International Journal of Food Microbiology 190 (2014) 84–96

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j foodmicro

African fermented foods and probiotics

Charles M.A.P. Franz a,⁎, Melanie Huch a, Julius Maina Mathara b, Hikmate Abriouel c,Nabil Benomar c, Gregor Reid d, Antonio Galvez c, Wilhelm H. Holzapfel e

a Max Rubner-Institute, Federal Research Institute of Nutrition and Food, Department of Safety and Quality of Fruit and Vegetables, Haid-und-Neu-Str.9, D-76131 Karlsruhe, Germanyb Department of Food Science and Technology, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, 00200 Nairobi, Kenyac Departamento de Ciencias de la Salud, Área de Microbiología, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071 Jaén, Spaind Lawson Health Research Institute and Western University, London, Ontario, Canadae Handong Global University, School of Life Sciences, Pohang, Gyeongbuk 791-708, Republic of Korea

⁎ Corresponding author at: Department of Safety and Qfax: +49 721 6625 453.

E-mail address: Charles.Franz@mri.bund (C.M.A.P. Fra

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.0330168-1605/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 April 2014Received in revised form 25 June 2014Accepted 23 August 2014Available online 30 August 2014

Keywords:African traditional foodsProbioticsFermentationMalnutritionGI infection

Africa has an age old history of production of traditional fermented foods and is perhaps the continent with therichest variety of lactic acid fermented foods. These foods have a large impact on the nutrition, health and socio-economyof the people of the continent, often plagued bywar, drought, famine and disease. Sub-Saharan Africa isthe world's region with the highest percentage of chronically malnourished people and high child mortality.Further developing of traditional fermented foods with added probiotic health features would be an importantcontribution towards reaching the UNMillennium Development Goals of eradication of poverty and hunger, re-duction in childmortality rates and improvement of maternal health. Specific probiotic strains with documentedhealth benefits are sparsely available in Africa and not affordable to themajority of the population. Furthermore,they are not used in food fermentations. If such probiotic products could be developed especially for householdfood preparation, such as cereal or milk foods, it could make a profound impact on the health and well-being ofadults and children. Suitable strains need to be chosen and efforts are needed toproduce strains tomakeproductswhich will be available for clinical studies. This can gauge the impact of probiotics on consumers' nutrition andhealth, and increase the number of people who can benefit.

© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851.1. Probiotics, definition and suggested health-improving properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851.2. A global perspective on the use of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851.3. Why Africa needs probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

2. An overview of African fermented foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862.1. Non-alcoholic cereal and vegetable fermentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

2.1.1. Examples of African fermented maize products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872.1.2. Examples of fermented sorghum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882.1.3. Examples of fermented millet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2.2. Starchy root crop fermentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882.3. Animal protein fermentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

3. Choice of multifunctional strains for African probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893.1. The search for potential probiotic strains to supplement African fermented foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913.2. Intervention studies with African potential probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

4. Further perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925. Recommendations and research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

5.1. Choice of multifunctional strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935.2. Choice of product for probiotic delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

uality of Fruit and Vegetables, Max Rubner-Institut, Haid-und-Neu-Str. 9, D-76131Karlsruhe, Germany. Tel.: +49 721 6625 225;

nz).

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5.3. Improving research infrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

1. Introduction

1.1. Probiotics, definition and suggested health-improving properties

Kollath (1953) and Vergio (1954)were probably the first to intro-duce the term probiotic (Holzapfel and Schillinger, 2002), while abeneficial association of lactic acid bacteria (LAB) with the humanhost was already suggested in Biblical times and by Metchnikoff(1908). The latter considered the longevity of Bulgarian peasants tobe related to their high intake of fermented milk products, as he con-sidered gut microbes detrimental rather than beneficial to humanhealth (Metchnikoff, 1908). While documentation of said longevityis scant, recent large studies in Scandinavia support at least the abil-ity of regular intake of fermented foods to reduce the incidence of se-rious disease (Larsson et al., 2008; Keszei et al., 2010; Sonestedtet al., 2011). In this context the LAB and their production of lacticacid as a result of sugar metabolism were suggested to be health-promoting agents. Originally defined as microorganisms promotingthe growth of other microbes (Vergio, 1954; Lilly and Stillwell,1965), probiotics have been re-defined a number of times. In 1989,Fuller referred to the animal host when he defined a probiotic as ‘alive microbial feed supplement which beneficially affects the hostanimal by improving its intestinal microbial balance’ (Fuller, 1989).Havenaar et al. (1992) defined probiotics as ‘mono- or mixed cul-tures of live microorganisms which, when applied to animal orman, beneficially affect the host by improving the property of theindigenous flora’, while in relation to food, probiotics were consid-ered as ‘viable preparations in foods or dietary supplements to im-prove the health of humans and animals’ (Salminen et al., 1998).The German Federal Institute for Health and Consumer Protectionand Veterinary Medicine (currently the Federal Institute for Risk As-sessment) defined probiotics as ‘specific live microorganisms whichreach the intestinal tract in active form and in sufficient numbers topositively affect the health of the host’ (Franz et al., 2011a). Current-ly the most common definition is that from the FAO/WHO whichstates that probiotics are ‘live microorganisms which when adminis-tered in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2002).

The mechanisms of health-improving properties of probioticsare still not completely understood, but are commonly suggested torelate to pathogen interference, exclusion or antagonism, immune-modulation, anticarcinogenic and antimutagenic activities, alleviationof lactose intolerance symptoms, reduction in serum cholesterol levels,reduction in blood pressure, prevention and decreasing incidence andduration of diarrhea, prevention of bacterial vaginosis and urinarytract infection, maintenance of mucosal integrity, and improved peri-odontal health (Erbringer et al., 1995; Reid et al., 1995; Salminenet al., 1996; Klaenhammer and Kullen, 1999; Burns and Rowland,2000; de Vrese et al., 2000; Saarela et al., 2000; Holzapfel andSchillinger, 2002; Ouwehand et al., 2002; Isolauri, 2004; Hummelenet al., 2010; Gupta, 2011; Kumari et al., 2011). Many studies, whichwere also subsequently evaluated by meta-analyses, have clearlyshown that positive effects can be observed with specific strains,especially in the treatment of diarrheal disease among children (vanNiel et al., 2002; Allen et al., 2004, 2010; Szajewska et al., 2007a,b;Guandalini, 2008, 2011; Salari et al., 2012). Likewise, there are gooddata for the treatment of some cases of antibiotic associated diarrhea(D'Souza et al., 2002; Johnston et al., 2006, 2011; Hempel et al., 2012;Videlock and Cremonini, 2012). Especially important for the African

scenario with its high infant/childmortality, there is also good evidencefrom meta-analysis of intervention studies, that probiotics can preventnecrotizing enterocolitis, prevent infant sepsis and decrease mortalityin pre-term neonates with low birth weight (Deshpande et al., 2010;Bernardo et al., 2013).

1.2. A global perspective on the use of probiotics

In theWestern dietary culture, it is common to fermentmilk into yo-gurt, sour milk or cheese. This long-held custom of fermenting milk ex-plains the high prevalence of probiotic products delivered as fermentedmilk, particularly sourmilk and yogurt in Europe. It is noteworthy, how-ever, that industries in the developed world are creating other deliveryvehicles for probiotics, including cheese, whey dairy products, desserts,ice cream, breads, confectionary and soy products as well as meats andfermented vegetables (Gardiner et al., 1998; Ravula and Shah, 1998;Stanton et al., 1998; Hagen and Narvhus, 1999; Lee et al., 1999; Shahand Ravula, 2000; Haynes and Playne, 2002; Ross et al., 2002;Työppönen et al., 2003; Medici et al., 2004; Alamprese et al., 2005;Donkor et al., 2005; Madureira et al., 2005; Kröckel, 2006; Ong et al.,2006; Wang et al., 2006; Alagron-Alegro et al., 2007; Nebesny et al.,2007; Bernardez et al., 2008; Burns et al., 2008; De Vuyst et al., 2008;Sharp et al., 2008; Cruz et al., 2009; Mäkeläinen et al., 2009; Zollneret al., 2009; De Bellis et al., 2010; Gawkowski and Chikindas, 2013).Theworldmarket for ‘functional foods’, i.e. foodswhich contain ingredi-ents that optimize beneficial properties such as probiotics, prebiotics,vitamins and minerals, had an estimated size in 2003 of ca. 33 billionUSD, while the European market estimation exceeded 2 billion USD inthe same year. This rose in 2005, to 50 billion USD for functionalfoods. In western Europe, the consumer market for probiotic foodswas N1.4 billion euros (Saxelin, 2008). Probiotics and prebiotics accountfor the most important fraction of the overall market for functionalfoods (Figueroa-González et al., 2011), and the world probiotic marketshare has risen to an estimated 15 billion USD (Bhadoria andMahapatra, 2011).

