Isolation and Identification of Microorganisms Encountered in Traditional Fermentation of Soya Beans and Roselle seeds for the production of Food Tasty condiment (Dawadawa)

^1&2Adamu, Shahidah A.; ²Farouq, A. A.; ³Magashi, M. A.; ⁴Sokoto, A. M.; ^*5Salau, I. A.

¹Department of Microbiology, Sokoto State University, Sokoto, Nigeria

²Department of Microbiology, Usmanu Danfodiyo University Sokoto, Nigeria

³Department of Microbiology, Bayero University Kano, Nigeria

⁴Department of Pure and Applied Chemistry, Usmanu Danfodiyo University Sokoto, Nigeria

^*5Department of Biology, Shehu Shagari College of Education, Sokoto, Nigeria

INTRODUCTION

Fermentations are enzyme-induced chemical alterations in food or substrate. The enzyme involved may be produced by microorganisms or they may be indigenous to the food or substrate (Ihekoronye and Ngoddy, 1985). Generally, fermentation results in the breakdown of complex organic substances into smaller ones through the action of catalysis. Fermentation is one of the oldest methods of food preservation known to man. In Africa, the art of fermentation is wide spread including the processing of fruit and other carbohydrate source to yield alcoholic and non-alcoholic beverages, the production of sour tasting Ogi - the fermented cereal product which provide instant energy in breakfast and convalescent diets (Adewusi et al., 1991; 1992) oil seed such as African locust bean, melon seed, castor oil seed mesquite bean and soybean are also fermented to give condiment.

Production of fermented tasty food condiment has remained a traditional family art in homes with rudimentary utensils (Audu et al, 2004). Adewumi and Olalusi (1995) reported that women mainly do processing of Soya Beans and Roselle seeds locally. Methods used vary from one locality to another depending on the culture of the people, their beliefs, taste and practice of the fore-parents who were involved in the same vocation. These variations in processing techniques in turn bring about variations in the quality of iru (Sadiku, 2010).

A substantial literature documents the successful fermentation of vegetable proteins for condiment production. Both microbial and biochemical changes involved therein have received much attention.  A further understanding about the interactions of the microorganisms and plant materials is necessary to improve the quality of fermented condiments. Such understanding will aid in further development and control of the fermentation process (Dakwa et al, 2005)

MATERIALS AND METHODS

Dried sample of soybean (Glycine max) Roselle seeds (Habiscus sabdariffa.) were obtained from Sokoto central market located in Sokoto metropolis.  All the materials used in dawadawa production (Basket, Calabash, Sieve ,pawpaw /banana leaves, Wood ash/Potash, jute bags, plastic buckets) were purchased from Sokoto central market.  The samples were taken to laboratory for analyses.

Traditional fermentation of dawadawa from Soya bean              

To make traditional soy –dawadawa, soybean are cleaned, soaked overnight in tap water, dehulled manually and cooked by boiling for two  hours (Omafuvbe et, al,. 2000). The dehulled cooked beans are then placed in calabashes or bamboo baskets lined with banana or plantain leaves and left to ferment spontaneously in a worm place for up to 72 hours                   

Traditional fermentation of daddawan botso from Rosselle seed

Seeds fermentation was done in two phase, the cooked seeds were allowed in the pot to ferment naturally for two days by closing the pot tightly to prevent aeration. After the first fermentation, the cooked seeds were pounded nearly to paste in a mortar with the addition of ash leachate and mixed. This was returned back to the pot for second fermentation which lasted for one day and the pot was tightly closed. At the end of the second fermentation, the ammonia – like flavor condiment was sun dried by repeated turning to form balls and to enable good drying for 2 – 3 days according to the intensity of sunshine and packaged in polythene bags (Ibrahim et al., 2011).

