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Article

Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs

by
Thandiwe Semumu
1,2,
Nerve Zhou
2,
Lebani Kebaneilwe
2,
Daniel Loeto
1 and
Thando Ndlovu
1,*
1
Department of Biological Sciences, Faculty of Science, University of Botswana, Private Bag UB, Gaborone 0022, Botswana
2
Department of Biological Sciences and Biotechnology, Faculty of Science, Botswana International University of Science and Technology, Private Bag 16, Palapye 10071, Botswana
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(8), 417; https://doi.org/10.3390/fermentation10080417
Submission received: 25 June 2024 / Revised: 2 August 2024 / Accepted: 8 August 2024 / Published: 11 August 2024
(This article belongs to the Special Issue Advances in Yeast Biotechnology from Genomics to Industry)
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Abstract

:
Sourdough is one of the oldest technologies employed by humans to leaven bread because of its ability to enhance the flavour and structure of bread using micro-organisms. However, there is a lack of comprehensive information in Botswana regarding the diversity of sourdough starters and the fermentative micro-organisms responsible for spontaneous fermentation. The present study aimed to explore the microbial species diversity of sourdoughs in Botswana and gain insight into the unique microbial communities involved in sourdough production. A total of nine samples were collected from different areas in Botswana. The microbial diversity in sourdoughs was characterized through the sequencing of amplicons of the 16S ribosomal DNA and internal transcribed spacer regions. In silico polymerase chain reaction–restriction fragment length polymorphism and phylogenetics were utilized to determine the genetic diversity among the isolates. The dominant yeast species identified were Saccharomyces cerevisiae, Wickerhamomyces anomamlus, Pichia kudriazverii and kazachstania humilis. Additionally, the presence of Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Liquorilactobacillus nageli and Bacillus cereus was also detected. It is worth noting that two species of acetic acid bacteria (AAB), namely Acetobacter pasteurianus and A. indonesiensis, were isolated, though in low levels, but the finding is significant in sourdough fermentation. The low occurrence of AAB (acetic acid bacteria) species observed in this study could be an important finding, as these bacteria are considered understudied, yet they are known to contribute significantly to the final product.

1. Introduction

Sourdough is one of the oldest technologies employed by humans to leaven bread. This technology can be traced back to ancient times (1500 BC). It is well known for enhancing the flavour and structure of bread [1]. In the past decade, there has been a shift towards the industrial production of wheat bread using monocultures of baker’s yeast [2]. However, there is a renewed interest in traditional sourdough bread making due to the growing demand for tastier and healthier food, particularly those that are spontaneously fermented [3]. Sourdough is made by mixing water with ground cereal, which promotes the growth of naturally occurring micro-organisms that spontaneously ferment the sugars in the flour, resulting in leavened dough. The technique involves backslopping the spontaneously fermenting water–flour mixture. This allows the naturally diverse microbiota to be maintained and shared amongst households, with the culture being refreshed in a new water–flour mixture within predetermined time intervals (days, months or even years). Sourdoughs are highly complex biological ecosystems due to the presence of micro-organisms from the flour, environment and other ingredients [4,5]. The sourdough microbiota consists of heterofermentative lactic acid bacteria, acetic acid bacteria and yeasts. The bacteria are responsible for the pleasant, sour taste of the end product, while the yeasts help by producing carbon dioxide, which leavens the dough [6]. The most commonly isolated bacterial species in sourdough belong to the genera Lactiplantibacillus, Pediococcus, Leuconostoc and Weissella, with Lactiplantibacillus strains being the most frequently observed [7]. However, acetic acid bacteria remain understudied. Despite limited documentation, Acetobacter strains commonly found in sourdough contribute to the fruity taste and unique aromas. The majority of yeasts found in sourdoughs are from the species Candida milleri, C. holmii, Saccharomyces exiguous and S. cerevisiae [8,9,10]. The diversity of the microbiota influences the quality of the end product. For thousands of years, humans have relied on sourdough starter microbial communities to make leavened bread and other sourdough-based products, such as Botswana’s traditional sorghum ferment known as Ting [11]. However, only a small fraction of the global biodiversity found in sourdough and other sourdough-based products has been studied and characterized [12].
The microbial ecology of sourdough fermentations has been studied and documented in many European countries [13,14]. However, there is limited information about the diversity of microbial communities in sourdough and the specific fermentative micro-organisms responsible for spontaneous fermentation in Africa. In Botswana, the production and consumption of sourdough have declined due to the increasing use of single strains of baker’s yeast in industrial bread production. Nevertheless, there are still a few bakers in Botswana who continue to make sourdough bread, primarily using type I sourdough. This type of sourdough is initiated by the microbial strains already present in the original dough (mother dough) and is maintained through well-established processes, similar to the production of Botswana’s traditional sorghum ferment, Ting. The present study aimed to explore the species diversity of traditional sourdoughs in Botswana. Sourdoughs in Botswana are made through spontaneous fermentation and consistently maintained through backslopping. This is a similar process used by other sourdough producers around the world. Botswana is characterized by a semi-arid to an arid environment, with a total of eight biomes, whose microbial diversity may contribute to the complexity of micro-organisms with the potential to influence sourdough quality. In this study, we investigated the species diversity of sourdoughs collected in different regions in Botswana. To obtain insight into the uniqueness of Botswana’s sourdoughs, we used molecular and bioinformatics tools to determine the identity and diversity of the isolates.

