Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages

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1 MiniReview Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages Lene Jespersen Department of Dairy and Food Science, Food Microbiology, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 1958 Frederiksberg C, Denmark Abstract Received 31 May 2002;received in revised form 30 August 2002;accepted 6 September 2002 First published online 28 October 2002 Indigenous fermented foods and beverages play a major role in the diet of African people. The predominant yeast species seen is Saccharomyces cerevisiae, involved in basically three groups of indigenous fermented products: non-alcoholic starchy foods, alcoholic beverages and fermented milk. These products are to a great extent made by spontaneous fermentation and consequently S. cerevisiae often coexists with other microorganisms even though a microbiological succession usually takes place both between and within species. The functions of S. cerevisiae are mainly related to formation of alcohols and other aroma compounds, but stimulation of e.g. lactic acid bacteria, improvement of nutritional value, probiotic effects, inhibition of undesired microorganisms and production of tissue-degrading enzymes may also be observed. Several different isolates of S. cerevisiae have been shown to be involved in the fermentations and some of the isolates show pheno- and genotypic characteristics that deviate from those normally recognised for S. cerevisiae. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Saccharomyces cerevisiae;indigenous fermented food and beverage;africa;fermented maize;sorghum beer 1. Introduction * Tel.: ;Fax: address: lj@kvl.dk (L. Jespersen). FEMS Yeast Research 3 (2003) 191^200 Fermented foods and beverages play a predominant role in the diet of African people. Most often these foods and beverages are produced at household level or at small industrial scale and are consequently often of varying quality and stability [1^3]. Cereals, legumes and tuber roots are the major raw materials in Africa, but other raw materials such as milk, sh and meat may also be fermented [4]. The numbers of African indigenous fermented foods and beverages are many and their production is often not described in detail. Depending on country and even local region various names may be given to the same product or to products that are basically similar but are produced with slight variations. Besides the bene cial e ects often mentioned for fermented foods and beverages, such as improvement of avour and texture, prolonged shelf-life, other e ects are of special importance when it comes to production of foods and beverages in developing countries. These e ects include reduced loss of raw materials, reduced cooking time, improvement of protein quality and carbohydrate digestibility, improved bio-availability of micronutrients and elimination of toxic and anti-nutritional factors such as cyanogenic glycosides [1,2,5^11]. Also the probiotic effects and the reduced level of pathogenic bacteria seen in fermented foods and beverages are especially important when it comes to developing countries where fermented foods have been reported to reduce the severity, duration and morbidity of diarrhoea [12^16]. Yeasts have been reported to be involved in several di erent types of indigenous fermented foods and beverages [3,17^23]. Despite this, the role of yeasts in these products is often poorly investigated. An overview of possible functions of yeasts in African indigenous fermented foods and beverages is given in Table 1. The yeast species most often reported in African indigenous fermented foods and beverages is Saccharomyces cerevisiae. In the following an overview of the contribution of this species / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi: /s (02)00185-x

