phenols exceeding the sensory threshold 16-fold. In contrast,

Brettanomyces spp. have been used for production of Lambic beers where they can produce

ethyl esters which contribute to the overall fruity or floral character of the beverage at a

lower sensory threshold (Capece et al., 2018; Schifferdecker et al., 2014).

Zygosaccharomyces bailii is a wine spoilage yeast associated with grapes whose spoilage is

characterized by generation of taints, odors, off-flavors, development of hazes, and

excessive gas production (Barata et al., 2012; James and Stratford, 2003). Literature

suggests that Z. bailii can improve the flavor complexity of alcoholic beverages either used

individually or as a mixed culture with S. cerevisiae producing flavor compounds such as

alcohols, acids, esters, ketones and aldehydes (Xu et al., 2017). A concern for

spontaneously fermented brews is the presence of clinically relevant species. Pichia

kudriavzevii (also known as Candida krusei (Douglass et al., 2018)) was the only

potentially opportunistic pathogen (Douglass et al., 2018) that was isolated and it was

found in a Letlhakane sample. However, P. kudriavzevii also has good fermentation

characteristics and it has been previously isolated from taruba, grapes and masau fruits

(Nyanga et al., 2013b; Ponzzes-Gomes et al., 2014; Ramos et al., 2015).

61
One of the expected features of a fermented brew is the presence of Saccharomyces yeasts.

The phylogenetic tree based on the Hasegawa–Kishino–Yano model in Figure 10 shows the

relationship among the S. cerevisiae isolates isolated from the khadi samples. The tree

(Figure 10) suggests that S. cerevisiae isolates isolated from different locations are diverse

as they cluster into 7 different groups. The results suggest that some brewers could be using

commercial ale, baker’s, lager and/or wine strains instead of spontaneous fermentation. For

example, Group 7 (Figure 10) isolates clustered with a control commercial baker’s yeast,

which is readily available in local retail shops. In addition, it was noted that some isolates

clustered with commercial ale and wine yeasts as shown in Group 4, which are not

available in the local supermarkets of Botswana, while two isolates from Maun (MA6) and

Letlhakane (L3) clustered in a separate group with the commercial lager yeast. These

clusters suggest that commercial yeasts strains have been used as a starter culture. Brewers

prefer the addition of starter cultures to circumvent the negative outcomes of spontaneous

fermentation, which produces an inconsistent quality of fermented products. Contrastingly,

we noted that S. cerevisiae strain isolated from G. flava fruits (from Letlhakane) clustered

with S. cerevisiae isolates from khadi from Serowe and Mmashoro in Group 5, suggesting

that use of spontaneous fermentation could be popular among these two locations. Use of

dried fruits from the same supplier in these three locations could also be a possible

explanation for the isolation of yeasts that were closely related to isolates from the fruits.

62
Figure 10: Maximum likelihood analysis of the D1/D2 domains of 26S rDNA of Saccharomyces yeasts isolated from khadi

samples. The evolutionary relationship between isolates was inferred using the maximum likelihood method based on the Hasegawa–

Kishino–Yano model (Tamura and Nei, 1993). Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2018). Color code:

purple = Letlhakane, sky blue = Maun, yellow = Mmashoro, red = Palapye, Black = Serowe, green = Tonota and navy blue =

Controls.
63
Table 10: Identity of yeast isolates from the khadi samples based on D1/D2 domains of 26S

rDNA.

Species Name Isolate Number Collection Number Accession Number Identity (%)
Saccharomyces yeasts
Saccharomyces cerevisiae Z2 Letlhakane 1 KX428522.1 99
Saccharomyces cerevisiae L3 Letlhakane 1 MG773372.1 99
Saccharomyces cerevisiae (2) L6 and L10 Letlhakane 2 MG017580.1 99
Saccharomyces cerevisiae L7 Letlhakane 2 LC336457.1 99
Saccharomyces cerevisiae L8 Letlhakane 3 MF979228.1 99
Saccharomyces cerevisiae L11 Letlhakane 3 HM191639.1 99
Saccharomyces cerevisiae LMA1 Letlhakane 2 MF979228.1 99
Saccharomyces cerevisiae LMA3 Letlhakane 2 KY109286.1 99
Saccharomyces cerevisiae LMA4 Letlhakane 1 CP022977.1 99
Saccharomyces cerevisiae LMA5 Letlhakane 2 KM234472.1 99
Saccharomyces cerevisiae LMB1 Letlhakane 2 KX119942.1 99
Saccharomyces cerevisiae MA1 Maun 1 KX428522.1 99
Saccharomyces cerevisiae MA6 Maun 2 HM107789.1 99
Saccharomyces cerevisiae AC1MIII Mmashoro 3 MG641152.1 99
Saccharomyces cerevisiae (2) AC2MIII and Mmashoro 3 and 2 HM101473.1 99
BC1MII
Saccharomyces cerevisiae AC3MIII Mmashoro 3 MF406146.1 99
Saccharomyces cerevisiae AC5MIII Mmashoro 3 JN225410.1 99
Saccharomyces cerevisiae BC4MII Mmashoro 2 JX141335.1 99
Saccharomyces cerevisiae MIAC1 Mmashoro 1 KM103041.1 99
Saccharomyces cerevisiae MIAC3 Mmashoro 1 MF406146.1 99
Saccharomyces cerevisiae MIAC2 Mmashoro 1 KM103042.1 99
Saccharomyces cerevisiae MIAC5 Mmashoro 1 MK027354.1 99
Saccharomyces cerevisiae P10 Palapye 1 KY109242.1 99
Saccharomyces cerevisiae P11 Palapye 1 HQ443692.1 99
Saccharomyces cerevisiae P13 Palapye 3 HM101472.1 99
Saccharomyces cerevisiae (2) P14 and P16 Palapye 2 KX098507.1 99
Saccharomyces cerevisiae P17 Palapye 3 GU080046.1 99
Saccharomyces cerevisiae (2) S2 and S7 Serowe 1 GU080049.1 99
Saccharomyces cerevisiae S6 Serowe 2 GU080046.1 99
Saccharomyces cerevisiae T5 Tonota 1 MF406147.1 99
Saccharomyces cerevisiae T6 Tonota 1 HM101472.1 99
Saccharomyces cerevisiae T8 Tonota 1 JX141335.1 99
Saccharomyces cerevisiae T9 Tonota 2 MK027354.1 99
Saccharomyces cerevisiae T11 Tonota 2 MF498873.1 99
Saccharomyces cf. cerevisiae/paradoxus (2) AC4MIII and Mmashoro 1 and 3 KY109333.1 99
MIAC4
Non-Saccharomyces yeasts
Brettanomyces bruxellensis S5 Serowe 2 KY107600.1 99
Candida ethanolica LMB2 Letlhakane 3 KY283163.1 99
Candida ethanolica (2) LMB4 and Letlhakane 1 FM180545.1 99
LMC1
Candida ethanolica LMC4 Letlhakane 1 JX880409.1 99
Candida ethanolica (5) T10, T14 T15, Tonota 2 and 3 JX880409.1 99
T17, T18
Candida sake (2) MA2 and MA3 Maun 1 JX880410.1 99
Curvibasidium pallidicorallinum MA7 Maun 3 JX188149.1 99
Pichia kudriavzevii L13 Letlhakane 3 AY529504.1 99
Pichia kudriavzevii S8 Serowe 3 AY529504.1 91
Lachancea fermentati MA4 Maun 2 KY108224.1 99
Lachancea fermentati MA8 Maun 3 KM234440.1 99
Lachancea fermentati P18 Palapye 3 KM234440.1 99
Pichia kudriavzevii L1 Letlhakane 1 KM234442.1 99
Pichia kudriavzevii (2) L2 and L4 Letlhakane 2 KF214396.1 99

64
 Pichia manshurica P15 Palapye 2 MK034750.1 99
Rhodotorula nothofagi Z1 Letlhakane 1 KJ794722.1 99
Saccharomycodes ludwigii (2) L9 and L12 Letlhakane 3 FM180540.1 99
Saccharomycodes ludwigii (2) S1 and S4 Serowe 1 FM180540.1 99
Saccharomycodes ludwigii (4) T7, T13, T12, Tonota 1 and 2 FM180540.1 99
T16
Schizosaccharomyces pombe P8 Palapye 2 KY296084.1 99
Zygosaccharomyces bailii T1 Tonota 1 GU080052.1 99
Zygosaccharomyces bailii T4 Tonota 1 KY296086.1 99
Note: The number in the bracket shows strains with the same accession number from the same

location.

Table 11: The number and percentage of yeast isolates from khadi samples.