2. Materials and Methods

2.1. Sample Collection and Preparation

A total of nine sourdough samples were randomly collected from individuals who make and sell sourdough bread in various villages and cities across Botswana. The samples were obtained from villages (Ratholo, Mokatse and Tsabong) as well as towns (Maun, Francistown and Gaborone) that were known by the local community to have active sourdough producers (see Figure 1). The samples were collected aseptically using sterile 50 mL Falcon tubes and transported to the laboratory at temperatures ranging between 0 and 4 °C. Analyses of the samples was conducted within 72 h after collection. In order to compare microbial diversity, a commercial sample from the United Kingdom was used as a reference. The control yeast used in this study was a commercial baker’s yeast (Saccharomyces cerevisiae) product obtained from a local supermarket. The specific brand of baker’s yeast used was Anchor Instant Yeast.

2.2. Culture Media

Selective media were used to select for growth of micro-organisms, including yeast, LAB and AAB. Yeast peptone dextrose (YPD) agar (2% glucose, 0.5% yeast extract, 2% peptone and 1.5% agar, at a pH of 6.2) (Sigma-Alrich, St. Louis, MO, USA) [15] supplemented with antibiotics (100 µg/mL streptomycin and 100 µg/mL ampicillin), both obtained from Sigma-Aldrich, was used for selective enrichment for yeast. To isolate LAB, de Man, Rogosa and Sharpe agar (MRS) (Sigma-Alrich) [16], with a pH of 6.4 ± 0.2, was supplemented with 0.1 g/L cycloheximide, Frateur medium [17], which contained ethanol and calcium carbonate [0.05% D-glucose, 0.3% peptone, 0.5% yeast extract, 2% (v/v) ethanol, 1.5% CaCO3, 1.5% agar] was used for the isolation of AAB.

Isolation of Micro-Organisms from Sourdough Samples

The isolates from the sourdough starter samples were serially diluted using sterile de-ionized water. Then, 100 µL of each dilution was spread plated on modified YPD supplemented with antibiotics (100 µg/mL streptomycin and 100 µg/mL ampicillin) [15], de Man, Rogosa and Sharpe agar (MRS) (Sigma-Aldrich) supplemented with 0.1 g/L cycloheximide (Sigma-Aldrich) for LAB [16], and Frateur medium [17] for AAB. All microbial plating was performed in triplicate. The yeast and bacteria plates were incubated aerobically at 30 °C for 72 h and 37 °C for 24 h, respectively. Colonies with the desired morphological characteristics were selected from the plates with the lowest countable dilutions and then sub-cultured to obtain pure cultures. The cultures were stored in cryovials with 25% glycerol (Sigma-Alrich) at −80 °C until used for subsequent steps.

2.3. Molecular Identification of Sourdough Isolates

2.3.1. Genomic DNA Extraction

To extract genomic DNA from yeast isolates, a cell lysis solution containing 0.2 M lithium acetate (LiOAc) and a 1% solution of sodium dodecyl sulphate (SDS) (Sigma-Aldrich) was used as described by Lõoke, Kristjuhan [18]. Briefly, a sterile inoculating loop was used to resuspend a loopful of pure culture in 100 µL of the lysis solution in 2 mL micro-centrifuge tubes. The tubes were then incubated at 70 °C in a water bath (MRC Scientific instruments, London, UK) for 15 min. After incubation, the micro-centrifuge tubes were centrifuged using a Heraeus Pico 17 micro-centrifuge (Thermo Fischer Scientific, Dreieich, Germany) at 15,000× g for 2 min after which the supernatant was discarded. This was followed by DNA precipitation by adding 300 µL of 96% (v/v) ethanol. The suspension was briefly vortexed and centrifuged under the same conditions as before, and then the supernatant was discarded. The pellet was washed with 300 µL of 70% (v/v) ethanol to dissolve any excess salts and further centrifuged at 15 000× g for 3 min before discarding the supernatant. The DNA was dissolved in 100 µL of nuclease-free water. The solution was centrifuged for 1 min at 15,000× g to separate the cell debris from the DNA. The supernatant was then used as a DNA template for amplification in the polymerase chain reaction (PCR). The quality and quantity of the extracted DNA was determined using a Nanodrop One Spectrophotometer (Thermo Scientific, Waltham, MA, USA). For bacterial isolates, genomic DNA was extracted using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich), following the manufacturer’s instructions. The extracted DNA samples were then sent to Inqaba Biotechnological Industries (Pty) Ltd. (Pretoria, South Africa) for polymerase chain reaction (PCR) and sequencing.