2 192 L. Jespersen / FEMS Yeast Research 3(2003) 191^200 to the production of African fermented products is given with special emphasis on its occurrence, functionality, biodiversity and taxonomic characteristics. 2. The occurrence of S. cerevisiae in African indigenous fermented foods and beverages 2.1. Occurrence and origin of yeast strains S. cerevisiae has been isolated from various types of African indigenous fermented foods and beverages as indicated in Table 2. The type of fermented products where S. cerevisiae has been reported to occur can basically be divided into three groups: fermented non-alcoholic starchy foods, alcoholic beverages and fermented milk. Most of these products are produced by spontaneous fermentation or by back-slopping, i.e. inoculation with a part of a previous fermentation, as in the fermentation of cassava for agbelima production [7]. For spontaneous fermentations yeasts have been reported to originate from raw materials and process equipment [24]. The use of puri ed starter cultures in the production of indigenous fermented foods and beverages has been reported in a number of publications [10,25^29] but their use in practice for industrial fermentation of indigenous products appears to be very limited. An exception is the production of sorghum beer where brewing cultures of ale yeasts (S. cerevisiae) originally used for production of malt beer have been introduced [30] Fermented non-alcoholic starchy foods Some of the most popular non-alcoholic starchy foods in West Africa are based on fermented maize dough that is used to prepare a variety of staple foods in among others Ghana, Nigeria, Benin and Togo, where they constitute a large proportion of the daily food intake. The traditional maize dough is spontaneously fermented and characterised by a moisture content of 50% (w/w) and a nal ph of 3.7. The production of fermented maize dough includes washing and steeping (24 h) of maize kernels, milling, addition of water, formation of dough and fermentation (48^72 h). The microbiology of fermented maize dough used for production of kenkey has been investigated in detail [24,31]. A mixed ora comprising Candida spp., Saccharomyces spp., Trichosporon spp., Kluyveromyces spp. and Debaryomyces spp. has been isolated from the raw maize, during steeping and early phases of fermentation. After 24^48 h of fermentation S. cerevisiae was dominating with counts exceeding 10 6 cfu g 31. The number of Candida krusei was found to increase during the fermentation and after 72 h C. krusei was the dominant yeast species. The ph of the fresh dough was 5.7, falling to approx. 3.7 after 24 h of fermentation, and the microbial succession for C. krusei at the advanced stage of fermentation is likely to be due to this species greater tolerance of high levels of organic acids [24]. In Benin, S. cerevisiae and Lactobacillus spp. are reported to be the dominant microorganisms in the production of mawe, a porridge made from dehulled and partially germinated white maize corn [21]. S. cerevisiae has also been reported to be involved in fermentation of ogi, another acid-fermented food based on maize, sorghum or millet that is used as a traditional weaning food all over West Africa, but is also eaten by adults [1,2,9,29]. Examples of other acid-fermented foods are uji, a sour cereal gruel made from maize or mixtures of maize, sorghum and millet in East Africa, and mahewu, a beverage or a porridge made from sour maize meal eaten in South Africa and Zimbabwe [9,32]. Some acid-leavened breads and pancakes are produced from fermented cereals such as kisra, a fermented sorghum bread with a very sour taste produced in Sudan [9]. The predominant microorganisms involved in production of kisra have been reported to be S. cerevisiae, Lactobacillus spp. and Acetobacter spp. [4]. A fermented and baked rice product named masa is produced in Nigeria and apparently S. cerevisiae and Lactobacillus spp. are the predominant microorganisms involved in the fermentation [33] Alcoholic beverages The most studied African alcoholic beverages are beers produced from guinea corn (Sorghum vulgare). In sub-saharan Africa sorghum beer is known under a variety of names such as pito in Ghana and Nigeria and dolo in Burkina Faso and Togo [30,34,35]. Burukutu and otika are other sorghum-based beverages produced in Nigeria and surrounding countries [1,2,9]. The brewing of indigenous sorghum beer involves malting, souring, boiling, mashing, straining and alcoholic fermentation [30]. Indigenous fermented beer produced from sorghum deviates from traditionally produced malt beer in several ways. Prior to boiling and mashing the wort is spontaneously fermented by lactic acid bacteria that give the nal beer a quite acid taste. The beer has a fairly thick consistency due to a large amount of solids (5^7%) and the alcohol content is rather low (2^3% v/v). The beer is consumed in an actively fermenting state and therefore has a quite short shelf-life [35]. Due to its low alcohol content and the large quantity of suspended solids, many consumers consider these indigenous fermented sorghum beers to be more a food than a beverage [30]. The microbiology of pito from Ghana and dolo in Burkina Faso has been investigated [35]. Samples of dried yeasts harvested from previous brews and used as inoculum in beer fermentation were found to contain 0.2^7.6U10 7 cells g 31. When the yeasts were identi ed, 99% of the isolates showed macro- and micromorphological characteristics typical of S. cerevisiae whereas 1% was identi ed as Candida kefyr. The domi-