Species Number of isolates % Letlhakane Maun Mmashoro Palapye Serowe Tonota

sequenced

Saccharomyces cerevisiae 38 51.4 12 2 10 6 3 5

Candida ethanolica 9 12.2 4 5

Saccharomycodes ludwigii 8 10.8 2 2 4

Pichia kudriavzevii 5 6.75 4 1

Zygosaccharomyces bailii 2 2.7 2

Candida sake 2 2.7 2

Saccharomyces cf. 2 2.7 2

cerevisiae/paradoxus

Lachancea fermentati 3 5.4 2 1

Curvibasidium 1 1.4 1
pallidicorallinum

Schizosaccharomyces pombe 1 1.4 1

Brettanomyces bruxellensis 1 1.4 1

Rhodotorula nothofagi 1 1.4 1

Pichia manshurica 1 1.4 1

Total 74 100 23 7 12 9 7 16

4.6.2 The fermenting microorganism behind khadi

Yeasts play a pivotal role (ethanol production) in fermentation of all alcoholic beverages (Piškur

et al., 2006). Although S. cerevisiae species were found in low frequencies on G. flava fruits

65
before fermentation, the species dominated the isolates from the fermented product, khadi. All

samples were collected from spontaneously fermented brews or from back-slopped brews,

therefore, the fermenting Saccharomyces spp. could be those that were introduced from the

ripened sun-dried fruits or previously fermented fruits (inoculum) as well as those resident on the

brewing equipment and the brown table sugar as in agreement with the following study

(Boekhout and Phaff, 2003).

The diversity of S. cerevisiae strains isolated from khadi in comparison to the fruits prompted us

to further investigate adulteration of the brewing process using readily available commercial S.

cerevisiae strains sold by retail stores in Botswana. The brewers may add commercial baker’s

yeasts during khadi production to give it ‘strength’ and ‘enhance its capacity’ to intoxicate,

instead of relying on spontaneous fermentations (Rudloff et al., 2012). An investigation into the

genetic diversity of the Saccharomyces spp. using in-silico PCR-RFLP suggests that most of the

isolates were not genetically distinct from the commercial baker´s yeast (Fig 11a). To be

specific, restriction fragment patterns of 67.5% of the strains of this species (27 out of 40

isolates) digested with HaeIII matched a characteristic restriction fragment pattern of the

commercial baker´s yeast strain (Fig 11a and see also Appendix C). On the other hand, 13 unique

restriction fragment patterns from Mmashoro (AC1MIII, AC2MIII, AC3MIII, AC5MIII, MIAC2

and MIAC3), from Palapye (P10, P14, P16 and P17) and from Letlhakane (LMA4, LMA5 and

Z2) which neither matched the S. cerevisiae isolate from fruits nor the commercial baker´s, ale,

lager and wine strains were observed (Fig 11a). This suggests that there are other sources of

fermenting yeasts unaccounted for in this study. One source of yeasts could be equipment used

for brewing and pre-processing. Another possibility is that the yeasts were present on the fruit

surfaces at lower frequencies to be isolated using the plate count method. The latter is not

66
surprising because different studies have shown that S. cerevisiae is usually found in low

frequencies during the beginning of spontaneous fermentations as compared to other yeasts, but

accounts for the highest densities at the end of fermentation (Dashko et al., 2014; Goddard,

2008). On the other hand, there is a possibility that some colonies that had similar macro and

microscopic morphology to the S. cerevisiae isolate from fruits were overlooked since in an

effort to minimize potentially repetitive sequencing.

67
Figure 11: PCR-RFLP for yeast isolates from khadi. (a) and (b) show restriction patterns of Saccharomyces yeasts obtained using

restriction enzymes HaeIII and HinfI. (c) and (d) show restriction patterns of non-Saccharomyces yeasts obtained using restriction

enzymes HaeIII and GluI. The red shows isolate’s similar to the control commercial baker’s yeast (white boundary) while the WT is

the wild type S. cerevisiae isolated from the G. flava fruits (Fig 11a and 11b). The red boundary shows C. ethanolica that have the

same restriction patterns while purple shows the Saccharomycodes ludwigii with the same restriction patterns (Fig 11c and 11d).

68
Digestion with another enzyme, GluI suggests that there are subgroups within the strains (such as

C. ethanolica, C. sake, P. kudriavzevii and Saccharomycodes ludwigii) (Figure 11b). There was

no restriction fragment pattern of isolates from khadi that matched the pattern of the S. cerevisiae

isolate from G. flava fruits. There are several possible scenarios, which can explain the absence

of this strain´s restriction fragment patterns after fermentation. That is, the strain could have

existed in low frequencies to be picked amongst other isolates especially if back slopping was

practiced because the back-slopped inocula (including the wild isolate S. cerevisiae isolate from

the fruits) could have outcompeted it during the fermentation. In spite the clustering of some

isolates with the commercial baker’s yeast (Figure 10), the prevalence of strains with a distinct

restriction fragment pattern from that of the commercial baker’s yeast suggests that Mmashoro

and Letlhakane are the only areas that rely on spontaneous fermentation for brewing of khadi

(Appendix C). The in-silico PCR-RFLP results further revealed that none of the khadi isolates

had a similar restriction fragment pattern to the selected ale and lager brewing yeasts and wine

yeasts used in the industry even though there were similarities in the sequencing data (Figure

10). These yeasts are not available in the local supermarkets of Botswana as compared to the

readily available baker’s yeast explaining why they are not prevalent.

Non-conventional yeasts could be responsible for fermentation of khadi rather than conventional

Saccharomyces yeasts alone. The fermentative lifestyle of species of the genus Saccharomyces is

not unique among yeasts of the Saccharomycetaceae family (Hagman et al., 2013; Piškur et al.,

2006; Rozpedowska et al., 2011) but may have coincided with the origin of angiosperms, about

125 million years ago (Hagman et al., 2013; Sun et al., 2011). Therefore, other non-conventional

yeasts in the Saccharomycetaceae family, which existed in the same time period, are also

capable of making and accumulating ethanol. In this study we identified a number of non-

69
conventional yeasts (Table 10) which could have also been responsible for fermentation of khadi.

We therefore further investigated the genetic diversity using in-silico PCR-RFLP of non-

Saccharomyces yeasts to reveal unique strains responsible for fermentation of different brews.

We observed that among the genetically diverse yeasts, there were several unique yeasts that

could be responsible for khadi fermentation (Figure 11c and 11d). We noted that C. ethanolica

strains isolated from Tonota (T14, T15, T17 and T18) had matching restriction fragment pattern

with C. ethanolica strains from Letlhakane (LMB2, LMB4, LMC1 and LMC4) from digestion

with HaeIII. These two locations are 200 km apart and therefore sharing of the back slopped

broth or dried fruits is highly unlikely although the ability of yeasts to be dispersed by insects is

well documented. Therefore, this suggests that these strains could be naturally occurring on G.

flava fruits. In agreement to our assertions, strains from a different genus, Saccharomycodes

ludwigii from khadi from the same locations Tonota (T13, and T16) and Letlhakane (L9 and

L12) also had a similar restriction fragment pattern when digested using HaeIII (Figure 11c).

Similar results can be observed when running the same experiment using GluI (Figures 11c and

11d) as seen in Appendix C. This suggests the primary source of the fermenting yeasts could

have been the fruits and not the fermentation vessels or the equipment used. It is noteworthy that

non-Saccharomyces yeasts are not commercially available in Botswana to further suggest their

role in spontaneous fermentation of khadi. There has been a rapid expansion of craft breweries

that utilise non-Saccharomyces yeasts in the modern brewing industry (Alfeo et al., 2019;

Callejo et al., 2017; Iattici et al., 2020). This strategy has gained special attention due to the

demand for innovative and aromatically diverse beers associated with the changing lifestyle in

humankind (Michel et al., 2016; Ravasio et al., 2018). Non-Saccharomyces yeasts have been

demonstrated to produce a wide range of aromatic compounds and other metabolites that known

70
to improve the sensorial profiles of alcoholic beverages (Andorrà et al., 2012; Callejo et al.,

2017; Dashko et al., 2015a). This work further increases the attention to exploit these yeasts to

define khadi traits towards the development of this traditional beverage. The characterisation of

these non-Saccharomyces yeasts (not presented in this paper) should be explored to understand

how these diverse yeasts reported here could enrich and diversify aromas and flavours. Such

studies would pave way into improvement of the sensory complexity of khadi towards its entry

into regional and international markets.

4.6.3 Bioprocess development initiative

The isolation of different microorganisms from khadi belonging to the Saccharomycetaceae

complex, which are possibly responsible for fermentation, is one of the crucial steps towards the

development of starter cultures for the safe and hygienic bioprocessing of khadi. Stress tolerance

tests were done to investigate the ability of the strains to be used as starter cultures. These stress

tolerance tests were meant to pave the way for the commercialization of khadi. The tests

included carbon assimilation, fermentation profiling, ethanol inhibition, osmo-tolerance, thermo-

tolerance, oxidative stress and inhibition due to pH. These tests were done because there are

several stresses that microbes encounter during fermentation thus, it is pertinent to select the best

performing strain to be used for the bioprocess development of khadi.

[Link] Carbon compound assimilation

Strain selection is pivoted on the ability of yeasts to utilize different carbon sources in many

industries, such as food, bioethanol and pharmaceutical (Batistote et al., 2012). In this work, we

sought to ensure the ability of the isolated yeast strains to assimilate other carbon sources for use

71
of inexpensive and alternative carbon sources during the production. The results in Table 12

below show all the yeasts could assimilate glucose and maltose as carbon sources. All the strains

could not use N-acetyl-glucosamine as carbon source, which is a monosaccharide and a

derivative of glucose. This is not surprising because N-acetyl-glucosamine is structurally very

similar to glucose, but it cannot be metabolized by yeast thus it provides a stable signal that

glucose is present in the culture, and triggers glucose repression (Jarosz et al., 2015). A typical

industrial fermentation media contains starch as well as sucrose, maltose, fructose, glucose, and

other oligosaccharides (Donalies et al., 2008; Randez-Gil et al., 2003) hence the most

appropriate production strain should be able to efficiently utilize a wide range of carbon sources

(Zhou et al., 2017b). The utilization of glucose is to be expected as the glucose was used in the

selection media.