2.3.2. Polymerase Chain Reaction and Sequencing

The identification of yeast isolates was carried out by sequencing the ITS1-5.8S-ITS4 rRNA gene fragment using universal PCR primers, namely ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), as described by White, Bruns [19]. The sequences were quality trimmed using SnapGene® Viewer software ver. 4.2.11 (GSL Biotech) (http://www.snapgene.com). Species identification was conducted using the BLAST nucleotide sequence analysis tool (http://www.ncbi.nlm.nih.gov/BLAST/). The optimal identity threshold to distinguish between species based on the internal transcribed spacer (ITS) was set at 98.41% following the method described by Vu, Groenewald [20]. Any species with a sequence identity lower than the threshold (98.41%) were denoted as “aff‘’ (Latin: affinis, meaning related to or neighbouring in taxonomy), indicating an uncertain identity that requires further taxonomic reviews to validate their identity. The identification of LAB and AAB was carried out by sequencing the 16S rDNA region using 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers [21]. The optimal identity threshold to distinguish between species using the 16S rDNA was set at 98.65% as described by Kim, Oh [22]. The sequences were edited and trimmed as above and were identified using the BLAST nucleotide sequence analysis tool (http://www.ncbi.nlm.nih.gov/BLAST/; accessed on 10 June and 31 July 2024). Subsequently, the sequences were submitted to the NCBI GenBank repository.

2.3.3. Phylogenetic Analysis of Sourdough Isolates

In order to determine the evolutionary relationships of the sourdough isolates (both yeasts and bacteria), a Molecular Evolutionary Genetic Analysis (MEGA X) ver.10.2.6 software for phylogenetics analysis was used [23]. In summary, the ITS1-5.8S-ITS4 and 16S sequences were aligned using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) tool and then trimmed to uniform sequence sizes using the MEGAX software ver.10.2.6. The sequences of type strains used for alignment and phylogenetic analysis were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/; accessed on 10 June and 31 July 2024). The aligned sequences were then used to construct a phylogenetic tree using the maximum likelihood method and the parameter model (Kimura 2-parameter models) [23], with 1000 bootstrap replications. For yeasts, Schizosaccharomyces pombe CBS 7264 was included as an outgroup, while Bradyrhizobium paxllaerii DSM 18454 was used for LAB and Gluconacetobacter liquefaciens NBRC 12388 was used for AAB.

2.4. In Silico PCR-RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism)

In silico PCR-RFLP was performed to determine the genetic diversity among yeasts or bacteria of the same species. This technique allowed for the genetic differentiation and identification of similarities for both the isolated yeasts and bacteria and their type strains obtained from GenBank. The SnapGene® viewer software ver.4.2.11 (http://www.snapgene.com; accessed on 10 June 2024) was used, and a 4% agarose gel was simulated using TBE buffer. The ITS1-5.8S-ITS4 sequences were first aligned using MEGAX software to trim and exclude overhangs. Then, they were digested simultaneously with three (3) restriction enzymes: Hae111, Hinfl and Cfol. The Biozym quantitas 25–500 bp was linearized and used as a molecular weight marker to estimate the weight of the restriction fragments. The same technique was used to investigate whether the Saccharomyces cerevisiae isolates from sourdough starters around Botswana are identical to the readily available commercial bakers yeast sold in retail stores. For the 16S rDNA gene fragments, Alu1, Hae111 and Hinf1 were used as restriction enzymes for LAB, while SspI, BsgI and KpnI were used for AAB. In order to identify the weight of the restriction fragments of bacteria, a 100 bp ladder was used as a molecular weight marker.