3 L. Jespersen / FEMS Yeast Research 3(2003) 191^ Table 1 Possible functions of yeasts in African indigenous fermented foods and beverages Fermentation of carbohydrates (formation of alcohols, etc.) Production of aroma compounds (esters, alcohols, organic acids, carbonyls, etc.) Stimulation of lactic acid bacteria providing essential metabolites Inhibition of mycotoxin-producing moulds (nutrient competition, toxic compounds, etc.). Degradation of mycotoxins Degradation of cyanogenic glucosides (linamarase activity) Production of tissue-degrading enzymes (cellulases and pectinases) Probiotic properties nance of S. cerevisiae in the fermentation of pito and other indigenous fermented sorghum beers seems to be a general observation. However, the occurrence of other yeast species seems to vary. S. cerevisiae was found in a number of Nigerian cereal-based beverages including pito, burukutu and sekete [36]. Further, S. cerevisiae accounted for 55^90% of the yeast population in samples from ve out of six di erent production sites in Togo and Burkina Faso, whereas C. krusei was dominating (70%) in the sixth sample [34]. A number of yeast species was found to be involved in the fermentation of pito from various locations in Accra, Ghana [28]. S. cerevisiae was the dominant species [37] followed by Candida tropicalis, Kloeckera apiculata, Hansenula anomala (now recognised as Pichia anomala [38]), Torulaspora delbrueckii, Schizosaccharomyces pombe and Kluyveromyces africanus. The above-mentioned investigations imply that the composition of the yeast population responsible for the fermentation of African sorghum beer may vary, depending on regional area and local deviations in the production including the use of di erent types of ingredients. Also remarkable variations in biochemical characteristics have been found among different sorghum cultivars that could a ect the amount and Table 2 The occurrence of S. cerevisiae in some African indigenous fermented foods and beverages the quality of available substrates and thus in uence the micro ora [39,40]. Ka r beer, the traditional beverage of the Bantu people of Southern Africa, and doro, brewed in Zimbabwe and Botswana, are perhaps the only indigenous fermented beverages produced by modern industrial processes [1,30,32]. Ka r beer is made from malted sorghum and unmalted maize. Sometimes millet is used in place of these. The beer is opaque with a yeasty odour and a fruity avour and an average alcohol content of 3.2% (v/v). Wort souring is by lactic acid bacteria and ale yeast, S. cerevisiae, is used as starter culture for both ka r beer and doro [30,32]. The use of commercial starter cultures of S. cerevisiae has also been reported for production of chikokivana, a 1-day brewed beverage based on malted millet and maize meal [32]. S. cerevisiae has been reported to occur spontaneously in low-alcoholic beverages produced from maize such as busaa in Kenya [41], malawa beer in Uganda [9] and munkoyo in Zambia [3]. An alcohol content of 2^4% (v/v) has been reported for busaa. Besides S. cerevisiae, C. krusei, lactic acid bacteria and Pediococcus spp. have been reported to be involved in the production of busaa [9]. Palm wine is another alcoholic beverage where S. cerevisiae is implicated in the fermentation. Palm wine is known over major parts of West Africa under various names such as nsafufuo in Ghana, emu in Nigeria and akpeteshi in Ghana and Nigeria. The wine is made from fermented palm sap from the coconut palm, the oil palm, the wild date palm, the nipa palm or the kithul palm [1,42,43]. Palm wine is a mildly alcoholic beverage similar to beer. The alcohol content of palm wine may vary from 1.5 to 7.1% (v/v) and the ph from 4.0 to 5.5. S. cerevisiae and S. pombe have been reported to be the dominant yeast species [9]. Lactic acid bacteria and acetic acid bacteria are also present at high levels and other yeast species such as Type of product Raw material Name of product Country/region Reference Non-alcoholic starchy food Maize kenkey Ghana [24,31,73] Maize mawe Benin [21] Maize ogi West Africa [1,2,29] Rice masa Nigeria [33] Sorghum kisra Sudan [4,9] Alcoholic beverage Maize munkoyo Zambia [3] Maize busaa Kenya [44] Maize/millet chikokivana Zimbabwe [32] Plantain agadagigi Nigeria/Cameroon [36] Palm sap palm wine Nigeria [43,77] Sorghum pito Ghana/Nigeria [28,35,37,39,88] Sorghum dolo Burkina Faso/Togo [34,35,88] Sorghum burukutu Nigeria [36] Fermented milk Cow amasi Zimbabwe [23,47] Cow mbanik Senegal [50] Cow nono Nigeria [49] Cow/sheep/goat rob Sudan [46,48]