72
 Table 12: Carbon assimilation of the yeast isolates from G. flava fruits and khadi samples.

Substrate D2 D3 D4 MA1 MA2 MA4 MA7 P8 S1 S5 T1 T17 Z1

D-Glucose + + + + + + + + + + + + +

Glycerol + + - + + + + + + + + + -

2KG - + - + - + + - - + - + -

L-Arabinose - + - + - + - - - - + + -

D-Xylose - + - + - + - + - + - - -

Adonitol + + - + - + - - - - - - -

Xylitol - + - + - + - - - - + - -

D-Galactose + + + + + + - + + - + + +

Inositol - + - + + + + - - - - - -

D-Sorbitol - + - + - + + - - + - - -

MDG - + - + - + + - - + - - -

NAG - - - - - - - - - - - - -

D-Cellobiose + + - + - + + + - - + + -

D-Lactose - + + + - + - - - - - - -

D-Maltose + + + + + + + + + + + + +

D-Saccharose + + + + + + + + + + + + -

D-Trehalose - + + + - + - - - + + - -

D-Melezitose + + + + - + - - - + + - -

D-Raffinose + + + + - + + + + + + + -

Notes: - = no growth, + = growth, 2KG = Calcium 2toketotoGluconate, MDG = Methyl-α-

Glucopyranoside and NAG = N-Acetyl-Glucosamine.

Khadi brewing process involves the use of local commercial brown sugar from local

supermarkets. To investigate the possibility of using an inexpensive carbon source as an

alternative for sucrose, the isolates were grown on different carbon sources namely maltose,

fructose, lactose and sucrose at different concentrations as a control. Thus, the aim of this

73
experiment was to check if the isolated strains can utilize other carbon sources other than

sucrose. Most of the isolates grew well at 10 % sucrose except D4 which is an isolate from G.

flava fruits thus the result is not that much surprising as the fruit is not expected to have such

high level of sucrose. The isolates D2, D4 and MA2 did not grow on 20 % sucrose while MA1,

P8 and T1 showed poor growth (Figure 12). The control baker’s yeast strain had an average

growth at 20 % sucrose which is to be expected as the strain is used in high sugar concentration

doughs with an average sucrose level of approximately 30 % (Verstrepen et al., 2006).

The yeast isolates were also tested for their utilization of different sugars namely maltose,

fructose and lactose at a 2 % level. The isolates D3 and control baker’s yeast strain grew very

well on the lactose media while D2, D4, MA4 and Z1 showed a poor growth. Lactose is a

disaccharide, made up of galactose and glucose, whose utilization depends on the presence of the

LAC4 genes which can break lactose into its monomers and research has shown that most yeasts

reported in alcoholic milk fermented products lack genes encoding β-galactosidases, suggesting

that they must rely on free monosaccharides or lactic acid (Hittinger et al., 2018). Fructose is

found in many food items that could be used as substrates such as marula fruits (Sclerocarya

birrea), watermelon juice (Citrus lanatus), masau fruits (Ziziphus mauritiana), to name a few

(Dlamini and Dube, 2008; Nyanga et al., 2008; Ogodo et al., 2015). As such, a good strain

should be able to utilize fructose and the results below shows that all the strains utilized fructose

except for MA2 and MA7. The isolates D4 and T1 were not able to grow on media with maltose

while the control strain, D3 and S5 grew very well.

74
Figure 12: Ability of the isolated yeast strains to utilize different carbon sources. The

baker’s yeast (S. cerevisiae) was used at the control since it is the strain that khadi brewers use.

The black triangle shows concentrations of the dilutions from left to right (0.5, 0.25 and 0.125).

[Link] Fermentation capacity

The purpose of this experiment was to determine the amount of CO2 produced per isolate, with

CO2 production signifying progression of the fermentation process in comparison to the control,

S. cerevisiae (local commercial baker’s yeast). The experiment was done using synthetic media

prepared using G. flava fruits as a source of nutrients and brown sugar as a carbon source

mimicking the khadi brewing process. Our results showed that C. pallidicorallinum performed

exceptionally well followed by A. leucospermi, N. diffluens and C. sake while strains, A.

melanogenum and S. cerevisiae had a long lag phase (Figure 13). The fermentative ability of

some of these yeasts, such as C. sake and S. cerevisiae, was not surprising as they have been

75
reported in many traditional beverages such as tej of Ethiopia (Bahiru et al., 2006), palm wine of

Nigeria (Amoa-Awua et al., 2007; Stringini et al., 2009; Tapsoba et al., 2015), mukumbi and

kachasu of Zimbabwe (Mpofu et al., 2008; Nyanga et al., 2013b, 2007; Okagbue and Siwela,

2002).

30

Aureobasidium leucospermi

Naganishia diffluens

Saccharomyces cerevisiae
25
Candida sake

Lachancea fermentati

Curvibasidium pallidicorallinum
20
Schizosaccharomyces pombe
Carbon dioxide accumulation (mL)

Saccharomycodes ludwigii

Brettanomyces bruxellensis
15
Zygosaccharomyces bailii

Candida ethanolica

Rhodotorula nothofagi

10 Aureobasidium melanogenum

Baker's yeast

Ale yeast

5 Lager yeast

Wine yeast

0
0 100 200 300 400 500 600 700 800 900
Time (mins)

76
Figure 13: Fermentative capacity of yeast isolates. The graph shows the carbon dioxide

produced by the yeast isolates at an interval of 60 minutes. This experiment was performed three

times with two replicates each.

CO2 production rate which directly translates to fermentation rate is an additional critical

attribute of yeast isolates (Donalies et al., 2008; Johnson and Echavarri-Erasun, 2011). As such,

we investigated the CO2 production rate. The results below (Figure 14) show that the control (S.

cerevisiae) performed better than all the test strains isolated from khadi. The CO2 production rate

of L. fermentati (0.076 ± 0.004 mL/min) was higher than all the 4 controls. Our results suggest

that there was a significant statistical difference between all samples when each of them was

compared to the commercial baker’s yeast (t-test; p < 0.001), except for C. sake. The C. sake

could be one of the ideal strains to use for the fermentation of khadi but a valid conclusion will

be drawn once the strain chemical analysis of its fermentation broth has been done.

77
 0.09
***
Carbon dioxide production rate (mL/hr)

0.08

0.07 ***

0.06 *** ***

0.05

0.04 *** **
*** ** *** ***
0.03
***
0.02

0.01

Figure 14: Carbon dioxide production rate of yeast isolates. The average carbon dioxide

production rate (mL/hr) of the yeast isolates with the controls (ale, baker’s, lager and wine

yeasts). The t-test results (isolated yeasts vs baker’s yeast) are presented as with asterisks where

significance is a * (P<0.05), high significance is ** (P<0.01) and great significance is ***

(P<0.001).

[Link] Ethanol stress tolerance

Ethanol is one of the metabolites produced during khadi fermentation. As such, the fermentation

process requires a strain that can withstand elevated ethanol concentrations without its growth

being inhibited or impaired. The amount of ethanol produced depends on many attributes such as

the fermenting microorganism, the amount and types of sugars used as a substrate, and the

conditions at which fermentation is carried out, to list a few. Utilization of an organism with
78
higher resistance to ethanol is important for an efficient fermentation process. All potential

isolates were tested for growth on YPD supplemented with different concentrations of ethanol

(Figure 15). All isolates were resistant to 3 % ethanol and exhibited growth comparable to when

grown in the absence of ethanol except MA2. This ethanol concentration was chosen because

ethanol concentration of khadi is 3.69 % (v/v) as previously reported by Mapitse et al., (2014).

However, as observed in this work, ethanol concentrations in different khadi were on average,

5.27 ± 2.02 % (Figure 8). All isolates were resistant to this amount of ethanol except Z1 and

MA2. It was noted that ethanol concentrations in different khadi could be higher up to about 9%

(Figure 8). All the isolates remained resistant to 9 % ethanol except, MA2, T1, and Z1

suggesting that all the resistant isolates have potential to be used as starter cultures to produce

higher amounts of ethanol without a decrease in productivity.

79
Figure 15: Growth inhibition of the yeast strains due to different ethanol concentrations.

The baker’s yeast (S. cerevisiae) was used at the control since it is the strain that khadi brewers

use. The black triangle shows concentrations of the dilutions from left to right (0.5, 0.25 and

0.125).

[Link] Osmotolerance

Sugar content of fruits is subject to change every season (Yahia and Kader, 2011). The change

could bring about high sugar concentrations, which are associated with a higher osmotic stress.