3. Results and Discussion

3.1. Microbial Communities Found in Traditional Sourdough in Botswana

The composition of microbial communities in traditional sourdough, including yeasts and bacteria (LAB and AAB), can greatly influence the fermentation process and the resulting bread [9,24]. A total of 42 microbial isolates were identified from the nine samples analyzed in this study. This included nineteen yeast isolates, nine lactic acid bacteria (LAB) isolates, three acetic acid bacteria (AAB) isolates and eleven other bacteria isolates (see Table 1 and Table 2). In comparison, the comparator sample (UK1) had two yeast isolates, one AAB isolate and two other bacteria isolates, resulting in a total of forty-seven microbial isolates with twenty-one yeasts, nine LAB, four AAB and thirteen other bacteria isolates across all samples. The majority (seven out of nine) of sourdough samples comprised of a single yeast species, while 55.5% (five out of nine) samples contained at least one LAB species, and 33% (three out of nine) had a single AAB species. The remaining 22% had two yeast species, each with varying numbers of LAB species, and one of these communities also had an AAB species. For detailed information, please refer to Supplementary Materials Tables S1–S3.
The study aimed to explore the diversity of yeasts and bacteria found in traditional sourdoughs in Botswana. The 21 isolated yeasts consisted of four genera and six species, namely Saccharomyces cerevisiae, Wickerhamomyces anomalus, Pichia kudriavzevii, P. membranifaciens, Kazachstania unispora and K. humilis (Table 1). Among the nine samples collected in Botswana, we identified a diverse range of yeast species, with S. cerevisiae being the most prevalent, present in 60% of the sourdough samples and was the only species in most of the samples (30%). Three samples did not contain S. cerevisiae, suggesting that it is not indispensable in sourdough production. In addition, we identified three genera of non-Saccharomyces yeasts associated with traditional sourdoughs in Botswana (Table 1). These genera included the three species, namely Kazachstania humilis, Wickerhamomyces anomalus and Pichia kudriavzevii [8,25,26], which have also been reported in previous sourdough studies [27,28,29]. However, we observed that some isolates had a very low similarity to the threshold, indicating that they might be novel strains denoted by “aff” (see Supplementary Table S1). In the Francistown sample (FJ2), we found two yeast genera represented by S. cerevisiae and W. anomalus. It is worth noting that W. anomalus was the only unique isolate in all other samples from different sampling sites. A similar result was observed in samples from Gaborone (GD1) and Mokatse (MN1), where only two yeast genera (S. cerevisiae and P. kudriavzerii) were found, while all other samples had only one genus. These sourdough isolates (Table 1) are believed to have the ability to withstand the stress conditions encountered during fermentation, including nutrient scarcity, as well as the effects of acidity, oxidation and thermal and osmotic stresses [8].
The co-occurrence of diverse LAB and AAB species is commonly associated with sourdough starters [12]. While yeasts are responsible for gas production to leaven the dough, bacteria play a role in enhancing the functional properties of sourdough by producing organic acids (lactic acid and acetic acid) that impart complex flavour profiles [12]. Most LAB strains are heterofermentative, meaning they can ferment diverse carbohydrates found in flour and produce volatile compounds. In addition, they can degrade gluten, contributing not only to the taste of the bread but also other health benefits [26]. A gluten-free diet is particularly important for individuals with celiac disease and other gluten-related disorders. It helps to ease digestion, mitigate adverse innate immune responses and reduce inflammation in these patients [30]. The current study presents a diverse array of LAB isolates, including L. plantarum, L. nageli and L.paracasei together with Bacillus and other bacteria isolates found in sourdoughs from various regions of Botswana. Notably, unique Bacillus species, Bacillus stratosphericus and Bacillus ginseng, were identified in a sourdough sample from Mokatse (MN1), a village not far from Gaborone (Table 2). B. stratosphericus is well known for being predominant in the stratosphere, but recent research [31] highlights its presence everywhere in the atmosphere. This is due to its ability to withstand unfavourable conditions, hence its growing interest in potential food biotechnology applications. Similarly, B. ginsengi is not commonly found in sourdough. Their presence, therefore, could be influenced by various environmental parameters such as temperature and pH, as well as pre-fermentation processes and handling practices, including the state of the sourdough medium and the maturity of the sourdough samples [32]. Bacillus licheniformis is also common in sourdough microbiota, which has been reported to contribute to the bread’s elasticity, volume and shelf-life. This is attributed to its maltogenic amylase and its ability to produce α-amylase, which is an anti-staling agent.
The AAB contribute to the fruity taste and unique aromas found in sourdough by converting alcohol, sugars and polynols into various organic acids, aldehydes and ketones [33,34]. This study presents only three (3) AAB strains isolated from sourdough samples of Botswana and one (1) AAB strain from the comparator sample (UK1). Across all the samples, only one genus, Acetobacter, was reported. Acetobacter pasteurianus and Acetobacter indonesiensis were isolated from Francistown (FJ1) and Gaborone (GA1 and GD1), whereas the comparator sample (UK1) had Acetobacter malorum. All these isolated species are known to be found in sourdough [28]. The presence, though at low occurrence, of AAB species could be a significant contribution of this work, considering that they are understudied and often absent from key studies of sourdough microbial diversity. Studies on AAB further explain that species require specialized culture conditions, and thus their absence could be based on cultivation biases [12].