Hibiscus sabdariffa raw seed

↓

Dry cleaning (pounding, winnowing and removal of stones

↓

Dry cleaned seeds

↓

Water washing

↓

Cleaned and selected seeds

↓

Cooking (8-10hr)

↓

Cooked seeds

↓

First fermentation (2days)

↓

Deep pounding

↓

Mixing with ash leachate

↓

Second fermentation (1day)

↓

Drying under sun for 2-3 days

↓

Dawadawan botso

Fig. 1: Flow chart for traditional fermentation processing of Hibiscus sabdariffa raw seed

(Ibrahim et al., 2011)

Isolation of the fermenting microorganisms

One gram [1g] of the fermented Soya bean Roselle seeds were taken and diluted serially. One (1ml) from different dilutions was streaked onto nutrient agar. Isolates from plate count agar (PCA) was picked and sub-cultured in nutrient agar and Bacillus species was characterized using morphological examination and biochemical test comprising of colony and cell morphology. The biochemical tests that were carried out are: anaerobic growth, acid production from D-glucose, hydrolysis of casein and starch, growth at pH 5.7, in 6.5% (w/v) NaCl and 10% (W/V) NaCl, at 37°C and 65°C (Claus and Berkeley, 1986).

Characterization of fermenting isolates from fermented Locust and Soya bean

Anaerobic Growth

The organism was isolated using method described by Claus and Berkeley, (1986) using standard isolation medium. 0.1ml of the test organism was spread out on the solid standard medium plates. The plates were incubated in anaerobic jar for 2 days at 35⁰C.

Acid production from D-glucose

Isolates were inoculated into nutrient broth sugars and examine daily for seven days for acid and gas production (Barrow and Feltham, 1993)

Hydrolysis of Casein

Plates of Casein agar were inoculated at intervals and examined for up to 14days for clearing of the medium around the bacterial growth (Barrow and Feltham, 1993).

Starch Hydrolysis

Isolates were inoculated onto nutrient agar plates containing 0.2% soluble starch and incubated at 30⁰C for 5 days. Plates were flooded with lugols iodine solution, the medium turned blue for negative hydrolysis. A clear colourless zone indicates hydrolysis (Barrow and Feltham, 1993).

Growth in Media with Increased NaCl Concentration

Required amount of sodium chloride 6.5% (wv) and 10% (wv) was added to broth and inoculated with organism to be tested. The tubes were incubated at 37⁰C for 24hrs (Barrow and Feltham, 1993

Determination of Hazard Analysis and Critical Control Point during the production of condiments

At each different stage of Daddawa production, sample was collected from raw seeds, fermented seeds, washed seeds, floor swab, mortar and pestle to determine critical control point, according to the method of the Food and Agricultural Organization (FAO, 1979).

RESULTS

Figure 2: Percentage frequency of occurrence of bacteria isolated from Dawadawa production

Figure 3: Viable colony count from Soya beans at each stage of production

Figure 4: Viable colony count from Habiscus sabdariffa at each stage of production

DISCUSSION

The microorganisms identified are from Bacillus genera which are the dominant organisms, other microorganisms isolated are from the genera of Staphylolococcus, Kurthia, Listeria, Micrococcus and Serratia .Similar result was also reported by Achi (1992) who investigated the microorganisms associated with the natural fermentation of Prosopis africana seed. A wide array of microorganisms including B. subtilis, B. megaterium, B. licheniformis, Staphylococcus spp, Micrococcus spp, Klebsiella spp, Enterobacter spp and Lactobacillus spp. The involvement of a variety of microorganisms in spontaneous food fermentation is normal and does not render the product unsafe for human consumption, especially when none of the microorganisms is pathogenic to man (Oyeyiola, 2002). The growth of microorganisms during the fermentation of daddawa is likely to have a significant influence on the quality and flavor of the final product.

Bacillus species were frequently isolated from traditional daddawa and this explain the reason why the organism was used as starter culture in the laboratory fermentation of daddawa. The presence of Bacillus species is expected since they have been found to be associated with fermenting legume seeds during the production of Okpehe (Achi, 1992, Ogunshe et al, 2007), Ugba (Obeta, 1983) Dawadawa (Odunfa, 1981, Antai and Ibrahim, 1986), Iru (Oyeyiola, 2002) and dawadawa botso (Ibrahim et al., 2011a) and soumbala (Ouoba et al, 2007).  The presence of these organisms which are proteolytic, am ylolytic and lipolytic may lead to an increased degradation of the major nutritional components of the seeds. Bacillus have been implicated in the fermentation of most vegetable oil protein seeds like African locust bean seeds, soybean seeds, African oil bean seeds e.t.c. (Odunfa, 1981, Antai and Ibrahim, 1986).