Osmotic stress reduces productivity of production strains. Utilization of osmotolerant isolates is

an important attribute in brewing. All isolates were tested for osmotolerance by growing them in

media supplemented with an osmolyte, in this case NaCl was chosen. The yeast isolates grew on

media with 0.5 M NaCl except for MA2 (Figure 16). Their osmotolerance was comparably to the

control yeast. However, increasing the osmotic pressure by growing isolates on 1.0 M NaCl was

inhibitory for growth of isolates except 5 isolates (D3, D4, MA4 and S1) and the control strain.

These results suggest that strains will be highly impaired or inhibited by media with high sugar

concentrations. It is noteworthy that S1 was the only strain to grow on 1.5M NaCl although there

was no growth on 2.0M NaCl suggesting this isolate as the only isolate of choice in the presence

of higher amounts of sugars.

80
Figure 16: Osmo-tolerance of the yeast isolates due to supplementation with various NaCl

concentrations. The baker’s yeast (S. cerevisiae) was used at the control since it is the strain that

khadi brewers use. The black triangle shows concentrations of the dilutions from left to right

(0.5, 0.25 and 0.125).

[Link] pH stresses

During the production of khadi or any alcoholic beverage, a variety of organic acids are

produced which lowers the pH of the alcoholic beverage. From this work, it was found out that

Khadi is an acidic alcoholic beverage, which was in agreement to previously reports by (Mapitse

et al., 2014). These results prompted an investigation into the performance of isolates subjected

to various acidic pHs. In addition, it was important to investigate the lowest possible pH at which

isolated strains could withstand because it would not be ideal to select a potential isolate that will

81
be inhibited at a lower pH during the course of fermentation. The results show that all the strains

could not withstand pH 1 (Figure 17) as expected. Although some isolates performed well at pH

2, this pH is not ideal for any alcoholic beverage. It should be noted that the average pH recorded

among khadi in this work was 3.1 ± 0.31 (Figure 8) in agreement to a range from 2.87 to 3.16

reported by Mapitse et al., 2014. All strains grew on media at pH 3 except T1 and Z1. This

suggests that most of these isolates could be ideal to withstand low acidic conditions during the

khadi fermentation. The acidity of alcoholic beverages is important for inhibition of growth of

spoilage microorganisms (Manel et al., 2011; Schifferdecker et al., 2014; Swiegers et al., 2009).

Figure 17: The pH stress tolerance of the yeast isolates. The baker’s yeast (S. cerevisiae) was

used at the control since it is the strain that khadi brewers use. The black triangle shows

concentrations of the dilutions from left to right (0.5, 0.25 and 0.125).

82
[Link] Thermotolerance

Khadi is produced under non-standardized conditions in which fluctuation of temperatures could

reduce fermentative capacity. This is because fermentation is an exergonic process and thus there

is increase in temperature of the fermentation broth as fermentation proceeds. Therefore,

uncontrolled fermentation of khadi requires the use of a thermotolerant isolate should there be

excessive production of heat during fermentation. In addition, when khadi fermentation is done

under controlled fermenters, this thermotolerant attribute could be very economic as the costs of

cooling the fermentation vessels will be reduced or negated. Therefore, the upper threshold

temperatures that the yeast isolates could withstand were investigated. The results suggest that

most of the strains performed well at 37 °C and 40 °C except for isolates T1 and Z1 (Figure 18).

Higher temperatures were inhibitory for most of the isolates except 5 (D2, D3, D4, MA4, P8).

The control yeast was also able to grow at temperature of 43 °C. These results suggest that the

isolates have an immense potential to be used for khadi production at higher temperatures. The

isolates Curvibasidium pallicorallinum (MA7), C. sake (MA2), L. fermentati (MA4),

Saccharomycodes ludwigii (S1) and C. ethanolica (T17) have potential for production of khadi.

83
Figure 18: The thermo-tolerance of the isolates. The baker’s yeast (S. cerevisiae) was used at

the control since it is the strain that khadi brewers use. The black triangle shows concentrations

of the dilutions from left to right (0.5, 0.25 and 0.125).

4.7 Safety in bioprocessing of khadi

4.7.1 Identification of molds contaminants of khadi

To check for molds contamination, the khadi samples were grown on molds isolation media. The

results showed that the majority of the samples were contaminated with phylogenetically diverse

molds species (Figure 19). A total of 14 molds isolates were identified, some of which include

Aspergillus ochraceus, Cladosporium sphaerospermum, Cladosporium cladosporioides, Phoma

eupyrena and Setosphaeria turcica, to list a few (Table 13 and Figure 19). The predominant

isolates were C. sphaerospermum (Serowe and Maun), A. ochraceus (Palapye and Tonota) and

84
Flavodon ambrosius (Mmashoro) (Table 13), each attributing 21.4 % of the total isolates. The

introduction of molds to khadi might be through the fruits or as a contamination from the

brewers or brewing utensils. Visible molds growth on khadi may reduce the shelf life of the

product and make it not fit for human consumption thus meaning molds are undesirables in

foods.

Molds isolates such as Aspergillus ochraceus in beverages can be an indicator of the possible

presence of mycotoxins. However, molds in most cases does not imply the presence of

mycotoxins. Sometimes it means the food item has necessary ideal conditions such as high water

activity, the presence of sugars, and the presence of organic acids capable of reducing pH

(Snowdon and Cliver, 1996) hence a possible explanation for the presence of these molds

isolates on the khadi samples. Aspergillus ochraceus is of great concern as it has been previously

reported to produce aflatoxins and ochratoxins (Kharayat and Singh, 2018) which are

carcinogenic, nephrotoxic, hepatotoxic and teratogenic to mammalian cells (Roseanu et al.,

2010). On the other hand, S. turcica is a hemi-biotrophic plant pathogen (parasitic on living and

dead tissue) and heterothallic (sexes that reside in different individuals) whose usual host is

maize and sorghum. Due to its hemi-biotrophic ability, hypothetically S. turcica could be passed

from G. flava fruits to the khadi. The other isolates of interest are C. sphaerospermum and C.

cladosporioides, which are filamentous molds previously isolated from flowers of edible fruit

plants as well as being isolated from honey, dairy products and grapes prior to harvesting

(Cosme et al., 2018; Hill, 2015; Moubasher et al., 2018; Snowdon and Cliver, 1996). The

Cladosporium species have been previously reported to be mycotoxin secreting and potentially

pathogenic fungus frequently occurring in outdoor environments (Hadi, 2017). With that said,

85
the presence of all these molds could be an indicator for mycotoxin presence in the khadi

samples.

Table 13: Identity of molds isolates from the khadi samples based on D1/D2 domains of 26S

rDNA.

Species name Collection number Accession number Identity %

Aspergillus ochraceus (2) Tonota 2 KX958037.1 99
Aspergillus ochraceus Palapye 1 KX958037.1 99
Chaetomium longiciliata Maun 3 KP336823.1 99
Cladosporium cladosporioides Palapye 1 KM246047.1 99
Cladosporium sphaerospermum Serowe 1 KY873376.1 99
Cladosporium sphaerospermum (2) Maun 3 KY873376.1 99
Flavodon ambrosius (3) Mmashoro 3 NG_060421.1 99
Periconia thailandica Serowe 2 KY753888.1 99
Phoma eupyrena Palapye 3 JF766684.1 99
Setosphaeria turcica Maun 1 LT715640.1 99
Note: The number in the bracket shows strains with the same accession number from the same

location.

86
Figure 19: A picture of the molds isolates from khadi samples namely: (a) Cladosporium sphaerospermum, (b) Periconia

thailandica, (c) Cladosporium cladosporioides, (d) Setosphaeria turcica (e) Flavodon ambrosius (f) Aspergillus ochraceus, (g)

Phoma eupyrena and (h) Chaetomium longiciliata.

87
4.7.2 Mycotoxins quantification: Evidence Investigator calibration

The presence of molds isolates (Figure 19) in the khadi samples prompted us to analyze this

brew for mycotoxins. Mycotoxin quantification was carried out using the Randox Evidence

Investigator. To optimize the sensitivity of the method and test its accuracy, the Randox

Evidence Investigator was calibrated using the provided calibrators of a known concentration.

The acquired data was then used to provide information of the limits of detection (LODs) and

limits of quantification (LOQs). LOD and LOQ are terms used to describe the smallest

concentration of an analyte that can be reliably measured by an analytical procedure with an

acceptable degree of accuracy and precision. The LOD and LOQ were calculated based on

threefold and tenfold signal to noise (S/N) ratios, respectively (Table 14). The LOD ranged from

0.13 to 101.33 while the LOQ ranged from 0.43 to 337.78 with DON having the highest LOD

and LOQ. The linearity was established by the calibration curve based on the expected values

and actual values from calibrators. The coefficient correlations R2 ranged from 0.97 to 0.99 thus

showing good linearity (Table 14) hence method validation.