4 194 L. Jespersen / FEMS Yeast Research 3(2003) 191^200 C. krusei, other Candida spp. and Pichia spp. have been found [44]. The microorganisms are reported to originate from the palm tree, the gourd used for sap collection and fermentation, or the tapping equipment [9]. Spontaneous fermentation of bananas and plantains is used to produce a plantain beer called agadagidi in Nigeria and Cameroon. Beside lactic acid bacteria, S. cerevisiae has been reported to be involved in the fermentation [36]. An example of a high-alcoholic drink similar to wine is muratina, produced by fermentation of sugar cane in Kenya. The product is a sweet-and-sour alcoholic beverage with an alcohol content between 9.4 and 13.4% (v/v) and a ph value of 3.0. No systematic microbiological studies have been conducted for this product but the fermentation is reported to be dominated by yeasts [44]. S. cerevisiae has been shown to be the prevailing yeast species for a similar product produced from sugar cane at the Philippines [45] Fermented milk Milk is by far the most abundant fermented animal product in Africa, even though the extent to which milk is used in the daily diet varies to a great extent. Fermented milk is mostly used in East Africa and up to 53% of the milk produced in the Kenyan highland has been reported to be consumed as fermented milk [9]. Also in Zimbabwe and Sudan fermented milk is an important part of the traditional diet [46,47]. In Zimbabwe products known under names such as hodzeko, mukaka wakakora or amasi are produced by spontaneous fermentation of unpasteurised or boiled milk. After fermentation for 2^4 days some whey is drained o to obtain a product with a thick consistency. High numbers of yeast up to 10 8 cfu g 31 were found in amasi with S. cerevisiae as the dominant yeast species. Also lactic acid bacteria, coliforms and other yeast species were detected [23,47]. Most of the identi ed yeasts would not grow alone in the milk, however, all S. cerevisiae isolates could utilise galactose and 32% of the isolates were able to assimilate DL-lactate. In Sudan, the major fermented milk product is rob, which is usually made from a surplus of milk occurring in the rainy season. Rob is mostly made from cow s milk but milk from sheep and goats is similarly processed. After souring of the milk, butter is separated and the remaining sour milk is known as rob [46]. Yeast counts of 10 6^10 7 cells ml 31 were found during the production of rob and S. cerevisiae, followed by C. kefyr, was the dominant yeast species [48]. S. cerevisiae has also been reported to be involved in fermentation of nono, a Nigerian fermented milk product [49], and mbanik a Senegalese cultured milk product [50]. Saccharomyces spp. have further been encountered in fermented milk made by the Masai in northern Tanzania [51]. 3. Functions of S. cerevisiae in indigenous fermented foods and beverages 3.1. Aroma formation The most intensively studied function of S. cerevisiae in the fermentation of foods and beverages is the conversion of carbohydrates into alcohols and other aroma components such as esters, organic acids and carbonyl compounds [17,52]. Especially in the brewing industry it is recognised that Saccharomyces yeasts have a great impact on the avour of fermented products [53]. However, the importance of S. cerevisiae for the formation of aroma compounds in indigenous fermented foods and beverages has not been investigated to the same extent. A large number of yeasts including S. cerevisiae have been found during fermentation of maize dough and it is therefore likely that they will in uence the organoleptic quality of the dough [24]. A total of 76 compounds have been identi ed in spontaneously fermented maize dough by gas chromatography/mass spectrometry [54]. These included 21 carbonyls, 19 alcohols, 17 esters, 12 acids, a furan, ve phenolic compounds, an alkene and one unidenti ed compound. The majority of the compounds showed a sharp increase after 24 h of fermentation, reaching a peak after 48 h. In general, alcohols and esters were produced in much larger amounts than other aroma compounds. The development