Development of Starter Culture for the Production of African Condiments and Seasoning Agents

As a process, food fermentation dates back at least 6000 years and is likely to be derived from microbial interactions of an appropriate nature. Fermentation has allowed our ancestors to survive the winter season in temperate and cooler regions and those in the tropics to survive the periods of drought by improving food shelf-life and safety. Traditional fermentation process is still used as a replacement where there is no refrigeration or other means available for food storage. In general, fermented foods can be defined as foods produced through controlled microbial growth and the conversion of food components through enzymatic actions. They are generally appreciated for characteristics such as pleasant taste, aroma, texture and improved cooking and processing properties. Microorganisms contribute to the development of characteristic properties such as taste, smell, visual appearance, texture, shelf-life and protection by virtue of their metabolic activities. Enzymes indigenous to the raw materials may play a role in enhancing these characteristics. The use of starter cultures is a hallmark of industrial food fermentation and their introduction has been followed by a continuous search to improve them. Examples of desired properties in starter cultures include robustness during manufacturing, fast growth, high biomass yield and product yield and specific organoleptic properties. Quality, safety and acceptability of traditional fermented foods may be significantly improved through the use of starter cultures selected on the basis of multifunctional considerations, also taking into account the probiotic concept and possibilities offered for improved health benefits. Focused studies toward the introduction of starter cultures for small-scale


Food Fermentation
Fermented foods are major sources of nutrition across Africa [1]. Traditional fermented food is an important part of the cultural heritage and diet that has been widely consumed since ancient times [2]. The process of fermentation involves the breakdown of organic compounds into acids or alcohol through enzymatic action of microorganisms particularly yeasts, bacteria and moulds under aerobic or anaerobic conditions [3]. It has been shown that fermentation enhances the nutritional, healthy, organoleptic and preservative properties of food [4]. As a technology that increases income sources, food availability, food diversity, reduces post-harvest losses and helps sustain food sovereignty, it plays an important role in developing economies. Most fermented foods are traditionally produced in Africa, resulting in differences in the substrate used, manufacturing conditions, packaging materials, handling and storage practices [5], and eventually significant differences in the final product.
Fermentation has been used as a means of preserving and improving the organoleptic properties of food for millennia [6]. Indeed, it has recently been revealed that more than 5000 varieties of fermented food and beverage are consumed worldwide [7]. Well-known examples include alcoholic beverages like beer and wine, dairy products such as yogurt and cheese, fermented vegetables such as pickles and sauerkraut, and fermented meat in various types of sausage.
Fermentations are carried out by a variety of microorganisms: alcoholic fermentations normally involve yeast; alkaline-fermented foods normally involve a mixture of Bacillus spp.; and lactic acid bacteria are most usually found in fermentations where lactic acid is the end product while fermented legumes and cheeses involved moulds [8].
Traditional diets in Africa often lack variety and consist of large quantities of staple food (grains and tubers/root crops) with supplements of plantain, cocoyam, rice and beans, based on seasonality and availability [9] [10]. Soups eaten with staples are an essential part of the diet and may contain a wide variety of seeds, nuts, pulses and leaves [11]. The staple food supplies calories but is poor in other nutrients. Soups are key sources of protein and minerals, and one way to improve diet has been to increase the nutrient content of soups [10]. Leguminous seeds can account for up to 80% of the dietary protein and can be the only protein source for certain groups. These are consumed as meals and are commonly used as condiments to improve the taste of food in fermented form [12] [13] [14] [15]. With a high protein content, leguminous condiments can serve as a tasty supplement to sauces and soups and can be used as a substitute for fish or meat, especially for the very poor [10].
Fermented foods are regarded as major dietary constituents in many developing countries, mainly because of their environmental quality, protection and conventional acceptability. Food fermentation as a process is at least 6,000 years old and is likely to be developed from sufficient microbial interactions. Fermentation has allowed our ancestors to survive the winter season in temperate and cooler regions and those in the tropics to survive the drought, improving shelf life and food safety. Over the years, fermentation has had a significant impact on dietary patterns and behaviors, on culture, and on the industrial processing and storage of food. Traditional fermentation processes also act as a replacement in the absence of refrigeration or other means of food safety [21].
Fermented foods can generally be defined as foods produced through controlled microbial growth and the conversion of food components through enzymatic actions. These are generally appreciated for characteristics such as good taste, flavor, texture and enhanced properties for cooking and storage. Microorganisms contribute to the development of characteristics such as taste, flavor, visual appearance, texture, shelf life and safety by virtue of their metabolic activity. Indigenous enzymes or enzymes generated by fermenting organisms may play a role in improving these characteristics [22]. Modern skills have been developed to monitor technological parameters during fermentation processes through trial and error. Research has also shown that the back-slop or inoculation of raw materials with residues from the previous batch speeds up the initial fermentation phase and facilitates beneficial improvements during the fermentation process. Reviews of considerable depth on traditional fermented foods have been published by Odunfa [12], Iwuoha and Eke [23] and Achi [10]. Against the background of the importance, benefits and improvement of traditional fermented condiments, the aim of this review is to assess the current state of knowledge of African fermented food condiments, with particular emphasis on the development of starter culture, for the production of safe, acceptable condiments capable of meeting the GMP requirements for global trade food.