88
Table 14: The calibration results from the Randox Evidence Investigator.

Compound Linear equation R2 SD LOD LOQ

PAX Y = 0.9634x + 0.0252 0.99 6.48 20.18 69.39

FUM Y = 1.0061x - 0.1004 0.97 0.68 2.03 6.77

OTA Y = 1.0088x - 0.0131 0.98 0.27 0.81 2.70

AFG1 Y = 0.9883x - 0.0103 0.98 0.33 1.01 3.38

EA Y = 1.0056x - 0.1973 0.97 4.49 13.41 44.70

DAS Y = 1.0816x - 0.4864 0.97 7.33 20.33 67.76

DON Y = 1.0907x - 2.4123 0.98 36.84 101.34 337.78

T2 Y = 1.0935x - 0.0721 0.98 1.47 4.04 13.47

AFB1 Y = 1.0769x - 0.0023 0.98 0.05 0.13 0.43

ZEA Y = 1.0766x - 0.0459 0.99 0.72 2.02 6.73

Notes: PAX = paxilline, FUM = fumonisin, OTA = ochratoxin A, AFG1/G2 = aflatoxin G1/G2,

ERG = ergot alkaloids, DAS = diacetoxyscirpenol, DON = deoxynivalenol, AFB1/B2 = aflatoxin

B1/B2, ZEA = zearalenone.

Three samples from three different sampling areas were then spiked with a known concentration

of AFB1, FB1 and OTA to test the efficiency of the acetonitrile method and the Myco-10

method (according to the manufacturer’s instruction) (Table 15). The recovery of the samples

ranged from 12.05 % to 107.3 % using the acetonitrile method where the best recovery was

observed on FB1 and AFB1. The acetonitrile method failed to extract OTA from all the three

samples as the spike recovery was very low. The low recovery of mycotoxins OTA could

possibly mean the acetonitrile extraction method was not optimal for these mycotoxins and this

could be affecting the extraction of these toxins from the khadi samples thus giving inaccurate

89
results. Table 14 shows that the challenge is not with the biochip as the quantification of the all

control standards read values close to the true concentrations as stated by the manual provided by

the manufacturer. This is further confirmed by the R2 of the sample calibrators. The results in

Table 15 also show that the Myco-10 extraction method was not suitable for use in this

experiment. The Myco-10 extraction method was based on the use of chemicals namely

acetonitrile, methanol and water as the extraction solvents and no organic salts. The acetonitrile

method was probably more effective as it countered the presence of water in khadi through the

use of organic salts to bind to the water molecules (Font et al., 2017). This step was not there in

the Myco-10 extraction method. This method did not yield meaningful results and thus it was

deemed unsuitable for use in this work.

Table 15: Comparison of the product recovery from the spiked samples using the acetonitrile

method and the Myco-10 method.

Sample FB1 (1 ppm) OTA (2 ppm) AFB1 (0.1 ppm)

Maun 1 (ACN method) 91.40 % 18.80 % 53.10 %

Mmashoro 2 (ACN method) 103.70 % 12.05 % 45.80 %

Serowe 1 (ACN method) 107.30 % 23.50 % 61.90 %

Serowe 1 (Myco-10 method) -98.2 % -5.11 % -75 %

4.7.3 Mycotoxins from khadi

After the optimization of the acetonitrile method, the khadi samples were analysed for presence

of mycotoxins. Table 16 shows the mycotoxins from the khadi samples. All the samples had

fumonisin, ochratoxin A, diacetoxyscirpenol and zearalenone. The least detected toxins from the

90
brews were paxilline (detected from Mmashoro 1, Mmashoro 2, Palapye 1, Palapye 3, Serowe 1

and Serowe 3), deoxynivalenol (detected from Maun 1, Maun 2, Serowe 1, Tonota 1, Tonota 2

and Tonota 3) and aflatoxin B1/B2 (detected from Mmashoro 3, Serowe 1 and Tonota 2). The

Serowe 1 sample was observed to have all the mycotoxins detected from it. The overall average

quantification shows that the highest detected mycotoxin was DAS (22.9 ± 8.12 ppb) and the

least detected toxin was aflatoxin B1/B2 (0.0023 ± 0.0023 ppb). The average of the other toxins

being PAX (0.56 ± 0.47), FUM (1.27 ± 0.55), OTA (2.11 ± 0.92), AFG1/G2 (0.088 ± 0.02),

ERG (3.9 ± 0.68), DON (11.1 ± 5.81) and ZEA (0.13 ± 0.1).

Mycotoxins such as zearalenone have been detected from traditional opaque beers of Botswana

(Nkwe et al., 2005) while others such as ochratoxin A, deoxynivalenol, fumonisin B1 and

fumonisin B2 have been detected in many European beers (Bertuzzi et al., 2011). In another

study on the maize-based alcoholic beverage of South Africa, umqombothi, the authors detected

mycotoxins such as T2, DON, fumonisin, zearalenone and aflatoxins from the beverage with

evidence pointing at the raw material or substrate as the source of the mycotoxins (Adekoya et

al., 2018; Peters et al., 2017). Dried fruits have more mycotoxins than fresh fruits. Mycotoxins

accumulate during the harvesting, drying and storage due to conditions that favour mycotoxin

accumulation (Drusch and Ragab, 2003). The A. ochraceus produces ochratoxin A, which was

detected in all the khadi samples in this work. The detection of other mycotoxins such as

aflatoxins is characteristic of other molds such as A. flavus, A. parasiticus, A. bombycis, A.

ochraceus, A. nomius, and A. pseudotamar (Bircan, 2009; Kharayat and Singh, 2018; Kumari et

al., 2015; Lyumugabe et al., 2012; Roseanu et al., 2010; Zain, 2011). In this work, only A.

ochraceus was isolated from khadi and thus this is the only possible source of aflatoxins

91
 detected. Due to the limitation of the rapid analysis method and acetonitrile extraction method,

no comparison was made to the FDA or EC law on the mycotoxin maximum allowed limits.

Table 16: The quantification of mycotoxins from khadi samples in ppb.

Sample PAX FUM OTA AFG1/G2 ERG DAS DON T2 AFB1/B2 ZEA

Control 1.51 0.27 0.057 0.066 1.10 1.30 8.89 0.25 0.0081 0.14

Maun 1 0 1.30 2.53 0.092 4.57 27.06 5.57 0.24 0 0.16

Maun 2 0 1.48 2.26 0.1116 4.86 25.13 10.02 0.30 0 0.094

Maun 3 0 0.61 0.87 0.067 2.43 14.53 0 0.06 0 0.091

Mmashoro 1 0.23 1.14 1.97 0.12 4.16 21.12 0 0.20 0 0.11

Mmashoro 2 0.51 1.46 2.26 0.088 4.45 24.74 0 0.15 0 0.12

Mmashoro 3 0 1.90 2.79 0.096 4.10 29.23 0 0.079 0.0006 0.13

Palapye 1 0.27 1.55 2.22 0.060 3.60 25.21 0 0.13 0 0.19

Palapye 2 0 1.81 2.33 0.079 3.26 27.30 0 0.13 0 0.12

Palapye 3 0.33 2.27 3.52 0.072 3.78 29.91 0 0.093 0 0.068

Serowe 1 1.32 1.14 2.13 0.12 3.22 26.06 21.86 0.33 0.0049 0.49

Serowe 2 0 1.45 1.89 0.058 3.80 23.11 0 0.091 0 0.13

Serowe 3 0.16 1.15 1.76 0.074 3.35 22.19 0 0.12 0 0.11

Tonota 1 0 1.72 3.84 0.11 4.61 33.79 8.46 0.24 0 0.18

Tonota 2 0 1.38 2.82 0.12 4.93 29.54 10.95 0.26 0.0013 0.19

Tonota 3 0 1.26 2.58 0.078 3.84 26.51 7.09 0.27 0 0.13

Letlhakane 1 0 1.02 1.60 0.07 3.56 19.63 0 0.027 0 0.042

Letlhakane 2 0 0.18 0.44 0 0 4.34 0 0 0 0.009

Letlhakane 3 0 0.14 0.33 0 0 3.47 0 0 0 0.004

Notes: PAX = paxilline, FUM = fumonisin, OTA = ochratoxin A, AFG1/G2 = aflatoxin G1/G2,

ERG = ergot alkaloids, DAS = diacetoxyscirpenol, DON = deoxynivalenol, AFB1/B2 = aflatoxin

B1/B2, ZEA = zearalenone.

92
 Chapter 5: General discussion and conclusion

5.1 Summary of study investigations and findings

Khadi is a homemade traditional beverage, produced from the spontaneous fermentation of G.

flava fruits, whose fermentation is carried out by unknown microbiota. Khadi fermentation

process varies based on the preference of the brewers. As such, there is no standard procedure of

producing this beverage. Therefore, there are a lot of product inconsistencies, which have led to

negative health implications. Thus, this work sought to document the step-by-step process of brewing

khadi using this traditional beverage and its substrate as a source of fermentative microbes. This was

done by sequencing 16S rDNA for bacterial identification and 26S rDNA for yeasts identification.

The source of these microorganisms could be the G. flava fruits and the fermentation equipment

used. The isolates identified from the G. flava fruits were not limited to yeasts only, we also

identified some bacteria. The Bacillus spp. were predominant species in the G. flava fruits. The

dominance of Bacillus species was not surprising as they are ubiquitous microbes that are

common in soil, air, dust, water as well as dried and processed foods of plant origin.

The microbial diversity of khadi was mostly made up of phylogenetically diverse consortium of

yeast and molds. Interestingly, no bacteria survived the fermentation process. The yeast diversity

of khadi showed that this beverage was produced by a mixed culture of yeast. The consortium

identified from khadi was made up of both conventional yeasts and non-conventional yeasts.

