Abstract: In this study African yam bean Seed (cream colour variety) were fermented naturally at 30±2°C for 72, 96 and 120 h, respectively. The bacteria isolated during the process include Lactobacillus jensenii, Bacillus coagulans, Aerococcus viridans and Pediococcus cerevisiae while the yeast were Saccharomyces cerevisiae and Candida mycoderm. Aspergillus niger was the only mould isolated from raw seed which disappeared after dehulling. The microbial loads increased with period of fermentation up to 72 h but decreased at 96 and 120 h of fermentation. Before fermentation, the bacterial count was 6.0x102 cfu g-1. While at 24, 48 and 72 h the bacterial load increased to 7.0x103, 7.8x103 a n d 8.5x103 cfu g-1, respectively. The highest bacterial load was observed after 72 h of fermentation. The load decreased to 4.3x103 and 2.1x103 cfu g-1 at 96 and 120 h of fermentation. In addition the yeast cells load increased up to 72 h of fermentation and the load decreased at 96 and 120 h, respectively.
INTRODUCTION
New food processing technologies can provide alternative for improving the nutritional quality of food plants (Paul and Southgate, 1978). Plant foods are the most important dietary sources for meeting the nutritional needs of majority of the population in Nigeria. This is because they are readily available, low in cost and acceptable. Legumes, cereals, vegetables, roots and tubers form this source of nutrients. Fermentation is identified as an economic processing method that could be used in the homes to improve the nutritional quality of plant foods (Obizoba and Atti, 1991; Obizoba and Nnam, 1992; Apata and Ologhobo, 1990; Olowoniyan, 1994). In some parts of the world, huge amounts of fermented foods are produced and used in the daily diet of the people Steinkraus (1995).
Man has practiced processing food by fermentation for centuries (Apata and Ologhobo, 1990). It has been used quite extensively in various parts of the world, especially in the orient (Bressani, 1983). It is probably the oldest method of processing legumes (Steinkraus, 1994). It involves the activities of microflora (bacterial and fungi) in the production of food. Fermentation could be done at home or in industry. Home fermentations are not dependent on industry because the raw materials and the simple equipment are all that are needed of produce a product. The new foods thus produced do not require major educational efforts to get them acceptable (Isichei and Achinewhu, 1988).
In Nigeria, fermented foods play a major role in the diet of the people Steinkraus (1994). Some of these include Ogi from corn and gari from cassava, Pito beer from millet/sorghum and different kinds of condiments. Some fermented food legumes in Nigeria include the African oil bean seed (Pentaclethra macropyhlla), which is widely eaten in southern Nigeria, especially in the eastern states.
It is popularly known as ugba and could be eaten alone or mixed with other food ingredients. Castor oil seed (Ricinus communis) is another plant food that undergoes the fermentation process to produce a condiment known as Ogiri, Melon (Citrullus vulgaris) and fluted pumpkin (Telfaria occidentalis) are fermented to produce different types of Ogiri. Currently soybean and baobab fermented products, dadawa (in Hausa, iru (in Yoruba) and Ogiri okpai (in Ibo) are replacing the locust bean seeds in the preparation of flavorings (Aderiye et al., 1991). Dawadawa is predominant in the northern states of Nigeria where soybean and baobab production is highest. Most of these fermented foods are either used as a major part of the main dish or as a soup ingredient, to enhance flavor (Rombouts and Nout, 1995).
Agronomy of African Yam Bean
Authority (Hochst. ex A. Rich.) Harms (Duke et al., 1997)
Family Magnoliophyta: Magnoliopsida: Fabales: Fabaceae (Allen and Allen, 1981)
Common names African yam bean, Afrikanische yambohne, girigiri, tuber bean,otili
(yourba)
The African yam bean is grown in West Africa, Anonymous (1979) particularly in Cameroon, Côte d’Ivoire, Ghana, Nigeria and Togo (Popoola and Akueshi, 1985). In Nigeria it is found in localized areas in the Volta Region, where peasant farmers grow it as a security crop. The African yam bean is grown for both its edible seeds and its tubers.