Substrates Used in Major African Fermented Condiments
In Africa, many names are used for the multitude of fermented food condiments. Table 1 indicates the variety of names attributed to the items in various parts of the continent. The precise origin of these names could be traced to 1) the region or area of manufacture 2) the type of legume or oilseed used and 3) the spelling of the product by region or area or even food use. Conventional substrates for condiment production are varied and can be produced from more

Dawadawa
Dawadawa is one of the most common fermented alkaline condiments produced in various parts of Africa [12] [25] [37]. It is derived from African locust bean seeds (Parkia biglobosa) and is known by different names in various parts of Africa [38]. However it can also be produced from soybeans [25]. Soy-dawadawa is a food condiment produced from the fermentation of soybean (Glycine max (L.) Merr.) seeds [39]. Dawadawa is the name widely used in Nigeria and Ghana, while in Nigeria there are other regional names such as iru, subbala, netetu and afitin or sonru [40], Burkina Faso [41], Senegal [42], and Benin [43], respectively. Dawadawa is used to enhance the taste of soups, sauces and other dishes prepared [44]. It is also reportedly often used by low-income families as a low-cost meat substitute [45] [46], In addition to its flavoring qualities, it contributes significantly to the intake of protein, essential fatty acids and group-B vitamins [47] [48] [49] [50]. The African locust bean seeds are rich in proteins (30% -40%).
Dawadawa processing operations include cleaning, boiling, de-hulling, washing, re-cooking and spontaneous fermentation for the production of food condiment [53]. Dehulling is considered to be very tedious, extremely fuel-consuming and least hygienic of the operational phases for the conventional dawadawa production. The beans are boiled for 8 to 12 hours using firewood as fuel to dehull the seeds [54]. The boiled locust bean is then dehulled by bare feet mashing usually near riversides or using mortar and pestle [55] or rarely by minimal picking. This allows the cycle time to be partially reduced with the seeds [52].
Different studies have been done to develop the current process and make it less labor-intensive [10] [48] [56]. In an act to improve dawadawa production technology, studies have been conducted in several sub-regional West African countries, including the development of a dehuller for locust bean seed [57] [58]. The design and implementation of a dehulling device in the soumbala production process in Burkina Faso significantly reduced the cooking time from 24 hours to 6 hours [52].

Okpehe
Okpehe or Okpeye is a popular fermented soup condiment made from seeds of Fermentation occurs at temperatures of 30˚C -35˚C within 3 -5 days, resulting in a sticky, dark brown and pungent-smelling mash. The condiment is generously added to soups in many homes as a flavoring agent, but in some instances, it is also used by both urban and rural people as a meat substitute [30] [62].