Some of the identified isolates are spoilage yeasts such as B. bruxellensis and Z. bailii (Barata et

al., 2012; Chatonnet et al., 1995; James and Stratford, 2003; Jeffries et al., 2015; Licker et al.,

1998; Schifferdecker et al., 2016). Even though khadi is supposed to be produced through

spontaneous fermentation, it is noteworthy that our results also showed some of the brewers

inoculated using the locally available commercial baker’s yeast.

93
As already stated above, the khadi consortium also included some molds. The identified were

Cladosporium sphaerospermum, Periconia thailandica, Cladosporium cladosporioides,

Aspergillus ochraceus, Phoma eupyrena, Setosphaeria turcica, Chaetomium longiciliata and

Flavodon ambrosius. The predominant molds isolates were C. sphaerospermum, A. ochraceus

and Flavodon ambrosius. The detection of these molds could be a possible indication of the

presence of mycotoxins thus we quantified mycotoxins from all the khadi samples. The

mycotoxins quantification results showed that, on average, the highest detected mycotoxin was

DAS and the least detected toxin to be aflatoxin B1/B2. Some of the mycotoxins in khadi have

previously been reported in beers from Africa and Europe, and these include aflatoxins,

deoxynivalenol (DON), fumonisin, ochratoxins, ergot alkaloids, and zearalenone (Ezekiel et al.,

2015; Fernández-Cruz et al., 2010; Galvis-Sánchez et al., 2008; Inoue et al., 2013; Kirui et al.,

2014; Richard, 2007; Zain, 2011).

To select a possible starter culture for khadi fermentation, the yeasts were characterized and

exposed to many strenuous conditions which included different concentrations of ethanol,

sodium chloride, carbon sources, different pH and different temperatures. On average, the results

showed that isolates C. pallicorallinum (MA7), C. sake (MA2), L. fermentati (MA4),

Saccharomycodes ludwigii (S1) and C. ethanolica (T17) performed well on all these experiments

including the fermentation profiling. These are the strains that further research could be

conducted in search of a potential starter culture.

5.2 The significance of the findings to food hygiene and safety

Spontaneous and uncontrolled fermentation of khadi could lead to many health implications.

This fermentation technique leads to the production of undesirable metabolites some of which

94
could have health implications on the khadi consumers. As such, our findings could be used to

develop a reliable starter culture which could be sold to khadi brewers. Khadi is a source of

income for many households thus a starter culture could benefit many families whose lives

depend on khadi sales. Our characterization results showed that the best strains to use for khadi

fermentation are C. pallicorallinum, C. sake, L. fermentati, Saccharomycodes ludwigii and C.

ethanolica. These strains are all non-conventional yeasts whose fermentation processes could

lead to the production of unique flavors. With these strains, the people could be able to produce a

flavor rich product. Future-work will include the chemical profiling of the beverages produced

using yeast isolated from khadi to confirm their selection for commercialization.

5.3 The major strengths and weaknesses of study methodology

Documentation of khadi preparation methods was based on scientifically accepted methods. The

methods used during the enumeration, isolation and identification of bacteria, molds and yeasts

from the G. flava fruits and khadi were scientifically accepted methods. The characterization of

bacterial isolates and yeasts were studied using scientifically accepted methods. The methods of

genetic characterization of yeasts from khadi may need improvement which could be done not

only through in-silico analysis but also the actual RFLP to confirm the results from the in-silico

work. There is need to conduct antibiotic susceptibility of the bacterial isolates from G. flava

fruits after identification by sequencing of 16S rDNA

95
5.4 Conclusion

This study presents the first process documentation and microbiological characterization of a

prominent traditional alcoholic beverage in Botswana., The survey questionnaire revealed that

khadi is brewed from ripened sun-dried G. flava fruits, an inoculum of previously fermented G.

flava fruits, brown table sugar and warm water only. The survey revealed that khadi fermentation

employs spontaneous of these ingredients to produce khadi. The microbial diversity of G. flava

fruits and khadi showed that there was predominance of non-fermentative Basidiomycetous

yeasts and bacteria on the ripened sun-dried G. flava fruits while the khadi samples harbored

fermentative Saccharomyces yeasts and non-Saccharomyces yeasts as well as molds. The results

presented in this work suggest that khadi is a product of mixed culture fermentation. It is

plausible that local commercially available baker’s yeast is regularly used as part of the

fermenting starter culture whereas some brewers employed spontaneous fermentation.

Evaluation of potential starter cultures suggests that C. pallicorallinum, C. sake, L. fermentati,

Saccharomycodes ludwigii and C. ethanolica isolated from khadi are able to withstand various

stressful conditions. Since stress resistance is also key to the bioprocess development of khadi

for commercialization the thesis highlights that these isolates have potential as starter cultures.

Further studies to test for the functionality of the mixed consortia could be useful for

development of starter cultures for a consistent product quality towards commercialization of

khadi. Like most naturally fermented beverages, khadi was found to contain mycotoxin

producers and thus further investigation to quantify mycotoxins was deemed necessary to

determine the safety of this alcoholic beverage. The rapid analysis of mycotoxins from khadi

revealed the presence of mycotoxins such as DAS and DON. These mycotoxins can be harmful

96
to the khadi consumers thus need for the detoxification of the raw materials and the final product

before it is consumed.

5.5 Recommendations

To improve this work, yeast isolates from khadi could be used to brew khadi under controlled

and standardized conditions and then subsequent characterization of the product using advanced

chromatographic methods with hope to produce a high-quality product. There is need to do a

thorough investigation of the presence of other microorganisms through use of culture

independent methods such as microbiomics. This is important to reveal other microorganisms

that could have been important in improvement of the quality of khadi. Chemical profiling of

khadi to quantify sugars, organic acids, aroma compounds and alcohols should could be

instrumental to document different flavors of these different beverages as well as to detect

primary or secondary metabolites that could be of harm to the consumers. There is need to

evaluate the ethanol production capacity or the contribution of each of the yeast isolates as an

important attribute towards development of potential starter cultures. This work did not focus on

other sources of yeasts, which could have been responsible for khadi fermentation. For example,

the drums (vessels) used for brewing could have been the source of the microflora. In the future,

the is need to characterize the different and amount of sugars found in G. flava in order to

understand their contribution to the fermentation process of khadi.

97
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116
 Appendices

Appendix A

Questionnaire

I, Koketso Motlhanka, a Master’s student of Botswana International University of Science and

Technology, am conducting a research project on the Microbiological and Chemical characterization of
khadi, a reddish-brown alcoholic beverage made from brown sugar and Grewia flava fruits from
Botswana. The purpose of the study is to find out the preparation steps and consumption of khadi in
Botswana. You are requested to assist by answering the following questionnaire. Your participation will
be highly appreciated.

Please tick (√) in the boxes as appropriate.

Location………………………………………………………………………………..

Sex Male Female

Age

19-25

26- 35

36-45

45 and older

Occupation:

Unemployed

Self-employed

Retired

Farmer

Civil servant

Please assist by answering the following questions.

1) What ingredients do you use to prepare Khadi?

………………………………………………………………………………………………………
………………………………………………………………………………………
2) Do you add starter culture or inoculum when preparing khadi?

117
 Yes
No
3) If yes to the above question, what is the source of your inoculum?
………………………………………………………………………………………………………
………………………………………………………………………………………
4) If no to the above question, what do use as a starter culture of your fermentation?
………………………………………………………………………………………………………
……………………………………………………………………………………….
5) How much starter culture do you add to initiate khadi fermentation?
Half a cup
One full cup
One and half cups
Two cups
If more specify………………………………………………………………………………
6) At which temperature do you incubate your khadi to ferment?
………………………………………………………………………………………………………
……………………………………………………………………………………..
7) How long does your khadi take to ferment?
6 hours
12 hours
18 hours
24 hours
36 hours
48 hours
8) How do you know fermentation of the khadi is complete?
Production of bubbles
Taste
Production of foam
Depletion of ingredients
Other, specify………………………………………………………………………………...
9) What type of vessel do you use to ferment your khadi?
Plastic drum
Clay pot
Calabash
Other, specify……………………………………………………………….
10) What temperature do you store the khadi ingredients (especially Grewia flava fruits)?
………………………………………………………………………………………………………
………………………………………………………………………………………
11) How do you store the Grewia flava fruits?
………………………………………………………………………………………………………………
………………………………………………………………………………

118
Appendix B

Table B1: The yeast enumeration results in log10CFU/mL from the khadi.