3.2. Sourdoughs of Botswana Harbour Phylogenetically Diverse Yeasts

Genetic differences among yeast isolates were investigated using phylogenetics and in silico RFLP. In silico RFLP is important because it simulates restriction patterns generated by selected restriction enzymes, making it easier to compare different microbial isolates. Phylogenetic analysis and in silico RFLP-PCR indicate that there could be genetic differences between the same isolated species of S. cerevisiae and those of P. kudriavzevii (Figure 2). Samples containing only non-Saccharomyces yeast species were observed, and these include UK1 (K. humilis), ME1 (K. unispora), GT1 (P. kudriavzevii) and GA1 (P. kudriavzevii). These isolates are commonly found in sourdough, as reported in various studies [27,28,35]. Furthermore, some samples exhibited co-occurrences, where more than one yeast species were present in a sample. These were FJ2 (S. cerevisiae and W. anomalus), GD1 (S. cerevisiae and P. kudriavzevii) and MN1 (S. cerevisiae and P. membranifaciens). All of these yeast species, including the non-conventional yeasts, are known to be involved in spontaneous fermentations of sourdough [14]. However, based on the 98.41% ITS sequence identity lower threshold established by Vu, Groenewald [20], and since they are denoted as “aff” in Supplementary Materials Table S1, this indicates that there is a possibility of novel yeast strains isolated from traditional sourdough samples in Botswana (see Supplementary Materials Tables S5–S7 for more information).
The S. cerevisiae strains were compared to commercial baker’s yeast as well as type strains. Interestingly, the strains isolated in the present study showed significant genetic differentiation from both commercial and type strains as they formed three major clades, indicating high genetic diversity among Botswana traditional sourdough starters. These differences are further supported by the in silico gel results, as the same restriction digests of the same species resulted in different fragment patterns (Figure 2). P. kudriavzevii strains formed three distinct clades, which separated isolates from the same community (GD1-A2 and GD1-A3). Strains with the same banding pattern, indicating high similarity, are indicated by brown borders in Figure 2. The brown borders in Figure 2 indicate the high similarity between K. unispora ME1-A2 and its type strain K. unispora CBS 396, which was notably distinct from K. unispora ME1-A1 from the same sourdough sample. Proximal clustering of the isolates with their type strain indicates their similarity and the accuracy of the molecular identification.
This research examines the microbial communities found in sourdough starters from Botswana. The presence of S. cerevisiae strains in samples from locations separated by significant geographic distance suggests that there is no correlation between species distribution and local geographical distance. This widespread distribution of S. cerevisiae species locally could be attributed to human-mediated dispersal [38,39]. Research conducted by Landis, Oliverio [12] in North America, Australia and Europe showed that geographic location has little influence on the microbial diversity of the starter cultures. However, the absence of S. cerevisiae yeast in the comparator sample (UK1) compared to the local Botswana samples could suggest a possible correlation between the species distribution and the geographical distance between the locations. This discrepancy may also be due to the selection of micro-organisms for a sourdough starter, rather than relying solely on spontaneous fermentation. Several studies support the findings of this study, indicating that local geographical distances have a limited role in shaping microbial communities [40]. There is a need for extensive studies that cover global geographical distances, including understudied countries such as Botswana in order to confirm the impact of intercontinental distances on the microbial communities.
The genetic diversity observed in S. cerevisiae strains of the same species can be attributed to various factors, such as flour type, fermentation temperatures and hydration preferences. This diversity is evident from the close clustering of strains within the same microbial community (Figure 2). The FJ and MN isolates show how all these factors contribute to the overall diversity. On the contrary, strains of the non-conventional yeast, P. kudriavzevii, from the same microbial community (GD), formed separate clades. While this may not be enough to nullify the influence of geography, it could indicate how processing practices, such as spontaneous fermentations, shape the microbial community. The presence of diverse non-conventional yeasts suggests that local sourdough producers in Botswana may rely on spontaneous fermentation rather than commercial yeasts inoculation. This assumption is supported by the absence of commercially available non-conventional yeasts in Botswana. The use of the backslopping technique could be responsible for increasing the frequency of non-conventional yeasts to detectable levels and their ability to withstand stresses associated with baking.

3.3. Sourdoughs of Botswana Harbour Phylogenetically Diverse LAB

The LAB species clustered along with their type strains, indicating a close phylogenetic relationship. In silico PCR-RFLP and phylogenetic analysis allowed us to demonstrate the genetic relationships among the LAB isolates relative to their type strains. The similarities are shown by purple borders (Figure 3). However, there were distinctions among strains of the same species, as observed from cladal differences. In some cases, the in silico gel showed differences that were not visible on the phylogenetic tree (Figure 3). The current study presents the genetic similarities and differences in LAB species belonging to three (3) genera, namely Lactiplantibacillus, Liquorilactobacillus and Lacticaseibacillus together with other bacteria isolated alongside LAB. A few more bacteria species were found in more than one sample, namely Lacticaseibacillus paracasei (FJ2 and GA1), Lactiplantibacillus plantarum (GD1 and ME1) and Liquorilactobacillus nageli (RK1 and ME1) (see Supplementary Materials Tables S8–S10 for more information).
The results of the phylogenetic analysis show the evolutionary relationship of microbial communities found in sourdough samples. These findings are supported by the in silico RFLP-PCR gel results, which is a method used for identification and differentiation purposes. In silico RFLP is a method that is used to compare and show relations among isolates by simulating a gel and using specific restriction enzymes to excise sequences at specific points. Therefore, the similarities and differences in these excision points are then compared to demonstrate the relatedness between the isolates. The phylogenetic tree clearly displays the differences among Lacticaseibacillus paracasei, which were obtained from different samples. These differences are further confirmed by the fragment sizes observed in the in silico RFLP-PCR gel. Similarly, the tree and in silico gel fragment sizes demonstrate the similarity of Liquorilactobacillus nagelii strains from different samples. Lactiplantibacillus plantarum had four strains from the same sample and one from a different sample. The phylogenetic analysis reveals clear differences among the four strains from the same sample. GD1-B4 stands out from the rest, while the remaining three strains form two distinct clades. These differences are also confirmed by the differences in the fragment sizes. Among the Bacillus and other bacteria analyzed, three B. cereus isolates from distinct samples clustered closely on the phylogenetic tree but exhibited varying fragment sizes in the in silico gel. Previous studies have identified this group of bacteria (Group I), including Bacillus species, as constituents of sourdough. These strains are recognized for their contributions to enhanced bread volume, elasticity and extended shelf life [41].
Among the bacterial species, there was limited diversity observed among strains of the same species. The only notable case of high diversity was found in the strains of L. plantarum (Group II), which is a very common species associated with sourdough. When compared to the frequently isolated strains of B. cereus, L. plantarum shows a higher genetic diversity (see Figure 3). This high intra-species diversity suggests that sourdough cultures serve as a natural reservoir for L. plantarum strains and may be a good strategy for eliminating microbial competition and potential contamination. Therefore, this could be a good LAB to include in a commercial sourdough starter.

