1. Introduction
The sustainable production of traditional foods in sub-Saharan Africa offers a viable opportunity to fight increasing hunger and malnutrition [
1]. Cereals such as millet, sorghum, and maize are important sources of food in Africa [
2], and are predominantly cultivated for human nutrition, in particular, for children [
3,
4,
5]. The maize-based African fermented
kwete is a fermented beverage, which is traditionally produced by Luo communities, but now commercially available in many rural and urban areas in Uganda [
6,
7]. Kwete is a slightly alcoholic, with a thick consistency and a sweet–sour taste [
8]. Consumers use these beverages as social drinks, a source of energy, thirst quenchers, and weaning foods [
6,
8,
9]. However, cereals are highly susceptible to aflatoxin contamination. This could be attributed to their rich nutrient composition and relatively high humidity that favors fungal growth [
10,
11]. Aflatoxin contamination in cereals, such as maize, has been reported to be as high as 46 mg kg
−1 and 19 mg kg
−1, in Kenya and Uganda, respectively [
11]. These levels of aflatoxin contamination are of great concern considering that maize is used in all pre-primary, primary, and post-primary schools in Uganda for the preparation of breakfast (porridge) and lunch (posho or pap, which is a solid gelatinized product comprising of maize flour and water). The schools get the maize through parental in-kind contributions, direct procurement from the open markets and, to a small extent, from school gardening [
12]. None of these sources of maize are subject to quality control and could, therefore, be contaminated with aflatoxins.
There is an urgent need to decrease the risk of aflatoxins due to concerns over human health, food safety, and economic losses. Aflatoxins have created a lot of havoc, particularly in sub-Saharan Africa, where acute and mostly chronic aflatoxicosis has been reported. In 2004, approximately 317 cases of aflatoxicosis and 125 deaths were reported in Kenya [
13]. A pilot study in Uganda, which evaluated aflatoxin exposure in rural populations, reported that all 100 adults included in the study and 92 out of 96 children contained detectable levels of aflatoxin–albumin adducts, including five babies who were exclusively fed via breastfeeding [
14]. This could explain the high number of cases of liver cancer (estimated at 6.5 and 5.5 age-standardized incidence rate per 100,000 people annually for males and females, respectively) in Uganda [
15]. Furthermore, aflatoxins suppress the activity of the human immune system by significantly lowering the levels of perforin, perforin-expressing, and granzyme A-expressing CD8+ T cells. This results in impaired CD
8+ T cells which, in turn, affects cellular immunity against infectious diseases [
16,
17]. Aflatoxins also affect absorption of nutrients through alteration of intestinal integrity [
18], thus affecting child growth and development.
Therefore, the mitigation of aflatoxins in food is of great importance, and methods such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) have been developed to monitor their levels. However, these methods can only be operated in laboratories by well-trained personnel [
19,
20]. As a drive to reduce exposure to contaminated food, we developed an on-site detection method [
21] and validated this method for the analysis of maize flour from markets and households in Kampala [
22]. Using this innovative detection methodology, we found that the average aflatoxin contamination at household level was higher (22.2 ± 4.6 µg kg
−1) than at the markets (7.6 ± 2.3 µg kg
−1). About three out of four samples from households tested positive for aflatoxins and nearly half the samples contained higher levels than the East Africa acceptable limit of 10 µg kg
−1 [
23]. This is a strong indication that high levels of aflatoxins are consumed daily by the Ugandan population. A combinatory approach aimed at prevention of contamination, lowering the amount of aflatoxins in the food chain as well as decreased uptake after human ingestion, could be most effective in lowering the burden of aflatoxins in cereals.
Fermentation has been appreciated as means to bring down the concentration of aflatoxins in cereal-based foods [
24]. However, variations in product quality and safety associated with the undefined nature of starter cultures in traditional fermentations create a barrier for this approach. The application of defined starters with lactic acid bacteria could offer an easy and inexpensive method that could be adopted for detoxifying aflatoxins in food in a controlled and reproducible manner [
25,
26]. The potential of probiotic strains to reduce the risk of aflatoxins has been studied, both in vitro and in vivo [
27,
28]. Probiotics do not only mitigate aflatoxins but also convey other health benefits to consumers, such as the decrease of intensity and duration of diarrhea, which is a major disease burden, especially in developing countries [
29]; a promising candidate in this respect is the probiotic model strain
Lactobacillus rhamnosus GG [
30].