of alcohols and some esters coincided with high levels of yeasts particularly. The esters found in fermented maize dough have also been reported as a result of yeast activity during beer fermentation and during spontaneous fermentation of Danish rye sourdoughs [53,55]. In order to identify volatile compounds produced by the microorganisms associated with fermentation of maize dough, samples obtained by spontaneous fermentation and prepared by the use of starter cultures have been compared [56]. The starter cultures examined were S. cerevisiae, C. krusei and Lactobacillus fermentum. As expected maize dough prepared with lactic acid bacteria had a lower ph and higher levels of lactic acid than doughs prepared with yeasts or spontaneously fermented. Dough fermented with starter cultures of S. cerevisiae had increased levels of alcohols and esters while dough fermented with lactic acid bacteria and C. krusei in general had lower levels of most volatiles [56]. Likewise, inoculation of maize dough with 10 6 cfu g 31 of S. cerevisiae in pure culture or in combination with various Candida species was found to increase the organoleptic scores of the fermented dough signi cantly [25]. Mixed cultures of S. cerevisiae and Lactobacillus brevis used for fermentation of Nigerian ogi resulted in a product with an aroma pro le quite similar to that of traditionally produced ogi [29]. S. cerevisiae isolated from fermenting pito has successfully been used as starter culture for the production of pito, resulting in a product with a higher alcohol content

5 L. Jespersen / FEMS Yeast Research 3(2003) 191^ than the traditional brew [28]. The use of single cultures of S. cerevisiae or a crude mixed yeast culture isolated from pito has been investigated for controlled production of fermented sorghum beer [39]. The reduction in total sugar concentration and the production of ethanol were found to be higher for single cultures of S. cerevisiae than for the mixed yeast culture. Based on sensory analysis the mixed yeast culture gave an aroma typical of pito, whereas beer made with single cultures of S. cerevisiae had a dry and slightly bitter taste that deviated from that of traditionally brewed pito Microbial interactions In indigenous fermentations S. cerevisiae often coexists with other microorganisms. Depending on the type of product a selection for S. cerevisiae might take place. Examples of African indigenous fermented products where the coexistence of S. cerevisiae with other microorganisms has been reported are shown in Table 3. Saccharomyces yeasts have been found to stimulate the growth of other microorganisms, including lactic acid bacteria, by providing essential metabolites such as pyruvate, amino acids and vitamins. On the other hand, S. cerevisiae has been reported to utilise certain bacterial metabolites as carbon sources [23,57]. However, the mechanisms have not been described in detail. S. cerevisiae as well as several other yeast species have been reported to have pectinase activity that could be of importance for the substrate availability for other microorganisms and for the subsequent microbial degradation of complex molecules. For S. cerevisiae, pectinase activity has been ascribed to an endo-polygalacturonase encoded by the PGU1 gene on chromosome X [22,58]. The presence of pectinase activity in yeasts could especially be of importance in the fermentation of cocoa and co ee, as well as in other indigenous fermented products where degradation of pectic substances is desired [59]. S. cerevisiae together with C. krusei has been observed to have an inhibitory e ect on the growth of mycotoxinproducing moulds such as Penicillium citrinum, Aspergillus avus and Aspergillus parasiticus [19]. The inhibitory effects of the yeasts were shown to be mainly due to substrate competition, but inhibition of spore germination might also occur due to the production of high concentrations of organic acids [19]. The growth of Shigella exneri and enterotoxigenic Escherichia coli in fermented maize dough has been found to be inhibited compared to growth in unfermented dough. The mechanisms behind the growth inhibition are unclear but likely involve the decreased ph of the fermented dough and the production of antimicrobial compounds [15] In uence on nutritional value Table 3 Coexistence between S. cerevisiae and other microorganisms in some African fermented foods and beverages It is possible that S. cerevisiae may in uence the nutritional value of fermented products, but there are only