        The presence of Staphylococcus species in the samples was typical of the micro flora of fermenting beans (Obeta, 1983, Antai and Ibrahim 1986).  Staphylococcus species have been associated with fermenting foods of plant origin especially vegetable proteins (Odunfa and Komolafe, 1989). The presence of Staphylococcus may also be linked to the hands of the handlers/producers of the condiments.

The coagulase-negative staphylococcus species are non- pathogenic and safe organisms on vegetable proteins. These organisms may contribute to the flavor of Okpehe because of their lipolytic activity.

Kurthia zopfil was also isolated from traditional fermented daddawa, this result agrees with the work of Chotwanawirach (1980) who reported the presence of some strains of lactic acid bacteria in Kapi (a traditional fermented shrimp paste that serves as a flavoring/seasoning for many decades) are Staphylococcus, Micrococcus,  Kurthia and Bacillus. Listeria monocytogenes isolated in this work, has also been detected by other researchers, Dajanta et al; (2012) also isolated Gram positive that include S. aureus, S. epidermidis, M. luteus, B. cereus and L. monocytogenes by fermenting soybean in traditional production of thua nao.

Saccharomyces cererisiae was found only in one sample of traditional daddawa produced from locust beans. Similar result was obtained by Oyeyiola (2002)  S. Cerevisae is known to be able to grow well  in the complete absence or presence of very little oxygen as was likely to prevail during the fermentation. The reduced sugar released following the breakdown of the carbohydrates may also influence the presence of yeast in this dawadawa sample.

          Based on the critical control point decision tree (NACMCF, 1997) a step is considered as a critical control point if it involves a hazard of sufficiently likelihood of occurrence and severity to warrant its control, is necessary in order to eliminate or reduce the hazard to an acceptable level. Based on this there are two steps that need to be controlled (washed seed and water used for the washing) since there was high bacteria count of (1.7 x 10⁶) which was higher than acceptable limit of (10⁵ cfu/g) provided by the food and Agriculture organization (FAO, 1979) of the United Nations. The high count recorded from the washed seeds was due to the water used in washing the seeds. Edema and Fawole (2006) reported water used for washing the dehulled seeds may remove some of the contaminating microorganisms introduced during dehulling, but it may also add new ones.  Another reason for not having high count in other steps during production of traditional daddawa was the daddawa was carefully done by the researcher at home taking consideration of all sources of contamination and thereby producing the condiment under hygienic conditions.

The raw materials, (that is soya bean seeds and Roselle seeds) usually comes with a substantial microbial load comprising spores of aerobic spore-forming bacteria and mould spores, most of which are drastically reduced during the prolonged boiling to soften the testa as obtainable in related fermentation (Feng et al, 2005) potential pathogens like E. Coli, Clostridium spp were not isolated in this research, but higher counts of these organisms were obtained in similar production of daddawa by fermenting African locust bean seeds (Rabi et al., 2013).

CONCLUSION

The predominant microorganisms in the fermentation process of both samples were found to be Bacillus species of which Bacillus subtilis, B. pumilus and B. licheniformis were most involved. The study has shown that a daddawa analogue, comparable to well accepted locust bean daddawa in terms of microbiological characteristics, could be produced from soya bean and roselle seed an under-utilized legumes. There is need for clean environment and materials to be used during traditional fermentation, most especially the use of clean water as it contains high count of bacteria from the research.

REFERENCES

Achi OK. (1992). Microorganisms associated with natural fermentation of Prosopis africana seeds for the production of Okpiye Plant Foods. Human. Nutrition 42: 297-304.

Adewumi BA and Olalusi, AP (1995): Performance of a Manual operated concave-type Locust Bean Dehuller. Journal of Agricultural. Technology 2:23:30.

Adewusi SRA, Orisadare BO and Oke OL (1991). Studies on wearing Diet in nigeria, 1. Carbohydrate  source. Cereal chemistry., 165-169

Adewusi SRA, Orisadare BO and Oke OL (1992). Studies on wearing Diet in nigeria, 2. Protein  source. Plant food for Human Nutrition, 42: 183-192.