Ugba
Ugba, a result of alkaline fermentation of oil bean seeds (Pentaclethra macrophylla) is very common in Southern Nigeria among Igbos and other ethnic groups [63]. The product acts as a food flavoring agent as well as a delicacy. Ugba is very rich in protein as an important nutritional item [63]. It similarly plays an economic, social and cultural role among the Igbos in the Eastern part of Nigeria. The production of ugba is usually seen as a family business. However, it has grown to become a trend that is handed over from one generation to another [63]. Its production process has been described previously as alkaline fermentation of African oil bean seeds [64] [65] [66]. Although the production method differs from one group to another and from one processor to another, a similar end product is widely manufactured in Southeast Nigeria, which usually comes with pungent ammonia-like smell [67]. A significant finding in its development is that the boiling time and the seed dehulling method differ. Obeta [68] reported 16 -18 h of boiling, Odunfa and Oyeyiola [69] and Odunfa [48], reported initial 12 h boiling time, while Njoku and Okemadu [70], boiled the seeds for 5 -8 h. Sokari and Wachukwu [71] used toasting of the bean seeds in hot (ca.100˚C) sand and holding for a further 30 mins at 100˚C to dehull the seeds. Upon dehulling, the cotyledons were either cut or cooked for 30 minutes or longer.
In addition, various techniques are used in the fermentation process. Odunfa and Oyeyiola [69] stated that the cotyledons were placed in a clean pot, covered and fermented with or without salt for up to 5 days at room temperature. On the other hand, Sokari and Wachukwu [71] reported that sliced cotyledons were washed in a basket lined with banana leaves (Musa sapientum Linn.) and then wrapped (around 40 -50 g of slices per wrap) using another leaf (Mallotus oppositifolius) and incubated at room temperature for 72 hours.

Ogiri
Ogiri is an alkaline fermentation of melon seeds (Citrullus vulgaris) produced in Nigeria as a food condiment. It is used for flavoring and also as a protein supplement [72]. For ogiri production, shelled melon seeds are cleaned by sorting  [72] found the pH of the final product to be around 7.9.

Owoh
Owoh is a food condiment with a final pH of about 9.0 that is popular in mid-western Nigeria and is processed by alkaline cotton seed fermentation (Gossypium hirsutum L.) [37]. Seed in its natural state is not edible because of its anti-nutritional content, especially gossypol, which inhibits digestion, but the fermented seeds are edible [34]. Owoh is prepared by boiling cotton seeds for about 2 hours until they are soft. The seeds are then soaked overnight in water and then dehulled by hand. Wrapped in banana leaves, the cotyledons are boiled again for 1 -2 hours. The packages are placed in earthenware pots or calabash trays after draining, covered with jute sacks and allowed to ferment into Owoh for 2 -3 days. The mash may also be ground and moulded into balls. To extend its shelf life it is smoked over charcoal [73].

Bikalga
One of the most common condiments in Burkina Faso is Bikalga, also known as dawadawa-botso in Niger Republic, datou in Mali, furundu in Sudan and mbuja in Cameroon. It is a product of Hibiscus sabdariffa L, alkaline fermentation commonly referred to as Roselle or sorrel [37]. Bikalga is used to flavour most dishes and is a good source of protein (22 -30 per cent), lipids, carbohydrates, essential amino acids and fatty acids in the diet [74] [75]. Bikalga production includes seed washing, overnight cooking (12 -24 hours) with the addition of liquid ash as a softening and alkalizing agent, followed by 3 -4 days of fermentation, crushing, moulding, overnight steaming and finally sun drying. Bikalga is steeped in lukewarm water during the preparation of food for a few minutes and steep water is used to make stews, soups, sauces and other foods [37].

Biochemical Changes Accompanying Fermentation
Proteins, fats and carbohydrates are the major constituents of legumes and oilseeds used in the production of condiments. The organisms responsible for fermentation must therefore be capable of utilizing or hydrolysing these components [10]. These carbohydrates are associated with abdominal distention and flatulence in humans [77]. Fermentation reduces total soybean flatus [81] and groundnut [82] and maybe other seeds are used to produce condiments. Trypsin inhibitor activity of Bambara groundnut [19] was also reduced during fermentation. Consequently, fermentation of oilseeds to produce condiments usually leads to a reduction in seed antinutrient components.