The plant produces underground tubers that are used as food in some parts of Africa and that serve as organs of perennation in the wild (Oniofiok et al., 1996; Anonymous, 1979; Rombouts and Nout, 1995).
Malnutrition, in its various forms is widespread in the world today; especially in Africa. Dietry deficiencies in terms of both quality and quantity of food are still among the most pressing problem in many Africa countries (Anosike and Egwuatu, 1981). The World Health Organization (Ikemefuna, 1998) has given highest priority to overcoming nutritional disease.
The African Yam Bean seeds is well suited to form part of the campaign to promote thorough food self-sufficiency. The African Yam Bean seeds have nutritional qualities that more than justify the increasing research efforts being devoted to its improvement and promotion as food.
The aim of the study was to ferment the seeds naturally and know the bacterial responsible for its fermentation in order to provide information that can enhance the utilization of the seeds.
MATERIALS AND METHODS
Collection and Processing of African Yam Bean (Sphenostylis sternocarpa
Harms) Seeds
Raw seeds of African yam bean (Fig. 1) were harvested
from Ighoba farm in Akure, Nigeria and processed into fermented form at Microbiology
Laboratory of Food Science and Technology Department, Federal University of
Technology, Akure, Nigeria.
The raw seeds samples were sorted and cleaned, boiled for 1 h, dehulled and then washed thoroughly in clean water. It was fermented as described by (Ikemefuna, 1998) in Fig. 2 which involves hand sorting of the seeds, washing, soaking in water for 12 h, dehulling, washing, boiling for 2 h, draining, wrapping in plantain leaves and jute sacks, fermentation for 24, 72, 96 and 120 h.
Sterilization of Material Used
All glasswares were washed with detergent and rinsed with clean tap water,
air-dried and then oven sterilized at 160°C for 2 h. Innoculating lops,
used were usually flamed to red hot, dipped into 70% ethanol, reflamed and allowed
to cool before used. Laboratory benches were also swabbed with cotton wool moisten
with 70% ethanol before and after investigation Inoculating chamber was swarbed
with 70% ethanol, thereafter the UV light was on for 2 h before and after inoculation.
| Fig. 1: | Raw seeds of African yam bean |
| Fig. 2: | Processing of African yam bean seed into fermented seed |
Preparation and Sterilization of Culture Media
Nutrient Agar (NA), was the culture medium used for the investigation. NA
was prepared by dissolving 28 g of the dehydrated powder (Oxoid) into 1 L of
distilled water in a conical flask. Thereafter, the mixtures was placed on hot
plates for 20 min to ensure proper dissolution of the agar. Physiological saline
was prepared by dissolving 0.85 g of sodium chloride in 100 mL of distilled
water. These were autoclaved at 121°C for 15 min.
The microbiological analyses include isolation of microorganisms from the sample, determination of total viable counts (microbial load) from the samples, direct and microscopic observation of the isolates and biochemical identification of the isolates (Olutiola et al., 1991) (Which include gelatin hydrolysis, a starch hydrolysis, casein hydrolysis, catalyse test, coagulase test, indole test, urease test, nitrate reduction test, sugar fermentation test, oxidative fermentation (O/F) test, methyl red voges-proskaur test, citrate test and oxidase test and motility test.
RESULTS
The bacteria encountered during natural fermentation include Lactobacillus jensenii, Bacillus coagulans, Aerococcus viridans, Pediococcus cerevisiae. Raw seeds were found to contain Lactobacillus jensenii, Bacillus coagulans, Aerococcus viridans, Candida mycoderm. The characterictic feature of the isolates are presented on Table 1 and 2.
Occurrence of Bacterial Isolates in the Fermenting Samples
Of all the four bacterial isolated from naturally fermented sample, the
occurrence of Lactobacillus jensenii and Pediococcus cerevisiae
was very high. They occurred more frequently even until 120 h of fermentation.