In Europe, the largest segments of the functional food markets arerepresented by foods prepared with probiotics, prebiotics or synbiotics(a combination of probiotics and prebiotics). The probiotic market, es-pecially directed towards yogurts and fermentedmilks, has experiencedrapid growth in the past year. The most developed markets for suchproducts are situated in northern European and Scandinavian countries,where consumers have long tradition of consumption of fermenteddairy products (Bhadoria and Mahapatra, 2011). However, due to poorlegislative decision making in Europe, some markets have fallen in re-cent years (Pedretti, 2013).Meanwhile, probiotics have been gaining in-creasing importance in North America with it being the fourth largestmarket in theworld. More than 100 companies in the USA sell productsclaiming to be probiotic in supplement form (Sanders, 2008; Bhadoriaand Mahapatra, 2011).

Sparse data are available formarket share of probiotics in Africa, but itisminiscule in global terms. SouthAfrica appears to have a relativelywell-establishedmarketwith supplements (capsules), fortified food items (es-pecially baby cereals) and fermented dairy products (Brink et al., 2005).The need and interest appear to be there (Anukam et al., 2004; Anukamand Reid, 2005; Reid et al., 2005), in part based upon traditionalfermented foods (Anukam and Reid, 2009). However, in line with FAOpriorities for food safety in Africa, suggestions have beenmade for studieson functional (probiotic) properties of LAB strains involved in traditionalfermented foods, and to include developing in vitro selectionmethods for

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probiotic strains, followed by studies on technical performance of thesestrains in traditional substrates (Holzapfel, 2002).

In theory, established probiotic strains and products could be sold inAfrica, but a number of critical factors have to be considered. Localpopulations in developing countries, for example, may prefer a ‘local’solution to nourishment, health maintenance and restoration. Thismay reduce the chances that they would purchase a foreign company'smilk product on a regular basis, especially if this product is not part ofthe normal diet or too expensive in the rural third world setting (Reidet al., 2005). Although the African cultural arena is associated with agreat diversity of fermented foods, these are usually based on vegetableprotein, cereal or starchy root fermentations and, with some exceptions,less on dairy products, which are dominant in northern Europe andNorth America. Fermentation ofmilk in Africamainly occurs in northernAfrica, northernWest Africa, the Sahara and the Savannah and Hills andvalleys of East Africa. Such regional products are not typical Europeantype yogurts or cheeses but rather local variations, where variousadditives such as wood ash, blood or occasionally leafy vegetablesmay be added and the fermentation vessels are frequently being pre-pared by smoking (Abdelgadir et al., 1998; Odunfa and Oyewole,1998; Akinyanju, 1989; Gonfa et al., 2001; Olasupo et al., 2004, 2010;Karenzi et al., 2013).

For Africans, the importance of traditional food fermentation lies inproviding improved flavors to existing staples (e.g. cereals and rootcrops), and as a cheap way of food preservation and an enhancementof the nutritional quality and digestibility of the raw products(Olasupo et al., 2010). Frequently, fermented foods are considered tohave health benefits, and in many regions they are believed to aidin the control of some diseases, in particular intestinal disorders(Mathara et al., 2004). Traditional fermented products with improvedproperties may be more easily accepted by populations with longstanding societal structure. An example is an improved ogi, nameddogik, that has been developed by Okagbue (1995) using a lacticacid starter with antimicrobial activities against some diarrheagenicbacteria.

Generally, the typical delivery vehicles and flavoring for probioticfoods used in first world countries may not be appropriate for theAfrican situation, especially in a rural setting. Yogurt-type productswhen not consumed immediately require refrigeration to ensure stabil-ity of both the product and the probiotics, but the high ambient temper-atures of most regions on the African continent, the great distributiondistances and potential problems with maintaining the cold chainin rural areas present logistical problems.

1.3. Why Africa needs probiotics

The reasons why probiotic products containing well-documentedstrains such as Lactobacillus rhamnosus GG or Lactobacillus casei Shirotaare not yet sold in sub-SaharanAfrican countries are unclear, butmay berelated either to the problems mentioned before, or because the pro-ducing companies do not expect sufficient profit, or suitable distributorsmay not be available to justify sales (Reid et al., 2005). Sadly, it is inthese regions where perhaps the greatest need for probiotics exist(Reid et al., 2005). According to FAO estimates, 925 million people suf-fered from hunger in 2010, the majority of them living in rural areas.Sub-Saharan Africa is the region of the world with the highest percent-age of chronically malnourished people (OECD-FAO, 2011). The reasonsfor Africa's serious problem to sufficiently feed itself both quantitativelyand qualitatively are numerous, but are based mainly on agronomicconstraints and limitations of locally appropriate processing techniques,thus resulting in huge postharvest losses of 30 to 50% (Shiundu andOniang'o, 2007). It is a tragedy that 14 out of the world's 20 poorestcountries are in Africa, most of which face constantly increasing prob-lemswith under- or malnourishment of a major part of the population;moreover, chronic malnutrition has been a persistent problem especiallyfor young children in sub-Saharan Africa. Child mortality in sub-Saharan

Africa was reported to be 104 per 1000 live births in 1992, and to rangefrom 51 (South Africa) to 147 (Malawi) per 1000 childbirths in differentAfrican countries in 1994 (Kalipeni, 2000). The UN inter-agency groupfor child mortality estimation reported in 2013 that the highest rates ofchild mortality are still in sub-Saharan Africa, with an under-five mortal-ity rate of 98 deaths per 1000 live births, this being 15 times the averagefor developed regions (UN Interagency Group for Child Mortalityestimation, 2013). Important to the causal pathway leading to under-weight is the malnutrition–infection cycle, whereby underweightchildren, because of their poor physical stamina and weakened immunesystem, are at increased risk of infectious diseases such as diarrhea andare thus at substantially increased mortality risk (Mosley and Chen,1984; Black et al., 2003; Nannan et al., 2007). Feachem and Jamison(1991) estimated that anAfrican childwould have a 10% chance of suffer-ing from diarrhea on any given day and a 14% chance of dying from asevere episode. This is significant since diarrhea accounts for 37% of child-hood deaths in sub-Saharan Africa, being one of the foremost causes ofpoor health and childhood mortality (Kalipeni, 2000). Thus, Africa is acontinent with a high incidence of diarrheal diseases, especially amongyoung children, where child mortality of children less than five yearsold is extremely high. The utilization of fermented foods containingprobiotics would be one avenue by which the health of the childrenmay be improved. Fermentation could also contribute to preservingfoods and thus minimizing postharvest losses, as well as detoxifying theraw materials and increasing the intake of macro- and micro-nutrients,thus alleviating malnutrition (Holzapfel, 2002).

Why should fermented foods act as carriers of probiotics, and whichstrains should be used? Asmentioned before, animal protein fermentedfoods, including milk fermentations, are not widely used in Africa as awhole. While in regions such as Ethiopia and Kenya where milk isfermented and consumed regularly, the production of milk-based prod-ucts with probiotics would make sense, such products would probablynot find wide distribution across the rest of the African continent.Thus, the development of probiotic foods for Africa should ideally relyon traditional fermented foods typical of a region and the local dietarypreferences. Or, dried probiotic products should be developed foraddition to a variety of local foods, and be able to survive heating incertain cases. Strains used in food fermentations should bewell adaptedto the product and show good health-promoting activities. In ouropinion, this requires the development of new bacterial strains asmultifunctional starter cultures or the testing of Western strains in anAfrican setting (Holzapfel, 2002). The development of new probioticswould require investigations into the functional properties of the strain,which might include survival at the body target site, enhancement ofthe immune response, anti-disease effects, not adversely disrupt theautochthonous microbiota as well as being produced at low cost, beadapted to African food substrates, and retain good viability/survivalin the product during storage. Lastly, their effect on properties such asflavor or texture of the food product they will be used in, is certainlyalso important.