Proteins
The extent of protein hydrolysis is one of the most important factors associated with changes in the texture and flavor of the substrate in most fermented high-protein products [83]. This is a strong reason why fermentation is critically controlled away from traditional unregulated processes.

Lipid
Significant leguminous lipolysis yields primarily oleic, linoleic and linolenic acids [50] [85] [86]. These free fatty acids were associated with non-specific antitryptic activity, particularly oleic, linoleic and linolenic acids [81]. As such, extensive lypolysis could diminish nutritional quality. Although oil accounts for up to 40% of the legumes used in food fermentation, extensive lipolysis typically does not occur. Odunfa [12] during dawadawa production reported low levels of lipase activity in Parkia biglobosa. The same author had previously reported low lipase levels in fermentation of melon seeds [48]. Similarly, Njoku and Okemadu [70] observed minimal participation of lipase in Pentachletera macrophylla during ugba production. Low lipase activity in some fermented foods was deemed beneficial due to adverse taste problems and rancidity production [12] [48] [87].
However, there are reports of beneficial effects of lipase in the development of characteristic flavours and aroma [83] [88]. These results appear to be conflicting and require further study. An interesting area would be the effects of changes in lipid quantity and quality on the organoleptic properties of fermented condiments Darweesh et al. [89] reported that aldehydes resulting from oxidation of lipids cause off-flavour and odour problems. Nevertheless, the organic solvent treatment of fermented condiments from locust bean, melon seed and soybean showed no appreciation of the odor [90]. Not much can be said about the flavoring components of fermented vegetable proteins used as condiments, as li-

Nutritional Quality of Fermented Condiments
Classical nutrient bioavailability evaluation methods generally involve animal models in vivo experiments. The use of proteins as a ratio of protein efficiency (PER) and the digestibility of soybean and some legumes has hardly been improved by fermentation [86] [91]. It has been documented that rats do not make better use of proteins from fermented soybean than from the cooked substrate.
Nevertheless, some reports indicate otherwise. Achinewhu [85] reported that fermentation and heat treatment improved the apparent digestibility, feed quality, and protein production ratio when rats were fed on African oilseed rations diets.
Eka [17] studied the effect of fermentation on the nutrient content of locust beans and reported an increase in protein and fat when fermented, whereas the amount of carbohydrate decreased, this relative increase is due to a reduction in the percentage of carbohydrate content. Increased levels of amino acids except arginine, leucine and phenylalanine have also been reported. Similar studies have been reported for other seed legumes [12] [77] [85]. During the fermentation of melon seeds, soluble products increased and the fermented product was highly digestible. The main amino acids are alanine, lysine and glutamic acid, with small amounts of arginine and proline [13]. Improved nutritional values were attributed to increased amino acid profiles due to fermentation [10].

Starter Culture
Starter cultures or starters are individual or mixed formulations of selected strains of microorganisms with a particular physiological capacity to turn a substrate into a food product with specific characteristics when added at specified concentrations [92] [93]. Adapted to the substrate, the typical starter facilitates improved control of the fermentation process and the predictability of its products [94]. The development, improvement and standardization of microbial starters have been driving force for the transformation of traditional food fermentations in developing countries from an "art" to a science. The development of microbial starter culture has also been a driving force for innovation in the design of equipment suitable for the hygienic processing of traditional fermented foods under controlled conditions in many developing countries.
The development of starter culture has played a pivotal role in the production of high-value products such as enzymes, microbial cultures and functional food ingredients, together with the development and improvement of bioreactor technology to control fermentation processes in developed countries. As inputs for their food processing operations, these goods are primarily manufactured in

Traditional Approaches: Spontaneous Fermentations and Back-Slopping
Traditional starter cultures are picked either as single or multiple strains, primarily for adaptation to the substrate or raw material. Spontaneous fermentation, i.e. processes conducted without the use of starter inoculum, have been

Inoculation to Improve Process Control
It takes a relatively long time (24 -48 h)

Starter Cultures as Inoculants of Fermentation Processes
In the fermented food sector, "Appropriate" starter cultures are widely used as inoculants, from household to industrial, low-income and low-middle-income economies [94]. These starter cultures are generally produced using a back-slopping process that uses samples from a previous batch of a fermented product as in-  [95].