very few published investigations on this topic. S. cerevisiae and Lactobacillus plantarum have been used as starter cultures to ferment various composite blends of cereals and soybeans in the production of weaning foods [10,27]. The fermentation was found to increase the content of ribo avin, thiamine, niacin and ascorbic acid. The contents in some amino acids were improved while others were reduced. The total contents of polyphenols, tannins and phytate were reduced by the fermentation, resulting in a better bio-availability of micronutrients. Also better physicochemical properties such as improved starch stability and improved gelatinisation were obtained by the fermentation. The last-mentioned properties are important in the production of adult foods, but might be a drawback for weaning foods. The investigations did not clarify which Type of product Name of product Microorganisms a References Non-alcoholic starchy kenkey LAB b, Candida krusei, Debaryomyces spp., [24,31,73,89] food Kluyveromyces spp., Trichosporon spp. ogi LAB, acetic acid bacteria, Corynebacterium sp., [2,9,29] Candida vini, Zygosaccharomyces rouxii Alcoholic beverage dolo / pito LAB, Candida spp., Kloeckera apiculata, [28,34,35,37,39] Kluyveromyces spp., Pichia anomala, Schizosaccharomyces pombe, Torulaspora delbrueckii munkoyo / busaa LAB, Candida krusei [3,41] palm wine LAB, acetic acid bacteria, Candida spp., [9,44,77] Kloeckera apiculata, Pichia spp. Schizosaccharomyces pombe Fermented milk amasi LAB, Candida kefyr, Candida lipolytica, [23,47] Candida lusitaniae, Candida tropicalis, Dekkera bruxellensis, Saccharomyces dairenensis, Torulaspora delbrueckii rob LAB, Candida kefyr [48] a Microorganisms other than S. cerevisiae reported to occur during fermentation of the product. b Lactic acid bacteria.

6 196 L. Jespersen / FEMS Yeast Research 3(2003) 191^200 e ects could be linked to the activity of the yeast. However, the authors pointed out that the increase in vitamin B 12 content and the increase of some amino acids could be linked to the fermentation by S. cerevisiae. Fermentation of ka r beer has been reported to double the content of ribo avin and niacin, and fermentation of palm wine to increase the content of ribo avin and thiamine [60]. The use of the probiotic yeast, Saccharomyces boulardii (now recognised as S. cerevisiae [61]), in the production of indigenous fermented foods has been investigated [11]. A mixture of S. boulardii, Lactobacillus casei and Lb. plantarum reduced the content of anti-nutritional factors such as phytic acids, polyphenols and trypsin inhibitors and improved the in vitro digestibility of starch and protein in a mixed cereal product. However, therapeutic application of the product was not investigated Probiotic e ects Despite the fact that Saccharomyces yeasts have been reported to have probiotic e ects [62,63], the probiotic e ects of strains of S. cerevisiae isolated from indigenous fermented products have never been investigated. In clinical trials Saccharomyces yeasts have been reported to be e ective in the treatment of acute infantile gastroenteritis and diarrhoea following treatment with antibiotics [64], and have been shown to inhibit infections with C. albicans [65,66], Salmonella typhimurium and Shigella exneri [64] as well as Clostridium di cile [67]. For the latter the e ect has been shown to be due to a reduction in the binding of C. di cile toxins A and B by inhibition of toxin^receptor binding, probably due to secretion of a protease that digests both the toxins and the intestinal receptor for these toxins [68]. Also Saccharomyces yeasts have been found to protect against cholera toxin probably by adhesion of the toxin to receptors on the yeast surface [69]. Furthermore, Saccharomyces yeasts have been observed to modulate the host immune response by stimulating siga production and the phagocytic system in mice [70]. Unfortunately, S. cerevisiae has recently been associated with invasive infections in immunodepressed patients [71,72]. 