2. An overview of African fermented foods

According to Tamang and Samuel (2010), the world dietary culturehas three distinct traditional food habits based on staple cereal diets:(a) cooked-rice eaters of Eastern food culture, (b) wheat/barley-basedbreads/loaves of Western and Australian food culture, and (c) sorghum/maize porridges of African and South American food culture. Traditionalfermented foods still play a major role in the diet of numerous societiesworldwide. The African dietary etho includes both fermented and non-fermented sorghum, maize, millet and cassava products, wild legumeseeds and tubers, but also meat, milk products, and alcoholic beverages(Tamang and Samuel, 2010). Of importance to all rural societies arelow-cost and, where possible, “low-tech” food processing proceduresaffordable also by the poor.

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In African civilizations, food fermentation still plays a major role incombating food spoilage and foodborne diseases that are prevalent inmany of its resource disadvantaged regions. The lactic acid fermentationis probably the oldest and best accepted among the African people(Dirar, 1993), and is largely a home-based process used throughoutthe continent (Oyewole, 1997). In Table 1, examples of fermentationtypes and raw materials are given, exemplifying this diversity andexpansive nature of the traditions. The legume fermentations, althoughalkaline and typically dominated by Bacillus spp., are also characterizedby thepresence of LAB, in particular enterococci, albeit as aminor group.Fermented foods in Africa can be classified in the following majorgroups based on the raw material used in their production (Olasupoet al., 2010): 1) fermented non-alcoholic cereals, 2) starchy root crops,3) fermented animal proteins, 4) fermented vegetable proteins, and5) alcoholic beverages. The first group is predominant and relativelysafe, even for infants. Only the fermented non-alcoholic cereals, starchyroot crops and animal proteins will be considered below as potentialcandidates for probiotic foods.

Milk from cattle, sheep and goats is typically fermented in Easternand Southern Africa, and some regions in North and (more rarely)West Africa, where keeping of such livestock has a long tradition,while camel's milk is mainly fermented in Northern Africa and theSudan region. Typical for West African foods are the fermented starchyroots, mainly using cassava (Manihot esculenta Crantz), although cassa-va fermentation can be also be found throughout the continent (Okaforand Ejiofor, 1990; Kimaryo et al., 2000; Ray and Sivakumar, 2009).Specific to Ethiopia is the enset (Ensete ventricosum) (also called falsebanana, Ethiopian banana, or Abyssinian banana) plant, which, can beconverted into “kocho” by lactic fermentation (Gashe, 1987), while tef(Eragrostis tef), also typical of Ethiopia, is used for preparing thetraditional acid-leavened (sourdough) type of “pancake” called injera(Oyewole, 1997; Urga, 1997). Maize (corn), sorghum and millet aremost commonly fermented throughout Africa. In contrast to Asia andEurope, rice and wheat are only rarely used in African traditional foodfermentations (Oyewole, 1997). Of note, while all these processes in-volve fermentation, little is known about the range of bacterial strainsused to perform the process. This represents an untapped source forfuture probiotic products.

2.1. Non-alcoholic cereal and vegetable fermentations

Cereals, mostly processed by natural fermentation, account for up to80% of total calorie consumption inmany African countries and they arealso an important source of dietary protein. They are frequently comple-mentary foods for young children and as dietary staples for adults. Lacticfermented cereal-based foods in Africa can be classified on the basis ofeither the raw cereal ingredients used for preparation, or the texture

Table 1Overview of raw materials and fermentation types of African food fermentations.

Categories

A B C D E

Ogi1,2,3 Kenkey Dawadawa5 Palmwines13

Uji1,2,3,4 Uji1,2,3 Soumbala5 Injera1,2,3

Mawe1 Ben saalga2 Ugba5 Traditional beers3,2,1

Mahewu1 Ogiri11 Tej12 Kisra3

Gari4 Okpehe5 Sherbote14 Kocho9

Kivunde4 Mwenge3,15

Kule naoto10 Kishk10+ 7

Ergo10

Hulumur3

Raw materials: 1 = maize; 2 = millet; 3 = sorghum; 4 = cassava; 5 = legumes;6 = tef; 7 = wheat; 8 = vegetables; 9 = ensete; 10 = milk; 11 = water melonseeds; 12 = honey; 13 = palm juice; 14 = dates; 15 = bananas.A = single-step LAB fermentation; B = soaking followed by lactic fermentation;C = alkaline, bacterial fermentation dominated by Bacillus subtilis; D = lactic/alcoholicfermentation; E = dough with LAB dominant.

of the fermented product (Nout, 2009; Oyewole, 1997). These includea) maize-based foods such as ogi (Nigeria/West Africa), kenkey(Ghana) and mawe (Benin); b) millet-based foods such as kunuzaki(Northern Nigeria), mbege (Tanzania) and ben-saalga (Burkina Faso);c) sorghum based foods such as ogi-baba (West Africa), bogobe(Botswana), humulur (Sudan) and hussuwa (Sudan);and d) wheat-based foods such as bouza, kishk or kishj (Egypt).

Recent studies on fermentation of leafy vegetables (Kasangi et al.,2010) reported that fermentation of cowpea leaves coupled with solardrying could be of potential for small-scale producers as amethod of en-hancement of nutritive quality. Selected probiotic multifunctionalstrains could be used to add value to the African indigenous leafyvegetables (Kasangi et al., 2010). In a study by Muchoki et al. (2010),fermentation of Kenyan cowpea leaves followed by solar dryingproduced a product with reduced anti-nutrients and an increasedacceptability, thus potentially alleviating micronutrient malnutrition.

2.1.1. Examples of African fermented maize productsMawé is an uncooked fermented maize dough, and is an important

ingredient for the preparation of cooked beverages, stiff gels, andsteamed cooked bread in Benin (Nout, 2009). It is prepared by ‘natural’or spontaneous fermentation which may be supported by ‘recycling’ or‘backslopping’ of microorganisms in support of a relatively stable(predictable) process, dominated by heterofermentative LAB such asLactobacillus fermentum, Lactobacillus brevis, Lactobacillus curvatus,Lactobacillus buchneri, Weissella confusa and pediococci (Hounhouiganet al., 1993, 1994). Typical of most traditional lactic fermentations,yeasts such as Candida krusei, Candida kefyr, Candida glabrata andSaccharomyces cerevisiae may also play some role in the fermentation(Hounhouigan et al., 1994; Nout, 2009). Other examples of fermentedmaize products include the Nigerian ogi, the Kenyan uji, and theGhanaian aflata (for making kenkey), although variations occur in pro-cedures, and because millet and sorghum can also be used as raw fer-mentation materials for the former two (Oyewole, 1997; Nout, 2009).The acidity of fermented uji was shown to correlate with increasedshelf-life and to contribute to hygienic safety (Mbugua and Njenga,1992). Preparation of kenkey, ogi and fermented uji involves soaking ofthe grains until soft, wet grinding, and fermentation. Fermentation ofogi and uji differs from that of kenkey by the slurrying and removal ofcoarse particles and bran by filtration or sieving, followed by fermenta-tion of the filtrate (Onyango et al., 2004). The uji or ogi filtrates areallowed to ferment near a fire or at ambient temperature for 1 to3 days. Uji is diluted with water to 8 ± 10% w/v solids and then boiledand sweetened and consumed while still hot, while ogi is boiled to10% solids into a porridge. Ogi and uji may serve as complementaryfoods for infants (Onyekwere et al., 1989; Omemu, 2011). The fermen-tation of uji during sedimentation is predominated by LAB such asLactobacillus plantarum, L. fermentum, Lactobacillus cellobiosus andL. buchneri, Pediococcus acidilactici and Pediococcus (Ped.) pentosaceus(Mbugua, 1985). In the fermentation of ogi in Nigeria, Odunfa andAdeyele (1985) showed that L. plantarumwas the predominant micro-organism associated with the fermentation. Other studies showed thatbesides L. plantarum, the heterofermentative L. fermentum and L. brevisdominated in ogi prepared in Benin or Nigeria (Nago et al., 1998;Omemu, 2011). Omemu (2011) reported a continuous increasein yeast in the fermentation and predominant species includedS. cerevisiae, Rhodotorula graminis, C. krusei, Candida tropicalis, Geotrichumcandidum andGeotrichum fermentum. In our studies (Sanni et al., 2013) onNigerian isolates from fufu and ogi we could show that Pediococcuspentosaceus strains dominated (42.1% of isolates) while L. plantarum andL. fermentum strains were also isolated at a high frequency of 24.6 and26.3%, respectively.