3.4. Sourdoughs of Botswana Harbour Diverse AAB

The AAB species clustered along with their type strains, indicating a close phylogenetic relationship. Out of the nine (9) samples, only three (3) isolates from three (3) samples were identified as AAB species. Acetobacter pasteurianus was the most frequently isolated species, as it was found in two samples (FJ1 and GA1). This study presents the genetic differences and similarities of the AAB isolated species and their LAB type strains (See Supplementary Materials Tables S11–S13 for more information). There are only minor genetic differences among the AAB, which may be due to the small sample size (see Figure 4). Therefore, the sample size may be too small to draw a definitive conclusion.
Among the AAB species, two strains belonging to the same species, that is, A. pasteurianus, were identified. These two strains were from different samples. One strain was obtained from sample FJ1, while the other one came from the sample (GA1). The in silico fragment sizes on the gel revealed a minor distinction between the two strains that was not shown by the phylogenetic tree. All other isolated AAB species exhibited differences with their type material. Sample GD1 contained Acetobacter indonesiensis, which was present in the same community as Lactiplantibacillus plantarum (LAB species), and Saccharomyces cerevisiae and Pichia kudriavzevii (yeast species). In the comparator sample UK1, Acetobacter malorum was detected in the same community as Bacillus thuringiensis and Bacillus licheniformis (LAB) and K. humilis (yeast). AAB is known to be responsible for the variations in flavours and aroma profiles in fermented products [42]. AAB possesses a distinctive fermentation capability known as “oxidative fermentation.” This process involves the incomplete oxidation of substrates, where membrane-bound dehydrogenases catalyze the rapid and partial oxidation of various carbohydrates, particularly sugars and alcohols, resulting in the release of oxidized products.
The presence of AAB in sourdough could potentially induce oxidative stress on LAB and yeast during fermentation process. This can lead to the production of diverse volatile compounds in sourdough [43]. Therefore, incorporating AAB with yeast and LAB in sourdough production may enhance the flavour profile of the final sourdough bread. Although bread is widely consumed worldwide, it is prone to spoilage due to moulds [44]. The antifungal properties of LAB and AAB are linked to the synergistic action of their secreted metabolites, such as acetic, propionic and caproic acids. Among these, caproic acid has shown the highest efficacy against moulds [45]. The presence of lactic, acetic and phenyllactic acids in sourdough has the potential to reduce bread spoilage attributed to moulds.
Future research on microbial co-occurrence analysis and validation of sourdough communities is important for demonstrating how microbial ecosystems can help identify the processes that structure microbiomes. The sourdough microbial consortium derived from the identified isolates has the potential for the development of novel baked goods in Botswana to cater to today’s diversified market. However, this requires more work to determine the specific applications and functional characteristics of these identified strains. Future experimental approaches that manipulate additional aspects of sourdough, such as the nature of sourdough interactions for the stable community, genomic diversity and evolutionary dynamics, will further enhance our understanding of the mechanisms of microbiome assembly in ancient fermented food. While previous studies may have underestimated the abundance of acetic acid bacteria (AAB) due to the limited use of selective media for AAB and a lack of metagenomic approaches, our findings suggest that the relative abundance of AAB should be regarded as a crucial factor in predicting the functional attributes of sourdough.