The inoculation belt used in traditional fermentations in West
Africa serves as a carrier of undefined fermenting microorganisms, and is one example of an appropriate starter culture [94]. It usually comprises of a woven fiber or mat or a piece of wood or woven sponge, saturated with a previous fermentation batch's "high"-quality product. To act as an inoculant, it is immersed in a new batch. The inoculation belt is used for the production of native fermented porridges, "uji" and "mawe", as well as for the production of Ghanaian beer "pito." Another example of an "appropriate" starter culture produced by back slopping is Iru. This starter culture is produced from concentrated fermented dawadawa (a result of fermented legumes), mixed with unfermented legumes, vegetables such as pepper, and cereals such as ground maize. It is preserved in a dried form and is used as an inoculant in dawadawa fermentations in West Africa [94]. In Asian countries, a variety of suitable starter cultures are used as fermentation inoculants, either in a granular form or as a pressed cake.
Different names such as marcha or murcha in India, ragi in Indonesia, bubod in the Philippines, nuruk in Korea, koji in Japan, ragi in Malaysia, and Loog-pang in Thailand generally refer to these traditional mould starters. They generally consist of a mixture of moulds grown under non-sterile conditions.

Defined Starter Cultures as Inoculants of Fermentation Processes
Few specified starter cultures have been developed to be used as inoculants in commercial fermentation processes in developing countries. However, over the last ten years, a number of developing countries have witnessed the development and application of selected and pre-cultivated starter cultures in food fermentation.
"Defined starter cultures" consist of single or mixed strains of micro-organisms [94]. It is possible to incorporate adjunct culture preparations that serve a function of food safety and preservation. Due to their ability to inhibit pathogenic or spoilage organisms, adjunct cultures do not necessarily produce fermentation acids or alter texture or taste, but are included in the defined culture. An inhibitory function is ascribed to the development of one or more products, such as hydrogen peroxide, organic acids, diacetyl and bacteriocin [6]. Generally defined cultures are produced by the maintenance and spread of pure culture under aseptic conditions. They are generally marketed in a liquid or powdered form or else as a pressed cake.
Defined starter cultures are also commonly imported by developing countries for use in the commercial production of dairy products such as yogurt, kefir, cheese and alcoholic beverages as well as in bakeries. Many of these cultures are tailored to produce specific textures and flavours. Most yogurt cultures also include probiotic strains in response to increasing consumer interest in achieving wellness through diet. Probiotics are currently produced in India, South Africa and several other countries for use as food additives, dietary supplements and for use in animal [94]. Methodologies used in these starters' design and tailoring are  fermented foods, such as phytic acid and phenolic compounds, can degrade antinutritional factors. Therefore, the incorporation of these organisms into starting culture can help to improve the nutritional value of foods [94].

Selection of Starter Cultures and Their Application in
In recent times, there has been considerable focus on the inclusion of mycotoxin-degrading strains in starter cultures. The use of mycotoxin contaminated raw materials for fermentation in developing countries presents a particular challenge in the choice of strains that can detoxify mycotoxins [94] [95] [96] [97]. In respect of their possible contribution to improving overall health and well-being, the probiotic properties of strains involved in food fermentation are also being studied.