Antai SP and Ibrahim HM (1986) Microorganisms associated with African locust bean (Parkia filicoidea Welw) fermentation for dawadawa production . Journal of applied bacteriology, 61:145-148

Audu I, Oloso A. and Umar B.(2004).Development of a concentric cylinder Locust bean dehuller. Agricultural Engineering International: The CIGR Journal of scientific research and development manuscript PM 04003.1(11)

Barrow GI and Feltham RKA. (1993) Cowan and steel’s manual for the identification of medical bacteria. Cambridge, Cambridge University press.

Chotwanawirach TA. (1980). Microbiological study on Thai traditional fermented food products, M.Sc. Thesis, Kasetsart University, Thailand.

Claus D and Berkerly RCW (1986). The genus Bacillus in Bergey’s manual of  systematic Bacteriology, 2; 1105-1139

Dakwa S, Sakyi-Dawson E, Diako C, Annan NT and Amoa-Awua WK. (2005): Effect on the fermentation of soybeans into dawadawa (Soy-dawadawa). International journal food, microbiology. 104:69-82.

Edema MO and Fawole O. (2006). Evaluation and optimization of critical control points in the production of Iru. Research Journal of Microbiology, 26; 533-541

Feng  XM, Erikson ARB and Schurer J (2005). Growth of lactic acid bacteria and Rhizopus oligosporus during berley tempeh fermentation. International Journal of Food Microbiology, 104:249-256.

Food and Agricultural Organisation (1979). Manuals of food Quality Control. FAO Food and Nutrition Paper, United Nations, Rome 14(4) A1-F10.

Ibrahim AD, Sani A, Aliero AA. and Shinkafi SA. (2011) Biocatalysis of H. sabdariffa during dawadawan botso production and biogenaration of volatile compounds. International journal of Biology and Chemical  Science . 5(5):1922-1931.

Ihekoronye  AI and Ngoddy PO. (1985). Integrated food science and technology for the tropics. Macmillan Publishers Ltd, London and Basingstroke, pp 34-87.       

National Advisory Committee on Microbiology Criteria for Food (NACMF, 1997). Hazard Analysis and Critical Control Point, Principle and Guidelines.

Obeta JAN (1983) A note on the microorganisms associated with the fermentation  of seeds of African oil bean (Pentaclethra macrophyla) Journal of Applied Biotechnology 54:433-435.

Odunfa SA and Komolafe OB. (1989) Nutritional characteristics of Staphylococcus species from fermenting African locust bean (Parkia biglobosa). Nahrung. 33:607–615.

Odunfa SA. (1981). Microorganisms associated with fermentation of African locust bean (Parkia filicoidea) during iru preparation. Journal of Plant Foods. 3: 245-250.

Ogunshe AAO, Omotosho MO and Ayanshina ADV. (2007). Microbial studies and biochemical characteristics of controlled fermented afiyo- a Nigerian fermented food condiment from Prosopis africana (Guill and Perr.) Taub. Pakistan Journal of Nutrition 6 (6):620-627.

Omafuvbe BO, Shonukan OO and Abiose SH (2000). Microbiological and biochemical changes in the traditional fermntation of soybean for soy- daddawa -a Nigerian food condiment. Food Microbiology. 17: 469-474.

Ouoba LI, Diawara B, Jespersen L and Jakobsen M (2007). Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for soumbala production. Journal of Applied Microbiology 102: 963-970 147.

Oyeyiola GP (2002). Fermentation of the seed of Prosopis Africana to produce condiment . NISEB Journal 2 (2):287-299.

Rabi M, Mukhtar MD, Kawo AH, Shamsuddeen U and Aminu B.(2013). Evaluation of the critical control points (CCP) in the production of Daddawa (African locust bean cake) Bayero Journal of pure and Applied Sciences, 6 (1):46-51.

Sadiku OA (2010): Processing Methods of Influence, the quality of fermented African Locust Bean (Iru/Ogiri/dadawa) Parkia biglobosa. Journal of Applied Sciences Research 6(11):1656-1661.