4. Conclusions

This work presents the extensive microbial diversity found in traditional sourdough starters from Botswana. The complex composition of yeast and bacterial communities in these sourdoughs was elucidated through exhaustive isolation and identification, revealing a tapestry of species that contributes to the fermentation process. An increasing number of studies have shown the possibilities for creating novel bakery products beyond bread, such as sourdough cookies, waffles, pancakes, tortillas, croissants and pizza, to meet today’s diversified market. From this study, we conclude that S. cerevisiae is a dominant yeast species, along with a diverse array of non-Saccharomyces yeasts, including K. humilis, W. anomalus and P. kudriavzerii. Additionally, our research underscores the phylogenetic diversity that exists among S. cerevisiae strains, indicating novel strains and local adaptation within the Botswana sourdough microbiota. This study also notes the presence of LAB and AAB, each of which makes a distinct contribution to the sourdough fermentation process. The co-occurrence of yeasts, LAB and AAB indicates that sourdough communities are characterized by complex ecological interactions. In the future, it is important to functionally characterize the sourdough microbial communities of yeast, LAB and AAB, and explore their potential applications to design robust sourdough consortia for the desired product outcomes. Further studies are needed to assess the functionality of these mixed microbial consortia, such as their fermentative capabilities, stress tolerance abilities and resulting aromatic profiles in order to develop consistent and high-quality starter cultures. This may help enhance the sensory, nutritional and shelf-life properties of sourdough bread, as well as contribute to the preservation of culinary traditions and biodiversity by utilizing the extensive microbial diversity found in traditional sourdough starters.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation10080417/s1, Table S1. Identity of yeast isolates from traditional sourdoughs around Botswana using the ITS region. All sequences were last blasted on the 31 July 2024; Table S2. Identity of lactic acid bacteria from traditional sourdoughs around Botswana using 16S region. All sequences were last blasted on the 31 July 2024; Table S3. Identity of Bacillus and other bacteria from traditional sourdoughs around Botswana using 16S region. All sequences were last blasted on the 31 July 2024; Table S4. Identity of Acetic acid bacteria from traditional sourdough around Botswana. All sequences were last blasted on the 10th of June 2024; Table S5. Yeast isolates and sequences used for phylogenetic analysis and PCR-RFLP; Table S6. Sequences for type strains/materials used for phylogenetic analysis; Table S7. 26S rDNA region of isolated yeasts and their excision regions using three restriction endonucleases (HaeIII, HinfI and GluI) for PCR-RFLP; Table S8. Identity and sequences for lactic acid bacteria isolates used for phylogenetic analysis and PCR-RFLP; Table S9. Sequences for type strains/material used for PCR-RFLP of 16S; Table S10. 16S rDNA region of isolated lactic acid bacteria and their excision regions using three restriction endonucleases (HaeIII, HinfI and GluI) for PCR-RFLP; Table S11. Identity and sequences for acetic acid bacteria isolates used for phylogenetic analysis and PCR-RFLP; Table S12. Sequences for type strains/material used for PCR-RFLP of the 16S rDNA for acetic acid bacteria; Table S13. 16S rDNA region of isolated acetic acid bacteria and their excision regions using three restriction endonucleases (SSPI, BsgI and KpnI) for PCR-RFLP.