Selection Criteria for Starter Culture Development
Spontaneous food fermentations are neither predictable nor controllable.
Therefore, single-and mixed-strain cultures must be tested on the pilot scale before being used in small-scale operations. According to Holzapfel [94], the in- The primary screening is based primarily on the evaluation of candidate strains' ability to withstand the pressure conditions imposed by fermentation processes [98]. At the beginning of the process, microbial cells are impaired by osmotic pressure due to the high concentration of solutes in the fermentation medium, such as grape sugar, hydrolyzed malt or sugar cane, milk matrix proteins and lipids and vegetable sodium [99].
Cell exposition to such hypertonic conditions leads to an efflux of water from the cell, decreasing both turgor pressure and water availability [100]. It causes main microbial metabolism enzymes to be inactivated, in addition to increasing toxic ion concentration and eventually cell death. Nonetheless, through natural selection it is possible to distinguish strains that are well suited to these stressful environments [98]. These resistant strains respond to osmotic stress by accumulating specific solutes, such as potassium ions, amino acids (e.g., glutamine, glutamate, proline, y-aminobutyrate, and glycinebetaine) and sugar (sucrose, trehalose, and glucosylglycerol), which decrease the activity of water within the cytoplasm and increase both cell volume and turgor near their prestress values [101]. detoxification; 8) probiotic features [94].  [105]. This is quite possibly the case for root crops, while the initiation of milk fermentations is typically associated with Lactococcus lactis, followed by L. casei (paracasei) and other Lactobacillus spp. during maturation. Several LAB are associated with meat and fish fermentations. L. sakei and L. curvatus [92] have been determined to be superior starter cultures for meat fermentations. Leuconostoc mesenteroides frequently dominates the early stages of most spontaneous fermentations.

Technical Considerations
Plant materials containing fermentable sugars provide suitable substrates for the yeast species Saccharomyces, Candida, Torula and Hansenula. Although the growth rate of these yeasts is lower than that of bacteria, such as L. mesenteroides, strains of Saccharomyces cerevisiae eventually dominate most spontaneous alcoholic fermentations as in the production of African opaque beers, palm wine and Asian beverages, such as rice wines and Indonesian tape'. Indeed, most traditional fermentations result from the combined metabolic activities of different types of microorganisms. Hounhouigan et al. [106] [107] reported a stimulating effect of the yeast Candida krusei on L. fermentum and L. brevis during a mixed starter culture fermentation of the fermented maize product, mawe.

Antagonism
This is the combined effect of different biological factors, resulting from meta- Gram-negative bacteria, such as pathogens.

Bacteriocins
Bacteriocins are protein-like antimicrobials that are active against closely related bacteria. They exhibit a narrow range of activity but are not active against Gram-negative bacteria. In food substrates, proteolytic enzymes are capable of inactivating bacteriocins. LAB bacteriocins can be divided into three structural groups based on their physico-chemical and antimicrobial properties [108].

Approaches toward Starter Cultures with Improved Properties Genetic Improvements: Pro and Contra
It is doubtful whether the most modern techniques and selection procedures would result in a multifunctional strain having all desirable metabolic features.
Selected strains may be improved through the application of genetic technologies. Recombinant DNA engineering may be used in the development of tailor-made starter cultures that would meet the technological and metabolic specifications required for specific fermentation (e.g. accelerated acid output, enhanced wholesomeness, health-promoting properties, overproduction of bacteriocins or particular enzymes needed to remove unwanted factors). Undesirable products, such as mycotoxin and antibiotic development in food grade moulds, may be removed by strategies such as gene destruction [109]. A large number of such optimised cultures already exist. Regulatory issues, however, preclude their application.  [111] showed the ability of B. subtilis and B. pumilus to degrade African locust bean oil, proteins and carbohydrates, and also their ability to produce a pleasant aroma and antimicrobial substances against pathogens. They have proposed B. subtilis B7 and B15 as suitable starter cultures for the controlled production of soumbala. For ugba production, Sanni et al. [65] suggested strains of B. subtilis, especially B. subtilis MM-4:B12, as potential starter cultures based on their enzymatic activities and ability to produce stickiness. Amoa-Awua et al. [25] have screened 42 Bacillus isolates from soydawadawa and proposed B. subtilis 24BP2 and B. subtilis FpdP2 as the best starter cultures for soydawadawa production. Further work by Terlabie et al. [112] rated B. subtilis FpdP2 as the best starter culture.

Conclusions
Focused studies on the introduction of starter cultures for small-scale fermentations appear more than justified, and in fact, deserve the highest priority.
A number of aspects of relevance to starter culture production should, therefore, receive special attention. As examples, the following should be mentioned. 1) Amino acid decarboxylase activity as a negative selection feature for potential starter cultures and mono-amino-oxidase activity as a positive selection criterion to reduce biogenic amine levels in selected fermented foods. 2) Potential for mycotoxin fermentation, strain selection methods, and toxicological tests on potential degradation products.