Kenkey has a shelf-life of several days, and is consumed as “ready-to-eat” meal together with other foods (Nout, 2009). Dominant microor-ganisms during the fermentation are L. plantarum, L. fermentum,L. brevis, L. reuteri and Ped. pentosaceus (Olsen et al., 1995) and yeasts,

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mainly C. krusei and S. cerevisiae (Jespersen et al., 1994). Amahewu ormahewu is a sour maize-based fermented gruel or beverage consumedmainly by the indigenous people of South Africa. Amahewu is preparedby adding 9 parts of water to 1 part maize meal and boiling. The slurryis then cooled to 40 °C and a small amount of wheat flour is added assource of starter inoculum. The gruel is allowed to ferment for 1 to3 days in a warm place (Chelule et al., 2010). This product has been in-dustrialized in South Africa (Holzapfel and Taljaard, 2004) and, togetherwith its wide acceptability and the established retail distributionchannels, it provides a promising basis for an African probiotic product.

2.1.2. Examples of fermented sorghumHussuwa is a semi-solid, dough-like food of the Sudan. A liquid paste

of sorghum flour and water are mixed in equal volumes, cooked to thestiff ‘aceda’ porridge, and a further half volume of sorghum maltadded and left to ferment for up to 48 h. The resulting sourdough iscooked on a hot plate until all moisture is expelled (El-Nour et al.,1999; Yousif et al., 2005). After cooking, the hussuwa is fermented inan earthenware pot buried under the fireplace for up to two months,thereby ensuring a continuous warm temperature throughout the peri-od to promote themixed lactic and alcoholic fermentation (Yousif et al.,2005), finally resulting in a sweet–sour product (Dirar, 1993). A taxo-nomic study showed that the majority of the LAB strains typical ofhussuwa fermentation were heterofermentative lactobacilli, mainlyL. fermentum while pediococci also predominate. As mentioned above,ogi or kenkey can also be produced with sorghum.

2.1.3. Examples of fermented milletBen-saalga, a thin porridge is prepared by cooking the fermented

sediment of pearl millet (Pennisetum glaucum) in water, and is typicalof Burkina Faso (Nout, 2009). L. fermentum, L. plantarum and Ped.pentosaceus typically dominate the natural fermentation. SomeL. plantarum strains are able to degrade starch (Ben Omar et al., 2006;Songré-Ouattara et al., 2008) which can be beneficial as they canpositively contribute to increase the energy density of cereal basedfermented gruels through starch hydrolysis (Tou et al., 2007;Songré-Ouattara et al., 2009), although considerable losses of milletnutrients may occur, as a result of discarding the coarse particles duringpreparation and by leaching into the water fraction. However, anti-nutritional components of pearl millet such as phytate were reportedto be degraded by more than 50% in commercial ben-saalga, therebyfacilitating the dietary uptake of proteins and minerals (Lestienneet al., 2005; Nout, 2009).

2.2. Starchy root crop fermentations

Cassava is a major root crop processed by lactic fermentation, pro-viding a wide range of safe products such as gari, fufu and lafun orkokonte common throughout West Africa, and kivunde and cingwadain East Africa (Kimaryo et al., 2000; Padonou et al., 2009). The shelflife of cassava is less than 5 days with deterioration starting within24 h after harvesting. Lactic fermentation drastically prolongs the shelflife, and, for bitter cassava with high cyanogenic glucoside levels, thetoxicological safety. L. plantarum typically dominates the fermentationprocess and is inhibitory to many spoilage molds and bacteria, both inthe East African (Tanzanian) (Kimaryo et al., 2000) and West African(Ghanaian) fermentation process (Amoa-Awua et al., 1996).

Gari is obtained by washing and grating fresh cassava roots,dewatering and fermenting at ambient temperature for up to 72 h.The resulting mash is heated and roasted to evaporate the moistureand the resulting flour is the gari product (Onyekwere et al., 1989;Kostinek et al., 2005). In contrast to the solid-state fermentation ofgari, fufu and lafun are produced in a submerged fermentation of thecassava roots for 4 days, followed by washing, dewatering, drying andmilling to obtain a cassava flour. A study on lafun fermentation showedthat L. fermentum, L. plantarum and W. confusa dominated the LAB

population in the fermentation, while predominant yeasts wereS. cerevisiae, Pichia scutulata, Kluyveromyces marxianus and Hanseniaguilliermondii (Padonou et al., 2009). In a study conducted on cassavafermentation to produce gari in Benin, L. plantarumwas themost abun-dantly isolated species, followed by Leuconostoc fallax and L. fermentum(Kostinek et al., 2005).

In controlled studies, selected L. plantarum strains showed potentialas starter cultures for cassava fermentation in the kivunde process(Kimaryo et al., 2000) and for the production of gari (Huch et al., 2008).Strains of L. plantarum, L. fermentum and Weissella paramesenteroideswere also found suitable for distribution and application as freeze-driedstarter cultures for gari production (Yao et al., 2009). Indeed, lyophilizedcultures were used to start gari fermentations in a rural pilot plant inQuedo village in Benin run by a women's group of 13 members. Theuse of the starter cultures resulted in rapid acidification of the mash,ensuring a high safety of the final product, and in a reduction of thefermentation period from 24 to 12 h (Egounlety et al., 2007).

Some cereal-based, lactic acid fermented products are consumedwithout heat treatment after fermentation, for instance, the Tanzanianbeverage togwa, which is often made from sorghum or maize and isconsumed regularly by young children (6–60 mo of age). It has beenshown to decrease the occurrence of enteropathogens in the rectumand improve the barrier function of the intestinal mucosa in childrenaged 6–25 months with acute diarrhea (Kingamkono et al., 1999;Willumsen et al., 1997).

2.3. Animal protein fermentations

In Africa, animal protein fermentations are mostly related to milkfermentations, although some fish are also fermented. Keeping of live-stock with concomitant milk production has a long tradition in Southand East regions, where the utilization of fermented milk products isfrequently integrated into the societal culture, comprising an importantpart of the daily diet. Milk fermentation is mainly by traditional, small-scale ‘sour milk technologies’ in rural areas to convert milk into variousproducts for extending shelf life. Amasi is a traditional fermented milkconsumed in South Africa and Zimbabwe, and its preparation involvesfermentation for several days of raw milk in calabashes made ofgourd, or in stone jars (Chelule et al., 2010). The major fermentedmilk products produced in Ethiopia by small holder farmers using tradi-tional methods are: Ergo (fermented sour milk), ititu (fermented milkcurd), kibe (traditional butter), neterkibe (kibe or traditional ghee), ayib(cottage cheese), and arera (sour defatted milk) (Gonfa et al., 2001).Relatively little is known about the microbiology and presumeddominating LAB of these fermentations. Two studies of traditionallyprocessed ititu showed that it contains high microbial numbers oflactobacilli with L. casei and L. plantarum as dominant species (Kasseyeet al., 1991; Gonfa et al., 2001).

The traditional Maasai fermented milk, kule naoto, is an importantpart of the daily diet of the Maasai community in Kenya and Tanzania.These people rarely consume fruits or grains, while their standarddaily diet comprises on average 2–3 L of kule naoto per person. Kulenaoto is produced from unpasteurized whole milk from Zebu cows inthe rural areas, with spontaneous fermentation in a calabash occurringover at least 5 days. Kenyans appreciate the product for its excellentnatural taste and aroma. In addition, they ascribe functional benefitsto consumption of kule naoto because of protection against diarrheaand constipation (Mathara, 1999). In a study on 300 LAB strains isolatedfromKenyan traditional kule naoto, the genus Lactobacilluswas found todominate the fermentation, followed by Enterococcus, Lactococcus andLeuconostoc. Interestingly, L. plantarum was the dominant species,with numbers ranging from 107 to 108 cfu/ml, while L. fermentum,L. paracasei and the L. acidophilus ‘group’ were detected at a level of106 to 108 cfu/ml (Mathara et al., 2004). The microbiologial safety ofkule naoto was indicated by the absence (detection level b log 2.0/ml)of Enterobacteriaceae in all samples (n = 16 out of 22) with pH b 4.5.

89C.M.A.P. Franz et al. / International Journal of Food Microbiology 190 (2014) 84–96

Yeasts were present as a minor population at pH values b 4.5, but werenot detectable (b log 1.0/ml) in sampleswith pHvalues≥ pH 4.5, wherenumbers of up to log 8.0 per ml of Enterobacteriaceae were present(Mathara et al., 2004).