Total Viable Count of Bacteria During Fermentation of Samples
The changes in bacterial population during natural fermentation on African
yam bean seeds at different time intervals are shown on Fig. 2.
The initial bacterial count (6.0x102 cfu g-1) was lower
than the bacterial count obtained at 24 h of fermentation (7.0x103
cfu g-1) and at 48 h of fermentation (7.8x103 cfu g-1).
The highest count was recorded in sample fermented for 72 h (8.5x103
cfu g-1). There was a decrease in the bacteria count of the sample
from 8.5x103 to 4.3x103 and 2.1x103 cfu g-1
at 96 and 120 h of fermentation, respectively.
Generally, there was an increase in bacterial count after 24 h of fermentation, a slight increase between 24-72 h and a sharp decrease in the bacteria load between 96-120 h of fermentation.
| Table 1: | Morphology and microscopic characteristic of the bacterial isolates |
| 1 = Shape on plates, 2 = Chromogenesis, 3 = Opaccity, 4 = Elevation, 5 = Surface, 6 = Edge, 7 = Consistency, 8 = Gram reaction, 9 = Shapes, 10 = Arrangement of cells, 11 = Spore, 12 = Spore position, 13 = Motility, +ve: Positive, -ve: Negative | |
| Table 2: | Biochemical characteristic of the Bacterial and yeast isolates |
| ND: Not Determined, +ve: Positive, -ve: Negative, ASP: Ascospore, GA: Galactose, GL: Glucose, MN: manitol, SC: Sucrose, LA: Lactose, MA: Maltose, AR: Arabinose, XY: Xylose, RA: Raffinose, SO: Sorbitol, LM: Litmus Milk, GH: Gelatin, SH: Starch Hydrolysis, CA: Catalase, CO: Coagulase, UR: Urease, IN: Indole, CI: Citrate | |
The total counts of Lactobacillus jensenii was 2.0x102 cfu g-1 at initial stage of fermentation. The counts increased to 2.0x103 cfu g-1 at 24 h, 3.1x103 cfu g-1 at 48 h and 3.8x103 cfu g-1 at 72 h of fermentation. There was reduction in the counts at 96 h (2.1x103 cfu g-1) and at 120 h (1.1x103 cfu g-1).
Bacillus coagulans count increased from 3.0x102 to 2.0x103 cfu g-1 at 24 h, 3.6x103 cfu g-1 at 48 h and 3.5x103 cfu g-1 at 72 h. The isolate was not isolated at 96 and 120 h of fermentation.
There was increase in the count of Aerococcus viridans from 1.0x102 cfu g-1 at zero hour to 2.0x103 cfu g-1 at 24 h, also to 4.5x103 cfu g-1 at 48 h and 4.8x103 cfu g-1 at 72 h but the bacteria was not isolated at 96 and 120 h of fermentation.
Pediococcus cerevisiae was not isolated at zero hour but at 24 h the count ranged from 1.0x103 to 3.0x103 cfu g-1 at 48 h, 3.2x103 cfu g-1 at 72 h and decreased slightly to 2.1x103 and 1.1x103 cfu g-1 at 96 and 120 h of fermentation.
Occurrence of Yeast Isolates in the Fermenting Samples
Two type of yeast were isolated from the sample during natural fermentation.
Only one occurred until 120 h of fermentation, Saccharomyces cerevisiae
was present until the end of fermentation (120 h) while Candida mycoderm
was not isolated at 120 h of fermentation (Table 3).
The Total Viable Count of Yeast from the Samples
The changes in yeast population during natural fermentation of African Yam
Bean seeds at different time interval are shown on Fig. 3
the yeast count was not as high as bacteria count. Yeast count ranged from 2.0x102
cfu g-1 at 0 h and increased to 6.0x103 cfu g-1
at 24 h, 6.7x103 cfu g-1 at 48 h, 6.8x103 cfu
g-1 at 72 h and decreased to 3x103 and 3x102 cfu
g-1 at 96 and 120 h, respectively.