The probiotic
L. rhamnosus GG is now accessible in East Africa, under the name
L. rhamnosus yoba 2012, following the introduction of the concept of ‘generic probiotics’ [
31,
32]. The
L. rhamnosus yoba 2012 strain has been previously applied for the preparation of African traditional products like
uji (fermented maize),
mutandabota (fermented pulp of the baobab fruit and milk),
zomkom (fermented wheat), and the naturally fermented milk
lait caillé [
1,
31,
33]. Recently, Di Stefano et al. [
34] reported the growth parameters, organoleptic characteristics, and acceptability of fermented millet by use of
L. rhamnosus GR-1 and
S. thermophilus C106, which provided a good reference point for this study. The inclusion of probiotic starters in a product may affect the products’ sensory properties and, hence, acceptability [
35,
36,
37]. It is therefore not only essential to ensure growth of the probiotic, but to also compare sensory characteristics and consumer acceptability of the probiotic-enriched, traditional fermented products.
In this paper, we evaluated the ability of L. rhamnosus yoba 2012 and S. thermophilus C106 to propagate in kwete, a traditional maize-based drink. In addition, we studied survival of the probiotic during storage and compared consumer acceptability with commercial traditional products previously introduced on the market. We also monitored the effect of the probiotic starter culture on the levels of aflatoxins during fermentation, and confirmed the ability of the probiotic to bind aflatoxin B1. The results of this study demonstrate the potential of utilizing widely consumed locally available traditional foods as carriers for probiotics, which adds health benefits and improves product safety.
4. Discussion
Introduction of bacterial probiotic strains in traditional fermented foods can be used as a means to convey their health benefits [
41]. In this study, we used the probiotic model bacterium
L. rhamnosus GG, since there is a wealth of scientific evidence showing its beneficial effects in the prevention and treatment of gastrointestinal diseases, including rotavirus and
Clostridium difficile-associated diarrhea, and travelers’ and antibiotic-associated diarrhea (AAD) [
42,
43,
44,
45]. In addition, this strain is readily accessible in its generic form,
L. rhamnosus yoba 2012, packed in a lyophilized state together with
S. thermophilus C106 in a sachet as the yoba starter culture [
31].
The yoba starter culture bacteria successfully fermented the traditional maize-based food
kwete, as evident from the production of lactic acid shown by a decrease in pH and simultaneous increase in titratable acid. As required for microbiological safety and stability of lactic acid-fermented beverages [
46,
47,
48,
49], the observed pH values of probiotic fermented
kwete were ≤4.3, and the amount of titratable acid was at least 0.6% after 24 h of fermentation at 30 °C. It should be noted that in case of natural
kwete fermentations, it can take between 24 to 120 h to attain these pH and acidity values, while— in line with our findings—with defined starter cultures, these values are reached within 12 to 24 h of fermentation [
6]. The maximum acidity levels observed during storage of probiotic
kwete, of 0.7%, corresponded to the maximum levels of acid production previously observed with starters containing
L. rhamnosus GG for fermentation of maize porridge with added barley [
50].
In this study,
kwete was used as a substrate to enhance growth of the probiotic
L. rhamnosus yoba 2012, reaching a maximum of 1.0 × 10
9 cfu g
−1 after 24 h fermentation of
kwete at a temperature of 30 °C. These counts of colony forming units were similar to those reported for other traditional products serving as a substrate for the same starter culture, including
mutandabota (a dairy product containing baobab pulp),
uji (fermented maize and sorghum beverage), and
zomkom (a fermented sorghum beverage [
1,
31]. Maximum counts of
L. rhamnosus yoba 2012 in
kwete were also comparable to those reported for other starter cultures with lactic acid bacteria, such as
L. reuteri,
L. acidophilus (LA5 and 1748), and
L. rhamnosus GG in maize porridge [
50]. The ability of
L. rhamnosus yoba 2012 to grow in cereal bases, such as
kwete, is attributed to the availability of sugars such as glucose, fructose, and maltose from maize and millet/sorghum malt for
kwete [
8], in addition to free amino nitrogen from cereal malt [
51]. The traditional production of
kwete with an undefined mixture of yeasts,
Lactobacillus, and
Lactococcus species often results in poor product quality and short shelf life, requiring a consumption of
kwete within 24 h after production [
52]. In probiotic
kwete prepared by the use of the yoba starter culture, we did not identify any (harmful) coliforms, yeasts, and molds in the samples during 4 weeks of storage.