3. Choice of multifunctional strains for African probiotics

As noted earlier and shown in Table 2, the predominant LAB associ-ated with traditional African fermented foods generally includelactobacilli and pediococci. Among the lactobacilli, L. plantarum andL. fermentum strains are particularly predominant in many traditionalAfrican fermentations (Mbugua, 1985; Olsen et al., 1995; Hounhouiganet al., 1993; Kostinek et al., 2005; Abriouel et al., 2006; Vizoso Pintoet al., 2006; Padonou et al., 2009; Njeru et al., 2010; Yousif et al., 2010;Oguntoyinbo and Narbad, 2012; Owusu-Kwarteng et al., 2012). Thewell-documented L. plantarum strain 299 V (Probi AB, Lund, Sweden)has been sold in some African countries for a few years, albeit in expen-sive capsule form, while less documented L. fermentum VRI003 (PCC)(Probiomics, Eveleigh, Australia) is also commercially available(Bhadoria and Mahapatra, 2011). Since both these species are known

Table 2African fermented food products and the microorganisms associated with the fermentation (e

Product Area of production Fermentable substrate

African, non-alcoholic cereal based foodsMahewu (magou) South Africa Maize, sorghum or millet

Ogi Nigeria, Benin Maize, sorghum or millet

Koko and Kenkey Ghana Maize, sorghum or millet

Uji East Africa Maize, sorghum or milletKisra Sudan SorghumHussuwa Sudan SorghumInjera Ethiopia SorghumTing Botswana, South Africa SorghumObusera Uganda MilletMawe Benin MaizeKunu-zaki Nigeria Millet, sorghumBogobe Botswana SorghumPotopoto Congo Maize

Dégué Burkina Faso MilletBen saalga Burkina Faso Millet

African fermented starchy root productsGari West Africa Cassava

Lafun Nigeria Cassava

Fufu NIgeria CassavaKivunde Tanzania CassavaChikawngue Zaire CassavaCingwada East and Central Africa CassavaKocho Ethiopia Ensette or Abyssinian banana

(Ensette ventricosum)Agbelima Ghana cassava

African fermented animal proteinsNono (milk curd) Northern part of West Africa MilkMaziwalala East Africa MilkLeban (sour milk) Morocco MilkWara West Africa MilkErgo Ethiopia MilkKulenaoto Kenya Milk

Sethemi South Africa Milk

Guedj Senegal FishBonome (stink fish) Ghana Fish

Ent: Enterococcus, L.: Lactobacillus, Lact.: Lactococcus, Leuc.: Leuconostoc, Ped.: Pediococcus, W.: W

to be associated with the human gastrointestinal system, they may bea good choice for addition to fermented foods. There is no shortage ofwell-studied, commercial probiotic strains used successfully in theNorth which could be useful in Africa, such as L. acidophilus LA1,L. paracasei CRL 431, Bifidobacterium lactis Bb-12 and L. rhamnosus GR-1 from Chr. Hansen, L. acidophilus NCFM from Dupont, L. acidophilusDDS-1 from Nebraska Cultures, L. acidophilus NCFB 1748 andL. paracasei F19 from Arla, L. paracasei Shirota and B. breve Yakult fromYakult, L. casei Immunitas (DN 114-001) and B. animalis (DN 173-010)from Danone, L. johnsonii LA-1 from Nestec, L. plantarum 299 V andL. rhamnosus 271 from Probi AB, L. reuteri SD2112 from Biogaia,L. rhamnosus GG from Valio Dairy, L. salivarius UCC118 from UniversityCollege Cork, B. longum BB536 from Morinaga, or B. lactis DR10 fromFonterra (Bhadoria andMahapatra, 2011; Sanders, 2003). Perhaps socialbusiness models could be set up by these profitable companies or bylocal Africans who simply utilize the strains (Reid et al., 2013a). TheYoba-for-Life (www.yoba4life.com) is one such model in whichgenericized L. rhamnosus GG is used to produce healthy yogurt in acommunity kitchen in Uganda, with expansion ongoing to other regionsincluding Zambia (Kort and Sybesma, 2012).

xcluding alcoholic beverages) (adapted and modified from Olasupo et al., 2010).

Microorganisms reported to be involved in the fermentation

L. delbrueckii subsp. bulgaricus; L. delbrueckii subsp. delbrueckii; Leuconostoc spp.;heterofermentative lactobacilliPed. pentosaceus, L. fermentum, L. plantarum, yeast (Saccharomyces cerevisiae,Candida kruseii)W. confusa, L. fermentum, L. salivarius, L. vaccinostercus, L. pantheris, Pediococcus spp.and yeastL. plantarum, L. paracasei, L. fermentum, L. buchneri, Ped. acidilactici, Ped. pentosaceusLABL. fermentum, Ped. acidilactici, Ent. faecium (minor proportions)Candida guillermondiiL. fermentum, L. plantarum, L. rhamnosusLABLact. lactis, Ped. pentosaceus, L. plantarumL. fermentum, P. pentosaceus, W. confusa, Ent. faecalisUnknownL. gasseri, L. plantarum/paraplantarum, L. acidophilus, L. delbrueckii, L. reuteri,L. casei, Bacillus spp., Enterococcus spp.L. gasseri, L. fermentum, L. brevis, L. casei, Enterococcus spp.L. plantarum and other LAB

L. plantarum, L. fallax, L. fermentum (predominating) W. paramesenteroides,L. brevis, Leuc. pseudomesenteroides (minor proportions), Strep. lactis, Geotrichumcandidum, Corynebacterium manihot (also reported)L. fermentum, L. plantarum, W. confusa, yeast (Saccharomyces cerevisiae, Pichiascutulata, Klyveromyces marxianus, Hanseniaspora guillermondii), and Bacillus spp.Ped. pentosaceus, L. fermentum, L. plantarumL. plantarum, other LAB, yeastLAB, yeastUnknownLAB yeast

L. plantarum, L. brevis, L. fermentum, Leuc. mesenteroides, also Bacillus spp.,Candida tropicalis, Geotrichum candidum, Penicillium spp.

LAB“Strep.” (Lact.) lactis, Strep. thermophilusLactic streptococci (lactococci), Leuc. lactis, Leuc. mesenteroides subsp. cremorisLactococcus lactis, Lactobacillus spp.Lactobacillus spp., Lactococcus spp.L. plantarum, L. fermentum, L. paracasei, L. acidophilus, also lactococci,leuconostocs and enterococciLactobacilli, lactococci, yeast (Debaromyces hansenii, Saccharomyces cerevisiae,Cryptococcus curvatus)Lact. lactisUnknown

eissella.

90 C.M.A.P. Franz et al. / International Journal of Food Microbiology 190 (2014) 84–96

The studies on kule naoto described above seem to strongly supportthe ‘recycling’ concept of beneficial microbes between the humanenvironment and the GI tract. Kule naoto is typically produced in closeassociation with the traditional Maasai living and cultural environment.The studies by Mathara et al. (2008a,b) and Vizoso Pinto et al. (2007,2009) have shown strains of L. casei, L. rhamnosus, L. plantarum and, inparticular, the L. acidophilus “group” (including L. johnsonii) all typicalof the human GI tract (; Holzapfel et al., 2001; Heilig et al., 2002;Vaughan et al., 2005), dominate in the Maasai milk fermentation.Moreover, these studies have confirmed their ability to survive pas-sage of the upper GI tract in high numbers, and their potential forbeneficial interaction under simulated conditions typical of thehuman GIT. The potential of some strains for technical implementationhas been confirmed (Patrignani et al., 2006), thereby suggesting the‘multifunctionality’ of at least someof the LAB strains typically associatedwith fermented Maasai milk.

The Food and Agriculture Organization andWorld Health Organiza-tion (FAO-WHO, 2002) published a guidelinepointing out requirementsfor products to be classified as probiotics. While some of these need tobe revised, the basic ones remain important, namely accurate identifica-tion of the strains, verification of safety and non-toxicity and the abilityto provide tangible physiological/health benefits as shown in random-ized clinical trials. For testing safety, the European Food Safety Authority(EFSA) QPS system for safety assessment may be utilized (EFSA, 2005).The approach for determining safety according to this document isbased on a safety decision tree. The EFSA took on the task to developthe QPS system within an EU regulatory framework. For each strain,safety needs to be shown based on the ‘body of knowledge’ of the strain,including description of known virulence determinants, as well as anti-biotic resistance patterns, some of which are intrinsic and not generallytransferred to other strains.