Sample Experiment 1 Experiment 2 Experiment 3 Average Standard

deviation

Letlhakane 1 5.827 5.226 4.462 5.822 5.474 4.315 5.878 5.561 5.257 5.314 0.576

Letlhakane 2 5.948 5.374 4.609 5.986 5.419 4.447 6.030 5.399 4.452 5.296 0.650

Letlhakane 3 5.608 4.944 4.079 5.602 4.861 4.125 5.479 4.905 4.014 4.846 0.649

Maun 1 6.090 6.205 6.544 6.181 5.785 6.672 6.167 6.259 6.843 6.305 0.325

Maun 2 5.454 5.630 5.523 5.454 5.732 5.802 5.466 5.817 6.014 5.655 0.199

Maun 3 5.553 6.156 6.156 5.645 6.276 6.091 5.677 6.225 6.166 5.994 0.283

Mmashoro 1 5.995 5.353 4.294 5.930 5.312 4.315 6.035 5.260 4.426 5.213 0.716

Mmashoro 2 6.047 4.808 3.602 5.882 5.361 4.519 5.899 5.347 4.690 5.128 0.799

Mmashoro 3 5.633 5.137 4.336 5.437 5.154 4.301 5.540 5.170 4.519 5.025 0.514

Palapye 1 6.185 6.689 6.956 6.309 6.365 6.811 6.265 6.640 6.847 6.563 0.286

Palapye 2 6.327 6.540 6.751 6.269 6.560 6.568 6.286 6.598 6.693 6.510 0.176

Palapye 3 5.954 6.144 6.380 6.057 6.230 6.114 6.066 6.420 6.054 6.158 0.157

Serowe 1 5.780 5.877 5.426 5.773 5.756 6.472 5.750 5.748 5.222 5.756 0.340

Serowe 2 6.335 5.989 5.268 6.272 5.941 5.281 6.271 5.957 5.279 5.844 0.450

Serowe 3 5.987 5.346 4.771 5.848 5.377 4.748 5.922 5.359 4.687 5.338 0.514

Tonota 1 4.519 4.301 4.301 4.374 4.000 4.000 4.491 4.477 4.824 4.365 0.259

Tonota 2 6.029 6.242 6.487 6.023 6.365 6.564 6.022 6.246 6.431 6.268 0.209

Tonota 3 5.929 6.219 6.447 5.985 6.294 6.409 6.073 6.221 6.431 6.223 0.193

119
Appendix C

Table C1. S. cerevisiae yeast strains PCR-RFLP of 26S region of the rRNA using three

restriction endonucleases (HaeIII, HinfI, GluI and RsaI).

Isolate name Location Restriction patterns

designation APa HaeIII HinfI GluI RsaI

Commercial 582 322, 160, 97, 3 209, 179, 147, 47 345, 186, 29, 22 254, 225, 47, 41,

bakers’ yeast 15

Ale yeast, 555 322, 124, 109 209, 156, 145, 48 331, 186, 38 225, 218, 65, 47

Saccharomyces

cerevisiae

Lager yeast, 556 427, 129 209, 152, 148, 47 336, 186, 34 225, 223, 61, 47

Saccharomyces

pastorianus

Wine 1, 555 322, 124, 109 209, 156, 143, 47 331, 186, 38 225, 218, 65, 47

Saccharomyces

bayanus

Wine 2, 555 322, 124, 109 209, 156, 145, 48 331, 186, 38 225, 218, 65, 47

Saccharomyces

cerevisiae

AC1MIII Mmashoro 3 322, 134, 122 209, 179, 147, 47 329, 186, 63 225, 205, 90, 47,

11

AC2MIII Mmashoro 3 584 237, 163, 99, 85 264, 193, 127 370, 186, 28 584

AC3MIII Mmashoro 3 584 322,162, 72, 28 209, 181, 147, 47 345, 186, 24, 17, 256, 225, 56, 47

12

AC5MIII Mmashoro 3 587 322, 161, 81, 13, 209, 180, 151, 47 248, 219, 97, 23 255, 225, 60, 47

10

BC1MII Mmashoro 2 586 322, 161, 103 209, 180, 150, 47 345, 186, 32, 23 284, 255, 47

BC4MII Mmashoro 2 585 322, 161, 102 209, 180, 149, 47 345, 186, 31, 23 255, 225, 58, 47

L3 Letlhakane 1 580 322, 165, 98 209, 179, 145, 47 345, 186, 27, 22 279, 245, 47

120
L6 Letlhakane 2 587 322, 165, 100 409, 184, 147, 47 345, 186, 29, 27 259, 225, 56, 47

L7 Letlhakane 2 588 322, 166, 100 209, 185, 147, 47 345, 186, 29, 28 260, 225, 56, 47

L8 Letlhakane 2 587 322, 165, 100 209, 184, 147, 47 345, 186, 29, 27 259, 225, 47, 41,

15

L10 Letlhakane 3 587 322, 165, 100 209, 184, 147, 47 345, 186, 29, 27 259, 225, 47, 41,

15

L11 Letlhakane 3 591 322, 169, 100 209, 188, 147, 47 346, 186, 30, 29 225, 135, 128,

47, 40, 16

M1 Maun 1 588 322, 161, 101 209, 185, 147, 47 345, 186, 29, 28 260, 225, 47, 41,

15

M6 Maun 2 581 322, 161, 98 209, 180, 145, 47 345, 186, 23, 22, 255, 225, 47, 41,

5 13

LMA1 Mmashoro 1 588 322, 166, 100 209, 185, 147, 47 345, 186, 29, 28 260, 225, 47, 41,

15

LMA3 Mmashoro 3 586 322, 160, 104 209, 185, 147, 47 345, 186, 33, 22 254, 225, 47, 41,

19

LMA4 Mmashoro 1 582 450, 102, 30 582 582 582

LMA5 Mmashoro 2 590 332, 100, 85, 73 396, 194 375, 215 590

LMB1 Mmashoro 1 584 322, 161, 101 209, 180, 148, 47 345, 186, 30, 23 255, 225, 47, 41,

16

MIAC1 Mmashoro 1 585 322, 162, 101 209, 181, 148, 47 345, 216, 24 256, 225, 57, 47

MIAC2 Mmashoro 1 585 322, 162, 79, 19, 209, 181, 99, 49, 345, 216, 24 225, 135, 121,

3 47 57, 47

MIAC3 Mmashoro 1 586 322, 160, 94, 6, 4 209, 179, 151, 47 345, 219, 22 254, 225, 60, 47

MIIAC4 Mmashoro 2 587 322, 161, 104 209, 180, 151, 47 345, 219, 23 255, 225, 60, 47

MIIBC5 Mmashoro 2 582 322, 161, 104 209, 180, 99, 47, 345, 214, 23 280, 255, 47

47

P10 Palapye 1 583 322, 134, 124, 3 209, 184, 143, 47 333, 186, 64 225, 221, 90, 47

P11 Palapye 1 582 322, 134, 126 209, 184, 142, 47 332, 186, 64 225, 220, 90, 47

P13 Palapye 3 585 322, 161, 102 209, 180, 149, 47 345, 186, 31, 23 255, 225, 58, 47

P14 Palapye 2 581 322, 135, 124 209, 184, 140, 47 330, 186, 65 225, 218, 91, 47

121
P16 Palapye 2 580 322, 134, 124 209, 184, 140, 47 330, 186, 65 225, 218, 90, 47

P17 Palapye 3 586 322, 140, 125 209, 184, 140, 47 326, 186, 70, 5 225, 219, 96, 47

S2 Serowe 1 590 322, 169, 99 209, 188, 146, 47 345, 186, 31, 28 263, 225, 47, 41,

14

S6 Serowe 2 588 322, 165, 101 209, 184, 148, 47 345, 186, 30, 27 259, 225, 47, 41,

16

S7 Serowe 3 585 322, 163, 100 209, 182, 147, 47 345, 186, 29, 25 257, 225, 47, 41,

16

T5 Tonota 1 591 322, 165, 104 209, 184, 151, 47 345, 219, 27 259, 225, 47, 43,

17

T6 Tonota 1 594 322, 168, 104 209, 184, 151, 47 375, 186, 33 225, 135, 127,

47, 41, 19

T8 Tonota 2 584 322, 161, 101 209, 180, 148, 47 345, 186, 30, 23 255, 225, 47, 41,

15

T9 Tonota 2 583 322, 161, 100 209, 180, 147, 47 345, 186, 29, 23 255, 225, 47, 41,

15

T11 Tonota 3 592 322, 168, 102 209, 187, 149, 47 345, 186, 31, 30 262, 225, 47, 41,

17

Z2 Letlhakane 1 583 483, 100 209, 180, 147, 47 209, 186, 136, 225, 135, 116,

29, 23 56, 47, 4

WT Strain from the 580 322, 160, 104 209, 187, 137, 47 327, 187, 66 226, 215, 92, 47

fruits

122
Table C2. The non-Saccharomyces yeast strains PCR-RFLP of 26S region of the rRNA

using three restriction endonucleases (HaeIII, HinfI, GluI and RsaI).