Different probiotic starters uniquely affect the flavor profile, sensorial properties and, ultimately, the acceptability of products in which they are introduced. For instance, mild acidity, relatively high amounts of acetaldehyde, and the presence of the human isolate
L. plantarum NCIMB 8826 correlated with higher acceptability scores of barley- and oat-based probiotic beverages [
53]. In another study,
L. rhamnosus LRB and
L. acidophilus PRO produced probiotic
mageu (a fermented maize beverage), whose sensory properties and acceptability scores were closer to that of the control than the product produced by
L. casei BGP1 and
L. paracasei BGP93 [
36]. Therefore, it is necessary to establish the effect of the addition of probiotics on the acceptability of traditional fermented foods. Benchmarking the new probiotic product with existing related traditional products helps in gauging the success of the probiotic product [
49]. This study shows that use of the yoba starter did not significantly affect the acceptability of
kwete. The acceptability of sensory characteristics was comparable to the commercial product on the market. The consumers took note of a difference in the taste, but appreciated the probiotic
kwete for its sweet and sour taste with a mean score of 6.8 ± 1.4 compared to 6.5 ± 1.6 of commercial brand. Therefore,
kwete produced using the yoba sachet culture can be readily accepted and frequently purchased by consumers, thus increasing accessibility of probiotics in Uganda.
For a product to be considered probiotic, it should contain a minimum of 10
6 cfu per mL or gram of the probiotic microorganisms at the time of consumption [
54]. Consuming 100–1000 mL per day of such a product provides the recommended daily dose (10
8–10
9 cfu), essential for conveying the health benefits of probiotics [
54,
55]. Probiotic
kwete contained a minimum of 4.0 × 10
8 cfu g
−1 of
L. rhamnosus yoba 2012 during four weeks of storage at 4 °C. Thus, a minimum daily consumption of 10 mL of probiotic
kwete per day would be more than sufficient to meet the recommended daily intake of probiotics. With respect to shelf stability, probiotic
kwete generally remained stable and acceptable during the entire study period of four weeks. Traditional
kwete is normally produced and consumed within 24 h [
56]. However, the use of yoba starter culture made the product stable for four weeks, thus improving its shelf life.
Detoxification of aflatoxins in food prior to consumption is a novel approach to curb their toxic effects. Several technologies have been employed to eliminate or reduce levels of aflatoxins in food, but only a handful are accepted for use and, as of yet, none offer 100% efficiency [
57]. The use of microorganisms to detoxify aflatoxins has been given more consideration [
25,
27]. In this study, yoba starter culture bacteria, which are being used extensively in Uganda, Kenya, and Tanzania to produce fermented milk, demonstrated an excellent ability to reduce aflatoxins, during fermentation of
kwete base, to non-detectable levels. There was a notable reduction in total concentrations containing all four major aflatoxins (B
1, B
2, G
1, and G
2) from 7.0 ng mL
−1 to non-detectable levels (
Figure 3). The detoxification of aflatoxins in
kwete could be the result of binding as well as of degradation, as binding alone would not reduce the toxin from the food substrate to non-detectable levels [
58]. We speculate that aflatoxin degradation is a specific property of our starter culture, as other studies reported less than 100% removal by
L. rhamnosus strains [
59,
60,
61]. However, under the experimental conditions used so far, we have not been able to confirm degradation of aflatoxins by pure cultures of bacterial strains in the yoba starter culture.
Our in vitro fluorescence experiments did confirm binding of aflatoxin B
1 to a cell suspension of
L. rhamnosus yoba 2012 at OD
600 of 0.5, which reduced the aflatoxin B
1 concentration of 1.0 µg/mL to 17%. Preliminary results indicated that the binding of aflatoxin B1 to
S. thermophilus C106 was less efficient, with a reduction of aflatoxin B
1 to 86% at the same cell density. Aflatoxin binding to lactic acid bacteria was previously suggested as a safe means to reduce the bioavailability and enhance excretion of the toxin from the body [
62,
63]. Although the mechanism of binding is still poorly understood, cell surface polysaccharide, peptidoglycans, and teichoic acids have been suggested as the binding sites [
59,
64,
65]. Here, we show that the yoba starter, including
L. rhamnosus yoba 2012 and
S. thermophilus C106, were able to remove 100% of 120 µg kg
−1 total aflatoxins spiked in the water-soluble fraction of
kwete, which is highly relevant considering the range of aflatoxin concentrations we previously found in maize flour in households in Uganda [
22].