Studies that relate only to in vitro investigations on adhesion proper-ties, pathogen inhibition and stimulation of immune parameters incell culture, for example, are not sufficient to call a strain probiotic(FAO/WHO, 2002; Reid, 2005; Anukam and Reid, 2009). Nevertheless,in vitro investigations are good to inform about strain properties. Func-tional cell models of the gut can be very useful to investigate potentialprobiotic activities of strains. Simple cell cultures models involvingsmall intestine or colon enterocytes such as Caco2 or HT29 cells haveoften been used to investigate probiotic activities of LAB strains as relat-ing to adhesion or prevention of pathogen invasion. Improved function-al mammalian cell cultures are becoming more and more available,which consist of cells grown on a microporous membrane in a well,and which are underlaid with immune cells such as dendritic cells ormacrophages in so-called 3-D or trans-well cell cultures (Cencic andLangerholc, 2010). As these consist of three components (epithelia, im-mune cells andmicrobiota) themodels are closer to the in vivo situationand are thus better suited for studying probiotic activities such as adhe-sion or host/beneficial bacteria/pathogen interaction (Cencic andLangerholc, 2010), as well as for assessing aspects of strain safety.Validated, in vitro gastrointestinal models such as the TIM-1/TIM-2or the (M)SHIME models, are also excellent tools to study probioticactivities such as potential survival in the gastrointestinal tract andtheir effect on the gutmicrobiota homeostasis after antibiotic treatment(Rehman et al., 2012; Venema and van den Abbeele, 2013).

For a product to be classified as probiotic, however, it needs to havebeen proven to confer a health benefit. For this, well-controlled humanclinical trials are required (Anukam and Reid, 2009). In the age ofmetagenomics, these trials should ideally also include sequencing ofthe appropriate microbiome (gut, oral, skin, vagina) and determinationof the impact of the probiotic on microbiota homeostasis. The humangut microbiota has received considerable attention in recent years andmetagenomic studies have shown a large variability in the microbiotacomposition of individuals (Arumugam et al., 2011). Although sizeablevariation between individuals exists,most can be categorized as belong-ing to one of three groups, so-called ‘enterotypes’, based on the

predominance of either of the genera Bacteroides, Prevotella orRuminococcus (Arumugam et al., 2011). However, these groups maybe better characterized as the ratio of abundance of Bacteroides andPrevotella, with the Ruminococcus enterotype folded into the Bacteroidesgroup (Wu et al., 2011; Clemente et al., 2012). Host genetics seem toplay a role in establishment and shaping of the gut microbiota(Benson et al., 2010; Spor et al., 2011), but diet is the driving force forthe formation of the different enterotypes (Wu et al., 2011).

So far only limited studies have focused on the gut microbiota ofAfrican individuals, and usually not in the context of probiotic interven-tion trials. In one study, De Filippo et al. (2010) used ametagenomic ap-proach to compare the composition of the gut microbiota of children(1–6 y) from Italy, who consume a Western-style diet (high in starch,sugar and animal protein, low in fiber), and from rural Africa (BurkinaFaso), whose diet consists mainly of cereals, legumes and vegetables(high in fiber, carbohydrates and non-animal protein). The gutmicrobi-ota of the African children showed a highly significant enrichment inBacteroidetes and a depletion in Firmicutes, with a unique abundanceof bacteria from the genera Prevotella and Xylanibacter which containgenes for cellulose and xylan hydrolysis, that were completely lackingin the EU children. Furthermore, the gut microbiota was far morecomplex than that of EU children. It has been suggested that the gutmicrobial richnessmay have a healthpromoting effect by offering betterprotection against pathogens and gastrointestinal disease. The authorshypothesized that the African diet correlated with a gut microbiotathat allowed maximum energy intake from fibers while at the sametime protecting against inflammation and non-infectious colonicdisease (De Filippo et al., 2010).

A later comparative study on the gut microbiota of 6 month oldinfants from Malawi and Finland showed considerable differences(Grzeskowiak et al., 2012) with that of the former in that the gutmicro-biota of Malawian children was dominated by bifidobacteria and theproportion of Bacteroides–Prevotella group was also much higher. Onthe other hand, Bifidobacterium adolescentis, Clostridium perfringensand Staphylococcus aureus were absent in Malawian, but detected inFinish infants. The authors noted that B. adolescentis has been associatedwith inflammatory effects and that allergic infants are often colonizedby C. difficile and S. aureus, but less so by bifidobacteria (Collado et al.,2010; Grzeskowiak et al., 2012). Interestingly, higher levels of Clostridiumspecies and staphylococci have been reported in overweight infantsand adults (Ley et al., 2005; Collado et al., 2010; Grzeskowiak et al.,2012).

One study investigated the fecal microbiota of African children withhealth problems, and showed that anemic children from the Ivory Coastcarry an unfavorable high ratio of fecal enterobacteria to bifidobacteriaand lactobacilli, which was even increased by iron fortification(Zimmermann et al., 2010). A further study showed that the gut micro-biota was a causal factor in the development of kwashiorkor, an enig-matic form of severe acute malnutrition in Malawian children thatappeared to be characterized by Bilophila wadsworthia, a bacteriumlinked to inflammatory bowel disease, and Clostridium innocuum, a spe-cies which can function as an opportunist in immunocompromisedhosts (Smith et al., 2013).

From the above studies (De Filippo et al., 2010; Zimmermannet al., 2010; Grzeskowiak et al., 2012) it appears that in healthyAfrican children a diet high in fiber and carbohydrates appears to se-lect a microbiota that is highly diverse, protective against infectionand inflammation and which extracts maximum energy from thelow calorie food. This supports the potential use of indigenousfermented plant foods as vehicles for probiotics in the African set-ting. In terms of disease such as anemia or severe under nutritionin children, fermented foods may become more important to affectthe gut microbiota. Restoration of gut functionality by interventionsthat stimulate the diversity of bacteria, and through probiotics withimmune stimulatory activity, appears to hold great promise andshould be further investigated.

Table3

Ove

rview

ofstrainsstud

iedas

potentialc

andida

teprob

ioticstrainsforusein

diffe

rent

African

ferm

entedfood

.

Strains

Source

ofstrains

Inoc

ulated

/con

sumed

African

food

prod

uct

Region

/cou

ntry

inwhich

theferm

ented

African

food

prod

uctisco

mmon

lyco

nsum

edRe

ferenc

e

Enterococcus

faecium

Raw

cow

milk

fortheprod

uction

ofno

noOnlyin

vitrostud

ies

Nigeria,S

ub-Sah

aran

Africa

Banw

oet

al.(20

13)

L.plan

tarum,L.fermen

tum,

Ferm

entedmaize

doug

h,cassav

a,Gari

Onlyin

vitrostud

ies

Gha

naJaco

bsen

etal.(19

99)

L.plan

tarum,P

ediococcus

spp.

Fufu

andog

iOnlyin

invitrostud

ies

Nigeria

Sann

ietal.(20

13)

Pediococccus,Lactoba

cillu

ssp

p.,Lactoba

cillu

sferm

entum

pearlm

illet

slurry

Onlyin

vitrostud

ies

BurkinaFa

so,G

hana

Turpin

etal.(20

11)

L.ferm

entum

kimere:

pearlm

illet

doug

hOnlyin

vitrostud

ies

Ken

yaNjeru

etal.(20

10)

L.john

sonii(BF

E61

28,B

FE61

54),L.

plan

tarum

(BFE

5092

,BFE

5759

,BFE

5878

),L.

acidop

hilus,

L.pa

racasei,L.

rham

nosus,L.

ferm

entum

Kulena

oto:

Maa

saifermen

tedmilk

,Kwerionik:

ferm

entedmilk

prod

uct

Onlyin

vitrostud

ies

Ken

ya(K

ulena

oto)

Uga

nda(K

werionik)

VizosoPintoet

al.(20

06,2

007,

2009

),Ch

oet

al.(20

10),Nielsen

etal.,(2

010)

,Matha

raet

al.,(2

008a

,b)

L.acidop

hilus,L.

pentosus

Ferm

entedco

w'smilk

andcassav

aFe

rmen

tedcereal,m

aize

grue

lW

estAfrica

Ope

reet

al.(20

03)

Lactic

AcidBa

cteria,m

ainlyWeissella

confusa

andL.

ferm

entum

Kok

oan

dko

kosour

water:from

millet

porridge

Kok

osour

water

NorthernGha

naLe

iand

Jako

bsen

(200

4),Lei

etal.(20

06)

GR-

1Milk

yogu

rtan

dmilk

yogu

rtwithMoringa

powde

rEa

st-A

frica,

Tanz

ania

Wen

ner(2

009)

,Van

Tien

enet

al.(20

11)

91C.M.A.P. Franz et al. / International Journal of Food Microbiology 190 (2014) 84–96

A number of studies on the in vitro probiotic potential of predomi-nant LAB isolated from African fermented foods have been carried out,as will be discussed below. However, their potential health benefitshave unfortunately not been subject to many intensive investigationsin well-controlled, randomized clinical trials in Africa. To date, nometagenomic studies have accompanied such intervention studies.The severe lack of government investment in R&D and lack of industryand philanthropic funding makes this a major challenge. For selectingprobiotic foods in the African setting, both the probiotic strain(s) andthe food matrix need to be considered.