Isolate name Location Restriction patterns

designation APa HaeIII HinfI GluI RsaI

AC4MIII Mmashoro 1 590 322, 164, 81, 23 209, 183, 151, 47 345, 186, 33, 26 258, 225, 60, 47

L1 Letlhakane 1 572 237, 173, 159, 3 385, 100, 87 177, 169, 83, 59, 298, 274

39, 19, 14, 12

L2 Letlhakane 1 582 237, 176, 68, 65, 33, 3 392, 100, 90 169, 147, 67, 59, 305, 277

39, 32, 23, 20, 14,

12

L4 Letlhakane 1 575 237, 175, 68, 60, 33, 2 387, 100, 88 169, 147, 62, 59, 300, 275

39, 32, 23, 19, 14,

11

L9 Letlhakane 3 589 425, 164 210, 183, 147, 49 225, 188, 121, 29, 283, 259, 47

26

L12 Letlhakane 3 592 428, 164 225, 188, 121, 32, 286, 259, 47

210, 183, 150, 49 26

L13 Letlhakane 3 572 572 524, 48 396, 146, 30 516. 31, 25

LMB2 Letlhakane 3 576 177, 173, 162, 60, 4 388, 100, 88 183, 128, 79, 66, 301, 275

49, 39, 20, 12

LMB4 Letlhakane 1 574 177, 175, 162, 60 388, 100, 86 183, 128, 79, 66, 301, 273

49, 39, 18, 12

LMC1 Letlhakane 1 576 177, 176, 163, 60 389, 100, 87 183, 128, 79, 67, 302, 274

49, 39, 19, 12

LMC4 Letlhakane 1 572 177, 176, 159, 60 385, 100, 87 183, 128, 79, 63, 298, 274

49, 39, 19, 12

M2 Maun 1 569 237, 173, 91, 68 385, 101, 83 222, 128, 60, 59, 298, 271

50, 23, 16, 11

M3 Maun 1 568 237, 173, 90, 68 384, 101, 83 222, 128, 59, 59, 297, 271

50, 23, 16, 11

123
M4 Maun 2 579 419, 160 386, 146, 47 194, 188, 122, 28, 280, 252, 47

28, 26, 21

M7 Maun 3 597 410, 187 291, 193, 113 241, 214, 142 267, 204, 67, 46, 13

M8 Maun 3 581 418, 163 389, 145, 47 194, 185, 122, 28, 264, 255, 47, 15

28, 24

MIAC4 Mmashoro 3 587 322, 161, 104 209, 180, 151, 47 345, 219, 23 255, 225, 60, 47

P8 Palapye 2 605 586, 19 405, 200 325, 128, 93, 59 320, 271, 14

S1 Serowe 1 586 586 390, 147, 49 225, 188, 121, 29, 267, 256, 47, 16

23

S4 Serowe 1 587 587 210, 180, 148, 49 225, 188, 144, 30 284, 256, 47

S5 Serowe 2 573 369, 167, 22, 15 389, 102, 46, 36 450, 105, 18 573

S8 Serowe 8 572 572 524, 48 396, 146, 30 516, 31, 25

T1 Tonota 1 589 420, 169 256, 188, 89, 56 224, 186, 131, 27, 310, 225, 40, 14

21

T4 Tonota 1 595 423, 172 209, 191, 92, 56, 47 196, 186, 131, 30, 266, 225, 47, 41, 16

28, 24

T7 Tonota 1 591 427, 164 210, 183, 149, 49 225, 188, 121, 31, 285, 259, 47

26

T10 Tonota 2 571 177, 166, 159, 60, 9 385, 100, 86 183, 158, 79, 63, 298, 273

49, 39

T12 Tonota 3 587 587 390, 148, 49 225, 218, 144 303, 284

T13 Tonota 3 586 426, 160 210, 179, 148, 49 225, 188, 121, 30, 268, 255, 47, 16

22

T14 Tonota 3 568 177, 172, 159, 60 385, 100, 83 183, 128, 79, 63, 298, 270

49, 39, 16, 11

T15 Tonota 3 569 177, 173, 159, 60 385, 100, 84 183, 128, 79, 63, 298, 271

49, 39, 17, 11

T16 Tonota 3 584 423, 161 210, 180, 145, 49 225, 188, 121, 27, 267, 256, 47, 14

23

T17 Tonota 3 569 177, 173, 159, 60 385, 100, 84 183, 128, 79, 63, 298, 271

124
 49, 39, 17, 11

T18 Tonota 3 567 177, 171, 159, 60 385, 100, 82 183, 128, 79, 63, 298, 269

49, 39, 15, 11

Z1 Letlhakane 1 595 409, 186 291, 192, 112 241, 213, 141 266, 203, 67, 46, 13

125
Figure C1: PCR-RFLP for the all S. cerevisiae isolates from khadi samples. The red shows yeast isolates similar to the control

commercial baker’s yeast (white boundary) while the WT is the wild type S. cerevisiae isolated from the Grewia flava fruits.

126
Figure B2: PCR-RFLP for the all non-Saccharomyces isolates from khadi samples. The restriction patterns of isolates indicate

similarities among isolates from different regions of the country.

127
 Appendix D

Table D1: The stress tolerance of yeast isolates from Grewia flava fruits and khadi.

Samples Sugars (%) Ethanol (%) Salt (M) pH Temperature (°C)

YPS YPS YPL YPF YPM 3 5 7 9 0.5 1.0 1.5 2.0 1 2 3 5 37 40 41 42 43

(10) (20) (2) (2) (2)

D2 ++ - + ++ + ++ ++ ++ + + - - - - + ++ - +++ +++ +++ +++ +++

D3 ++ ++ +++ ++ ++ +++ ++ ++ + + + - - - + ++ +++ +++ +++ +++ +++ +++

D4 - - + + - + ++ ++ + + + - - - + ++ - + + + + +

MA1 +++ + - ++ + +++ +++ ++ + + - - - - ++ ++ + +++ + + - -

MA2 ++ - ++ - ++ - - - - - - - - - - + ++ + + - - -

MA4 +++ ++ + ++ ++ ++ ++ + + + + - - - + +++ +++ ++ ++ ++ + +

MA7 ++ ++ - - ++ +++ ++ ++ ++ + - - - - + +++ + +++ +++ +++ + -

P8 + + + + + ++ +++ ++ + + - - - - + +++ + ++ + + + +

S1 +++ +++ ++ +++ ++ ++ ++ + + + + + - - - ++ + + + - - -

S5 +++ ++ ++ +++ +++ ++ ++ + + + - - - - - ++ + - - - - -

T1 ++ + - ++ - ++ - - - + - - - - - - - ++ + + - -

T17 +++ +++ ++ +++ ++ ++ ++ ++ ++ + - - - - - +++ - - - - - -

Z1 ++ ++ + +++ ++ +++ + - - - - - - - - + + ++ ++ + - -

Ctrl + ++ ++ +++ +++ ++ ++ ++ ++ + + - - - - +++ +++ +++ +++ +++ ++ ++

128
Appendix E

Table E1: The ethanol assay test results for the khadi samples. The experiment was done

in duplicates and repeated twice.

Sample Experiment 1 Experiment 2 Experiment 3 Experiment 4 Ethanol % Standard

(v/v) deviation

Letlhakane 1 6.664 6.236 6.486 6.759 6.536 0.230

Letlhakane 2 5.272 5.367 5.676 5.653 5.492 0.203

Letlhakane 3 5.772 5.831 5.902 5.855 5.840 0.054

Maun 1 8.639 8.425 8.818 8.628 8.628 0.161

Maun 2 4.236 3.987 3.558 3.891 3.918 0.281

Maun 3 0.762 0.940 0.738 0.678 0.779 0.113

Mmashoro 1 6.140 6.093 5.117 5.236 5.647 0.545

Mmashoro 2 7.354 7.497 6.652 6.724 7.057 0.431

Mmashoro 3 7.259 7.116 8.318 8.830 7.881 0.829

Palapye 1 8.068 8.068 8.366 8.235 8.184 0.144

Palapye 2 6.343 6.033 6.533 6.462 6.343 0.221

Palapye 3 7.783 7.973 6.807 6.973 7.384 0.580

Serowe 1 2.249 2.297 3.046 3.296 2.722 0.529

Serowe 2 1.416 1.416 1.380 1.595 1.452 0.097

Serowe 3 1.166 1.523 0.916 0.893 1.125 0.293

Tonota 1 4.403 4.320 4.915 4.831 4.617 0.299

Tonota 2 7.342 7.402 7.426 7.426 7.399 0.039

Tonota 3 4.284 3.903 3.820 3.879 3.972 0.211

129
Table E2: The pH of the khadi samples which was done in triplicates.

SAMPLE TRIAL 1 TRIAL 2 TRIAL 3 Average Standard

Deviation

Letlhakane 1 3.540 3.540 3.540 3.540 0.000

Letlhakane 2 3.620 3.620 3.620 3.620 0.000

Letlhakane 3 3.320 3.320 3.320 3.320 0.000

Maun 1 3.020 3.020 3.030 3.023 0.006

Maun 2 2.740 2.740 2.730 2.737 0.006

Maun 3 2.970 2.970 2.970 2.970 0.000

Mmashoro 1 3.340 3.340 3.340 3.340 0.000

Mmashoro 2 3.310 3.310 3.310 3.310 0.000

Mmashoro 3 3.370 3.370 3.370 3.370 0.000

Palapye 1 2.710 2.710 2.700 2.707 0.006

Palapye 2 2.800 2.800 2.790 2.797 0.006

Palapye 3 2.640 2.640 2.650 2.643 0.006

Serowe 1 2.810 2.810 2.820 2.813 0.006

Serowe 2 3.610 3.610 3.610 3.610 0.000

Serowe 3 3.460 3.460 3.460 3.460 0.006

Tonota 1 2.820 2.820 2.830 2.823 0.006

Tonota 2 2.690 2.690 2.690 2.823 0.000

130