Saccharomyces cerevisiae was not isolated at zero hour but the count ranged from 0 to 4x103 after 24 h, 4.9x103 cfu g-1 at 48 h and increased to 6.3x103 cfu g-1 at 72 h which in turn decreased to 3x103 cfu g-1 at 96 h and further decreased to 3x102 cfu g-1 at 120 h.
The result shows that Saccharomyces cerevisiae count was high (6.3x103 cfu g-1) at 72 h of fermentation.
Candida mycoderma count increased from 2.0x102 cfu g-1 at 0 h and increased slightly to 2.0x103 cfu g-1 at 24 h. The count remain unchanged till 72 h of fermentation while Candida mycoderma was not isolated at 96 and 120 h of fermentation.
| Fig. 3: | African yam bean seeds fermented at 72 h |
| Fig. 4: | Effect of fermentation time on total viable count of the isolates |
| Table 3: | Total microbial load (cfu g-1) of raw and naturally fermented African yam bean samples |
| *Values are means of three determinations, 1 = Bacteria, 2
= Yeast, 3 = Mould, 4 = L. jenseni, 5 = B. coagula ns, 6 = A. viridas, 7 = P. cerevisiae cerevisiae, 8 = S. cerevisiae, 9 = C. mycoderma |
|
The textural characteristic of the fermented seeds of African yam bean at 72 h of fermentation is shown in Fig. 4 the result shows that the seeds became very soft and sticky with pungent smell.
DISCUSSION
The progressive increases in the counts of fermenting seeds has been similarly reported by Barber and Achinewhu (1992) on melon seeds who reported a range of less than 30 cfu g-1 on the St. day to 6.8x1011 cfu g-1 on the 7th day (Table 3). Ikenebomeh et al. (1986) Jeff-Agboola and Oguntuase (2006) also recorded high aerobic count during the fermentation of African locust bean (Parkia filicoides welw) to produce dawadawa. The high aerobic count might be due to high protein content of African yam been seeds (Antai and Ibrahim, 1986). As well as the exothermic reaction which increased the temperature of the fermenting seed leading to sharp increase in the bacterial population (Antai and Ibrahim, 1986). Similar results were reported by Alabama and Legged (1982). On locust bean and melon seeds, respectively. The bacteria and yeast counts decreased after the 72 h (Table 3) this may be due to increased acidity after the 72 h of fermentation (Fagbemi and Atum, 2001). Similar results were obtained during the fermentation of Hura crepitans seeds (Adeyokunnu, 1977) Reduced oxygen tension obtainable in solid substrate fermentation was also reported to reduce mould growth.
The results of the tests carried out on microorganisms isolated from the onset of the African yam bean seeds during fermentation (Fig. 3, Table 1 and 2) Barber and Achinewhu (1992). Reported the isolation of Saccharomyces sp. and Aspergillus niger among other fungi and Bacillus sp. from fermented locust beans Kolawole and Okonkwo (1985). Reported the isolation of Bacillus subtilis, Micrococcus varians, Enterobacter aerogenes and Corynebacterium sp. from traditionally fermented oil bean seeds for ukpaka production Nout and Sarkar (1999). Also isolated Bacillus sp. and Streptococcus sp. from fermenting melon seeds.
CONCLUSIONS
This study documents the successful fermentation of African yam bean seeds for condiments production. It also reveals the microorganisms that were isolated from the process because of their ability to degrade some and build up other nutrients contained in the seed, by some enzymes they produce, thereby giving the produced condiment a characteristic organoleptic quality, which is the most important factor for consumers. Taking into account the increasing demand particularly by urban populations in Nigeria, there are certainly prospects for industrialization of traditional fermented condiments. Commercial availability of ready to use fermented products saves much labour and time in the household (Barber and Achinewhu, 1992).