Author Contributions

Conceptualization, T.S., T.N., L.K., D.L. and N.Z.; methodology, T.S., T.N., L.K., D.L. and N.Z.; investigation, T.S.; resources, T.N., L.K., D.L. and N.Z.; writing—original draft preparation, T.S.; writing—review and editing, T.N., L.K., D.L. and N.Z.; supervision, T.N., L.K., D.L. and N.Z.; project administration, T.N.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Office of Research and Development, University of Botswana through Research Grant No. UBR/RES/ROUND 17.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and supplementary material. Further inquiries can be directed to the corresponding author. The nucleic acid sequences raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We extend our gratitude to all the sourdough producers who contributed to this work by supplying sourdough starter samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Map of Botswana showing sourdough sampling areas. The study area was made up of locations in the southern (Gaborone, Mokatse and Tsabong), central (Ratholo), northwest (Maun) and northern (Francistown) parts of Botswana where the sourdough samples were collected.
Figure 1. Map of Botswana showing sourdough sampling areas. The study area was made up of locations in the southern (Gaborone, Mokatse and Tsabong), central (Ratholo), northwest (Maun) and northern (Francistown) parts of Botswana where the sourdough samples were collected.
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Figure 2. Molecular Phylogenetic analysis and PCR-RFLP for yeast isolates from traditional sourdough. The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA 7 [37]. Red represents type strains for species isolated, black represents isolates being investigated, green represents other S. cerevisiae strains in the market and purple represents the outgroup. Numbers above branches are bootstrap values and GeneBank and accession numbers are given in parentheses. In silico RFLP shows restriction fragment patterns of yeasts obtained using restriction enzymes Cof1, Hae111 and HinfI. The brown boxes show restriction fragment patterns of similar isolates.
Figure 2. Molecular Phylogenetic analysis and PCR-RFLP for yeast isolates from traditional sourdough. The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA 7 [37]. Red represents type strains for species isolated, black represents isolates being investigated, green represents other S. cerevisiae strains in the market and purple represents the outgroup. Numbers above branches are bootstrap values and GeneBank and accession numbers are given in parentheses. In silico RFLP shows restriction fragment patterns of yeasts obtained using restriction enzymes Cof1, Hae111 and HinfI. The brown boxes show restriction fragment patterns of similar isolates.
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Figure 3. The Molecular Phylogenetic analysis by Maximum Likelihood method and PCR-RFLP for Bacillus and other bacteria (Group I) together with lactic acid bacteria (Group II) from traditional sourdough. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA7 [37]. Red = type strains for species isolated, black = isolates being investigated, purple = outgroup. Numbers above branches are bootstrap values and GenBank and accession numbers are given in parenthesis. Restriction fragment patterns of LAB, bacillus and other bacteria obtained using restriction enzymes Alu1, Hae111 and HinfI. Purple borders show similarities between isolates from closely related isolates and their type strains, whereas red borders show genetic similarities of isolates to each other.
Figure 3. The Molecular Phylogenetic analysis by Maximum Likelihood method and PCR-RFLP for Bacillus and other bacteria (Group I) together with lactic acid bacteria (Group II) from traditional sourdough. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA7 [37]. Red = type strains for species isolated, black = isolates being investigated, purple = outgroup. Numbers above branches are bootstrap values and GenBank and accession numbers are given in parenthesis. Restriction fragment patterns of LAB, bacillus and other bacteria obtained using restriction enzymes Alu1, Hae111 and HinfI. Purple borders show similarities between isolates from closely related isolates and their type strains, whereas red borders show genetic similarities of isolates to each other.
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Figure 4. The Molecular Phylogenetic analysis by Maximum Likelihood method and PCR-RFLP for acetic acid bacteria isolates from traditional sourdough. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA7 [37]. Red = type strains for species isolated, black = isolates being investigated, purple = outgroup. The numbers above branches are bootstrap values and GenBank and accession numbers are given in parentheses. Restriction fragment patterns of AAB were obtained using restriction enzymes SspI, BsgI and KpnI. Red borders highlight similarities of isolates from different sources/sampling sites to their type strain.
Figure 4. The Molecular Phylogenetic analysis by Maximum Likelihood method and PCR-RFLP for acetic acid bacteria isolates from traditional sourdough. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura–Nei model [36]. Evolutionary analyses were conducted in MEGA7 [37]. Red = type strains for species isolated, black = isolates being investigated, purple = outgroup. The numbers above branches are bootstrap values and GenBank and accession numbers are given in parentheses. Restriction fragment patterns of AAB were obtained using restriction enzymes SspI, BsgI and KpnI. Red borders highlight similarities of isolates from different sources/sampling sites to their type strain.
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Table 1. Species identification of isolates from Botswana’s traditional sourdough starters.
Table 1. Species identification of isolates from Botswana’s traditional sourdough starters.
Sample NameYeast Isolate CodeScientific NameLAB Isolate CodeScientific NameAAB Isolate CodeScientific Name
FJ1FJ1-A1Saccharomyces cerevisiaeNone FJ1-C1Acetobacter pasteurianus
FJ2FJ2-A1Saccharomyces cerevisiae
FJ2-A2Wickerhamomyces anomalus None
FJ2-A3Saccharomyces cerevisiaeFJ2-B3Lacticaseibacillus paracasei
GA1GA1-A1Pichia kudriavzeriiGA1-B1Lacticaseibacillus paracaseiGA1-C1Acetobacter pasteurianus
GD1GD1-A1Saccharomyces cerevisiaeGD1-B1Lactiplantibacillus plantarumGD1-C1Acetobacter indonesiensis
GD1-A2Pichia kudriavzeriiGD1-B2Lactiplantibacillus plantarum
GD1-A3Pichia kudriavzeriiGD1-B3Lactiplantibacillus plantarum
GD1-A4Saccharomyces cerevisiaeGD1-B4Lactiplantibacillus plantarum
RK1RK1-A1Saccharomyces cerevisiae
RK1-A2Saccharomyces cerevisiae None
RK1-B4Liquorilactobacillus nageli
MN1MN1-A1Saccharomyces cerevisiae
MN1-A2Pichia membranifaciensNone None
MN1-A3Saccharomyces cerevisiae
GT1GT1-A1Pichia kudriavzeriiNone None
TB1TB1-A1Saccharomyces cerevisiaeNone None
TB1-A2Saccharomyces cerevisiae
ME1ME1-A1Kazachstania unisporaME1-B1Lactiplantibacillus plantarumNone
ME1-A2Kazachstania unisporaME1-B2Liquorilactobacillus nageli
UK1UK1-A1Kazachstania humilisNone UK1-C1Acetobacter malorum
UK1-A2Kazachstania humilis
Table 2. Species identification of Bacillus and other bacteria isolates from Botswana’s traditional sourdough starters.
Table 2. Species identification of Bacillus and other bacteria isolates from Botswana’s traditional sourdough starters.
Sample NameBacillus and Other Bacteria CodesScientific Name
FJ1FJ1-B1Bacillus pumilis
FJ2FJ2-B1Bacillus zhangzhouensis
FJ2-B2Bacillus cereus
GA1None
GA1-B2Bacillus cereus
GD1None
RK1RK1-B1Bacillus carboniphilus
RK1-B2Bacillus siamensis
RK1-B3Bacillus cereus
MN1MN1-B1Bacillus stratosphericus
MN1-B2Bhargavaea ginsengi
GT1GT1-B1Bacillus cereus
TB1TB1-B1Lysinibacillus halotolerans
ME1None
UK1UK1-B1Bacillus thuringiensis
UK1-B2Bacillus licheniformis
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Semumu, T.; Zhou, N.; Kebaneilwe, L.; Loeto, D.; Ndlovu, T. Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs. Fermentation 2024, 10, 417. https://doi.org/10.3390/fermentation10080417

AMA Style

Semumu T, Zhou N, Kebaneilwe L, Loeto D, Ndlovu T. Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs. Fermentation. 2024; 10(8):417. https://doi.org/10.3390/fermentation10080417

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Semumu, Thandiwe, Nerve Zhou, Lebani Kebaneilwe, Daniel Loeto, and Thando Ndlovu. 2024. "Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs" Fermentation 10, no. 8: 417. https://doi.org/10.3390/fermentation10080417

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Semumu, T., Zhou, N., Kebaneilwe, L., Loeto, D., & Ndlovu, T. (2024). Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs. Fermentation, 10(8), 417. https://doi.org/10.3390/fermentation10080417

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