4. Biodiversity and taxonomic characteristics of S. cerevisiae strains isolated from indigenous fermented foods and beverages As starter cultures are infrequently used in the production of indigenous fermented foods and beverages, a variety of S. cerevisiae strains is encountered [24,35,73]. The diversity of S. cerevisiae strains isolated from fermented maize dough has been investigated [73]. All isolates had assimilation pro les typical for S. cerevisiae and were able to assimilate galactose, saccharose, ra nose and glucose. Of the isolates, 98% were able to assimilate maltose, 83% DL-lactate and 40% melibiose. In total eight di erent assimilation pro les were seen, of which the most common pro le was observed for 50% of the isolates. The isolates were further investigated by pulsed- eld gel electrophoresis (PFGE). All investigated isolates were found to have chromosomal band patterns with size distribution typical for S. cerevisiae. However, chromosome length polymorphism (CLP) was evident among the isolates and was seen both for isolates from di erent production sites and for isolates within the same batch of fermentation. Nearly all isolates displayed an individual chromosome pro le, although some pro les were closely related. The diversity was further con rmed by PCR ampli cation using primers against the 5P-termini of the delta elements anking the Ty1 retrotransposon. A few isolates that could not be separated by the PFGE method could be separated by the PCR method and vice versa [73]. The diversity of S. cerevisiae isolates from fermented sorghum beer produced in the northern part of Ghana and Burkina Faso has been investigated [35]. Sixteen different assimilation pro les were seen of which the most common pro le was observed for 53% of the isolates: these were only able to assimilate glucose and maltose and thus were not consistent with the accepted description of the species S. cerevisiae by Vaughan-Martini and Martini [74], but in accordance with the description given by Barnett et al. [75]. By sequence analysis of the D1/D2 domain of the large subunit (26S) rdna a deviation from the type strain of S. cerevisiae (CBS 1171) of three nucleotides, equivalent to 0.5% of the DNA, was found. A di erence in nucleotide sequence of 1% has been reported for di erent species, whereas a di erence of zero to three nucleotides corresponding to 0^0.5% has been reported for conspeci c strains and sister species [76]. Accordingly, the Saccharomyces isolates from African fermented sorghum beer should most likely be placed within the species S. cerevisiae. As reported for isolates from fermented maize dough, determination of CLP by PFGE revealed a pronounced diversity. All isolates had individual chromosome pro les even though some pro les were closely related. No correlation was seen between the clustering of the chromosome pro les and the assimilation pattern of the isolates. Also in the fermentation of palm wine several physiological variants of S. cerevisiae have been found to be involved [77]. The biodiversity seen for the isolates of S. cerevisiae from fermented maize dough, fermented sorghum beer and others is in agreement with observations on brewing, wine and bakery strains of S. cerevisiae, as well as on contaminants of S. cerevisiae seen in the brewing industry [78^82]. Also for non-african indigenous fermented beverages a pronounced biodiversity of S. cerevisiae has been found. By use of PFGE and RAPD-PCR using primers complementary to intron consensus splicing sites, various strains of S. cerevisiae were found during fermentation of cachaca, an alcoholic beverage produced from sugarcane juice in Brazil [83]. Similarly, several S. cerevisiae strains

7 L. Jespersen / FEMS Yeast Research 3(2003) 191^ Table 4 Occurrence of MAL loci in Saccharomyces yeasts isolated from various fermented foods and beverages were detected by use of PFGE followed by hybridisation with chromosome-speci c probes (ADE2 and YNL075W) during fermentation of aguardente, another alcoholic beverage produced from sugarcane juice in Brazil [84]. As maltose is the major carbohydrate present in cereals, the ability to utilise this carbohydrate is an important technological property when selecting appropriate starter cultures. The presence of the MAL gene encoding maltose permease has been investigated for isolates of S. cerevisiae from fermented maize dough [73], for isolates from fermented sorghum beer [35], and for brewing yeasts [81] and brewing yeast contaminants [82]. A comparison of the occurrence of various MAL loci in the above-mentioned isolates is presented in Table 4. For isolates of S. cerevisiae from fermented maize dough the dominant MAL genotype seen for 58% of the isolates was MAL11, MAL31 [73]. Besides, for 6% of the isolates the MAL61 probe hybridised to a position of about 950 kb that is not in accordance with the position of any previously described MAL locus [85,86]. All S. cerevisiae isolates from fermented sorghum beer were found to have the genotype MAL11,MAL31 [35]. Besides, the MAL61 