3.1. The search for potential probiotic strains to supplement Africanfermented foods

Table 3 gives an overview of strains studied as potential probioticcandidates for use in African fermented foods. Banwo et al. (2013)investigated Enterococcus faecium strains isolated from cow's milk fortheir technological properties and probiotic potential in a fermentedmilk product nono. Two strains were shown to be tolerant to bile salts,possess antagonistic activity against foodborne pathogens includingBacillus cereus and Listeria monocytogenes and showed strong acidifica-tion properties. The strains were non-hemolytic and exhibited no anti-biotic resistance. Although there are some well-known examples ofprobiotic enterococci on European markets, these bacteria are stilloften viewed as problematic becausemany strains belonging to specificgenetic subsets are known to be involved in nosocomial infections(Franz et al., 2011b).

Jacobsen et al. (1999) investigated L. plantarum and L. fermentummaize dough strains from Ghana and found that some were poorlyadherent to Caco-2 cells but survived 4 h at pH 2.5, while growth wasnot delayed in 0.3% oxgall. Some strains showed antimicrobial activitytowards L. monocytogenes, S. aureus and B. cereus strains, but productshave not been tested in humans.

In an investigation of potential technological/probiotic characteris-tics of LAB from fufu and ogi, Sanni et al. (2013) showed that manystrains, especially pediococci, produced H2O2, and were toleranttowards bile and low pH. Some of the L. plantarum strains showedgood adherence capacity to mucus-secreting HT29 MTX epithelial cellsin cell culture (Sanni et al., 2013). Another L. plantarum strain KCA-1,isolated from the healthy vagina of a Nigerian woman, is the firstAfrican strain to be genome sequenced (Anukam et al., 2013), and itholds promise for prevention of urogenital infections in women as avaginal probiotic.

Turpin et al. (2011) investigated LAB from African pearl milletslurries (a ben-saalga model) using a metagenomics identificationapproach of bacteria present in the fermented food, and found speciesof Pediococcus and Lactobacillus to predominate, in particularL. fermentum strains. Genes associated with survival through passagethrough the gastrointestinal tract were found in both themetagenomesand in the isolates, but only L. fermentum strains showed good surviv-ability in low pH. There was also a wide distribution of genes associatedwith bile salt tolerance. Genetic screening revealed a potential for folateand riboflavin synthesis in both the metagenomes and the isolates. Theidea is for LAB capable of producing B vitamins to be used to fortifycereal based foods (Turpin et al., 2011). A study by Njeru et al. (2010)showed that L. fermentum was also the dominant species isolated inkimere, a spontaneously fermented pearl millet dough produced inKenya. All the L. fermentum strainswere able to grow in vitro inmediumcontaining 0.3% oxgall and 12 strains even grew in the presence of 3%oxgall. Of the latter, 60% of the strains survived incubation at pH 3 for3 h.

Our studies on LAB from the Maasai fermented milk productkule naoto (Mathara et al., 2008a,b) aimed to investigate the functionalcharacteristics of the predominant bacteria. Lactobacillus paracasei,L. plantarum, L. rhamnosus and isolates of the L. acidophilus groupshowed resistance to gastric juice and bile, while some expressed bile

92 C.M.A.P. Franz et al. / International Journal of Food Microbiology 190 (2014) 84–96

salt hydrolase activity and the ability to assimilate cholesterol in vitro.Adhesion to HT29 MTX cells of up to 70% of total bacterial cells addedto the HT29 MTX cells was recorded after 1 h incubation, and also ahigh survival rate under simulated stomach acidic conditions and phys-iological bile salt concentrations (Mathara et al., 2008a,b). The choice ofcells used for adhesion studies in cell cultures is quite critical to deter-mine the adhesion capability of potential probiotic strains. Caco2 andHT29 cells do not produce mucus, while the HT29-MTX cell line is amucus-producing cell line which mimics the intestinal situation moreclosely. Thus, binding assays can obtain quite different results whenusing HT-29 and HT29-MTX cell lines, as reported by Turpin et al.(2012), who showed that some, but not all, strains from fermentedfoods have higher binding capability on HT29-MTX cells when com-pared to HT29 cells. Quite interesting, in that study, some of the strainsfrom African fermented pearl millet showed a far higher bindingcapability to HT29 and HT29-MTX cells than established probioticstrains that are on the market (Turpin et al., 2012).

In our studies (Mathara et al., 2008a,b) technological featuressuch as growth kinetics in fresh heat-treated whole milk medium andsurvival in the final product during storage at 4 °C were also studiedfor several strains and L. acidophilus BFE 6059, L. paracasei BFE 5264and Lactococcus lactis BFE 6049 from kule naoto showed potential foruse as starters for milk fermentation (Patrignani et al., 2006). Fivestrains identified as L. plantarum and two as L. johnsonii showed goodsurvival under simulated gastrointestinal conditions, and expressedantimicrobial activity. All strains exhibited bile salt hydrolase activi-ty, while, interestingly, only the L. plantarum strains showed β-galactosidase activity (Vizoso Pinto et al., 2006). The strain BFE6128 was identified as L. johnsonii, and confirmed to have interestingpotentially probiotic properties (Vizoso Pinto et al., 2007, 2009). It wasalso studied for modulation of signal pathways involved in innateimmunity in enterocytes. It sensitized HT29 intestinal epithelial cellstowards recognition of Salmonella enterica serovar Typhimurium by in-creasing the IL-8 levels released after challenge with this pathogen,and by differentially modulating genes related to toll-like receptor(TLR) pathways and innate immunity.

Other L. plantarum strains from kule naoto showed potential asprobiotics as they adhered well to enterocytes and prevented invasionof pathogens in cell culture. They expressed antimicrobial activity to-wards foodbornepathogens and stimulated IL-8 production in intestinalepithelial cells in vitro (Vizoso Pinto et al., 2009). One of these strains,L. plantarum BFE 5092, had a bacteriocin locus and the genes for produc-tion of plantaricins EF, JK and N (Cho et al., 2010), with antimicrobialactivity towards L. monocytogenes (Nielsen et al., 2010). In summary,the pool of potential probiotic strains is sizeable, at least based uponfeatures believed to be useful in the gastrointestinal tract.

3.2. Intervention studies with African potential probiotics

Animal experiments have provided some rationale for humanstudies in Africa. Opere et al. (2003) showed Lactobacillus startercultures of a fermented cereal gruel prevented shigellosis in a murineanimal model. Whereas all control mice died upon Shigella dysenteriaeinfection, 33 to 100% of mice previously receiving a diet of an Africanfermented complementary food for young children containingL. acidophilus and L. pentosus survived, depending on the starter strainor combination of strains used to ferment the gruel.

Lei and Jakobsen (2004) studied the microbiology of the millet por-ridge koko and koko sourwater (the fermented liquid top layer develop-ing during koko fermentation) produced in Ghana containing approx.108 live LAB perml and proposed it as a probiotic drink. An interventionstudy was undertaken in which children of less than 5 years of agecoming to northern Ghana health clinics for treatment of diarrheawere randomized, to receive treatment for diarrhea and in addition upto 300 ml fermented koko sour water daily or a control drink for5 days after enrolment (Lei et al., 2006). Among the 184 children

included, no effects of the intervention were noted for stool frequency,stool consistency and duration of diarrhea, however, the koko sourwater treatment was associated with greater reported well-being14 days after the start of the intervention.Many of the children receivedantibiotics as normal part of the treatment, and this was suggested tohave masked probiotic effects.