probe was found to hybridise to a position of 950 kb for 40% of the isolates. The band with a position of 950 kb could indicate a yet undescribed MAL locus evolved by translocation from telomeric regions of di erent chromosomes [35,85]. When compared to the results obtained for brewing yeasts and brewing contaminants (Table 4), it appears that the S. cerevisiae isolates from African indigenous fermented foods and beverages in general have a smaller number of MAL loci than brewing yeasts and brewing contaminants of S. cerevisiae. This could imply that the selection pressure for maltose utilisation has not been as pronounced as for commercial strains of Saccharomyces spp. used as starter cultures in industrialised fermentations. Interestingly, MAL41 was never seen for the S. cerevisiae isolates from African indigenous fermented foods and beverages, whereas MAL41 was nearly always present in the industrial strains of S. cerevisiae and S. pastorianus as well as in brewing contaminants of S. cerevisiae. MAL11 was found to be universally present for all investigated isolates, which ts with the theory the MAL11 is the progenitor of all other MAL loci [85,87]. 5. Future use of S. cerevisiae as starter culture for production of indigenous fermented foods and beverages The advantages obtained by fermentation of foods are many, especially in developing countries. Also the socioeconomic and cultural e ects of the production of traditionally fermented foods and beverages from local crops are important. Despite this and the fact that indigenous fermented foods and beverages form a great part of the diet in many African countries, by far the majority of these products are still produced by spontaneous fermentation at household or semi-industrial scale. Many of these fermentations are not even described in detail and the losses due to inadequate process equipment and uncontrolled fermentation are likely to be very signi cant. In order to maintain and sustain the production of indigenous fermented foods in Africa, up-scaling of production plants is required, as is the change from spontaneous to controlled fermentations and the introduction of puri ed indigenous starter cultures. The fact that isolates of S. cerevisiae from African fermented products have properties di erent from those of well-recognised starter cultures [35,73] demonstrates that starter cultures for indigenous fermented foods and beverages should be isolated from the products they are supposed to be used for, and selected according to the technological properties required for the actual type of product. Moreover, antimicrobial, probiotic and pathogenic properties as well as genetic stability should be taken into consideration [20]. Reported examples where strains of S. cerevisiae have been isolated from indigenous fermented foods and beverages and thereafter been successfully used as starter cultures are Ghanaian fermented maize dough for kenkey and koko production [26], Ghanaian fermented sorghum beer [28], Zambian munkoyo maize beverage [3], and Nigerian ogi based on maize [29]. Yeast/product MAL11 a MAL21 a MAL31 a MAL41 a MAL61 a S. cerevisiae ( kenkey ) b;c S. cerevisiae ( pito ) b;d S. cerevisiae (brewing contaminants) e S. cerevisiae (ale yeasts) f S. pastorianus (lager yeasts) f a Percentages of isolates with the indicated MAL loci. b kenkey : additionally for 6% of the isolates the MAL probe hybridised to a position of 950 kb; pito : additionally for 40% of the isolates the MAL probe hybridised to a position of 950 kb. c [73]. d [35]. e [82]. f [81].

8 198 L. Jespersen / FEMS Yeast Research 3(2003) 191^ Conclusion S. cerevisiae is the predominant yeast in many African indigenous fermented foods and beverages where it is mainly introduced by spontaneous fermentation. Its major functions include the production of alcohols and other aroma compounds, especially esters and organic acids, but other e ects such as stimulation of e.g. lactic acid bacteria, improvement of nutritional value, probiotic activity, inhibition of mycotoxin-producing moulds, and production of tissue-degrading enzymes may also be seen. A pronounced biodiversity is observed among the strains of S. cerevisiae isolated from African indigenous fermented foods and beverages, and studies have shown that these strains share common traits di erent from those of well-recognised starter cultures of S. cerevisiae. However, further studies are required to fully explore their phylogenetic relationship. 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