With an initial goal of alleviating gastrointestinal abnormalitiesamong HIV infected subjects in sub-Saharan Africa, a communitykitchen project was established in Mwanza, Tanzania in 2004. Localmothers were taught to produce yogurt supplemented with probioticL. rhamnosus GR-1 (named Fiti), with good sensory and anti-diarrhealproperties (Hekmat and Reid, 2006; Anukam et al., 2008). This grassroots initiative holds great potential to deliver health and nutritionalbenefits to a wide population (Wenner, 2009; Reid, 2010). It washoped the yogurt could improve gastrointestinal integrity and reducethe incidence and severity of opportunistic infections among peoplewith HIV. Irvine et al. (2011) performed a retrospective study, andpreliminarily results suggested that yogurt supplemented withL. rhamnosusmay effectively alleviate GI symptoms and improve work-place productivity, nutritional intake and tolerance to antiretroviraltreatment. Further support for these benefits has emerged (Reid,2010; Hummelen et al., 2011a; Whaling et al., 2011), but more studiesare needed. The addition of micronutrients to the probiotic Fiti wasdesigned to further improve gut health and nutrient status, and twodifferent formulations offer hope in that regard, especially locallysourced Moringa (Hemsworth et al., 2011, 2012; Hummelen et al.,2011b; Van Tienen et al., 2011).

4. Further perspectives

Despite arguably Africa having the greatest need for probiotics withanti-infective characteristics, the introduction of products remains a for-lorn hope more than a reality. The perception that the marketplace istoo small is short-sighted, and while cold chain networks and effectivetransportation systems are believed to be poor, they do not preventmajor soft drink producers from establishing vast product distributionacross the continent. Indeed, if sugar-laden drinks can be affordable tolarge populations, often being cheaper than bottled water, why can'tprobiotic drinks be made available? Companies need to ask thesequestions themselves, and recognize that the African market potentialis extremely large in the long term.

There is undoubted disparity of income in many African countries,like everywhere else, but the extreme poverty, malnutrition and expo-sure to malaria, tuberculosis and HIV make the plight of the poor allthe more catastrophic. Failure to transfer probiotic products to Africathat are currently improving the lives of hundreds of millions of peopleelsewhere, is tantamount to a neglect. Nevertheless, for those scientistsand businesses trying to make a difference, solutions are slowly beingexplored. These include: (i) the establishment of networks in whichlocal people can set up their own kitchens and produce probioticfoods; (ii) the isolation of African bacterial strains from traditionalfoods, anddeveloping them for probiotic applications; and (iii) attemptsto industrialize fermented food processing to account for regional varia-tions in raw materials, recipes and production methods (Nout, 2009),and starter culture technology (Holzapfel, 1997, 2002). In terms of gov-ernment engagement, the situation remains dire even as countriesemerge from extreme poverty and copingwith the HIV crisis. Infrastruc-ture development in South Africa, Zambia, Rwanda, Kenya and othercountries is clear for all to see, and investment especially from China,as well as philanthropic contributions from non-governmental organi-zations and the Bill and Melinda Gates Foundation and Welcome Trust,to name two, have made significant differences. Yet, governments arenot investing in funding their own scientists to develop novel foodsand health remedies. This needs to change. A roadmap for R&D inprobiotics was proposed 8 years ago, without success: a) funding of tri-als at the local level to assess the applicability to different populations,

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b) the introduction of proven products even if the price is not as high aswould be attained in the developing world, c) exchange of students toenhance training and eventually the development of new researchcenters focused on probiotic foods, d) introduction of the concepts ofprobiotics to healthcare professionals who are not aware of this fieldand lastly, e) connecting to local industries, especially dairies, to provideadvice and training on development, production and distribution ofprobiotic foods (Reid et al., 2005).

5. Recommendations and research needs

5.1. Choice of multifunctional strains

The probiotic strains chosen for African probiotic foods should bewell studied for safety, probiotic characteristics and health benefits.For this, in vitro tests and functional mammalian cell cultures could beemployed for initial screening, followed by controlled and randomizedclinical trials to assess health benefits. The strains chosen should ideallyhave multifunctional characteristics so that they can ferment food andpredominate in the product, while maintaining probiotic properties inthe human gastrointestinal tract. As many African fermented foods arepredominantly fermented by lactic acid bacteria and because theseorganisms are widely used in probiotic foods, such microbes could beused at the present time. Yeast strains also have a track record infermented foods, and even Bacillus spp. used for African alkaline plantprotein fermentations, so many options are available to try (Parkoudaet al., 2009). What is furthermore needed are production facilities forstarter cultures. The production of probiotic foods relies on the abilityto propagate cultures and to scale up starter production for producingsufficient material for controlled fermentations from household tosmall to medium enterprise scale.

5.2. Choice of product for probiotic delivery

In our view, cereal or starch/cerealmixed fermentations,with awidedistribution throughout Africa should be a priority and their consump-tion should be emphasized. To circumvent heat killing of the beneficialbacteria, a portion of the finished fermented product could be ingestedbefore heating (as in the case of koko sourwater), or products promotedthat do not require heating, like the South Africanmaize gruel amahewuthe Sudanese hussuwa or the Tanzanian cereal root crop beverageTogwa which is cooked before fermentation. It is worth investigatingalso whether a portion of the fermented material could be re-introduced to other fermented slurries or gruels such as ogi or uji.Furthermore, new products can be created, such as ‘sorghurt’ producedby steeping and malting sorghum grains, followed by heating, drying,mixing with water, inoculation with starter culture and fermentation(Sanni et al., 2013).

Products that use traditionalmilk fermentation similar to amasi, ergoor kule naoto can be produced immediately if there is a will in regions ofAfrica where milk is plentiful. Different products could take advantageof the rich variety of indigenous vegetables, many of which are abun-dant micronutrients, if suitable cooking and storage conditions can beresolved (Kimiywe et al., 2007). It is time to take advantage of highlynutritive plants that convey health benefits. In addition to Moringa,there is amaranth (Amaranthus spp.), African nightshades (Solanumspp.), spiderplant (Gynandropsis gynandra) and cow pea leaves (Vignaunguiculata). Results from the limited metagenomic studies on the gutmicrobiota of African children indicate a richness of microbiota inhealthy African children with a predominance of Bacteroides associatedwith the high fiber, non-animal protein and carbohydrate rich diet. Thefermentation of indigenous leafy vegetables with multifunctionalstarters would be especially interesting for further development.While there is always a need formore research, there is already substan-tial evidence for using fermented foods to impact the health of Africans,especially given the levels of disease and malnutrition.

5.3. Improving research infrastructure

There is certainly cause for optimism, with the presence of researchcenters in SouthAfrica, Kenya andNigeriawith expertise inmicrobiomeresearch, and many sites including government run Ministries of Agri-culture, where small strides are being made to merge local foods withhealth benefits. The establishments of probiotic and/or NutraceuticalCenters of Excellence are achievable,whether based at local universities,hospitals, research institutions or independent sites, and would giveAfrican probiotic research a needed boost. The problems ofmalnutritionand disease, child mortality, poverty and social inequity can only besolved by collective and collaborative ventures whose pre-eminentfocus is the betterment of fellow humans. As the Scottish Early YearsCollaborative is demonstrating, it is through broad societal partnershipsthat change can occur. The establishment and funding of further closescientific collaborations between Europe and Africa should be a priorityto address the problems of malnutrition, disease and child mortality. Itmight seem strange or even contradictory to some that microbes andfermented foods harbor such potential for paradigm shifting change.But, after all, humans are mostly microbial in content, food is their ne-cessity and as multiple recent studies are showing, these organismsare integral to all aspects of human and animal development, longevityand ultimate survival (de Vos and de Vos, 2012; Hughes, 2012; Trivedi,2012; Biagi et al., 2013; de Vos and Nieuwdorp, 2013; Fang and Evans,2013; Loscalzo, 2013; Pennisi, 2013; Reid et al., 2013b). We wonderwho is willing to join this friendly revolution.

Acknowledgments

The authors from theMax Rubner-Institute and Jomo Kenyatta Uni-versity of Agriculture and Technology would like to thank the GermanFederal Ministry for Education and Research for funding of the project:Diversifying Food Systems, Horticultural Innovations and Learning forImproved Nutrition and Livelihood in East Africa (HORTINLEA) (ProjectNo. FKZ 031A248I). The responsibility for the article content lies solelywith the authors.

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