Journal of Applied Bacteriology 1990.69, 321-325 322711 1/89

Microbiology of ‘obiolor’ : a Nigerian fermented non-alcoholic beverage

0 . K. A C H I Department of Microbiology, University of Nigeria, Nsukka, Nigeria

Accepted 12 March 1990

ACHI, O.K. 1990. Microbiology of ‘obiolor’: a Nigerian fermented non-alcoholic beverage. Journal of Applied Bacteriology 69, 321-325.

Obiolor is an acidic non-alcoholic beverage prepared by fermenting sorghum and millet malts. The traditional process for the production and microbiological charac- teristics of the beverage were investigated. Bacillus spp., hctobacil lus plantarum and Streptococcus l a d s were the associated micro-organisms most actively involved. Yeasts were present in low numbers towards the end of the fermentation. Other micro-organisms isolated did not appear to play a role in the fermentation process. Variations in the important microbial groups involved and their metabolic products were studied. Titratable acidity increased gradually until the end of the fermentation while the total soluble solids and pH declined. Acetobacter spp. were probably responsible for the unacceptability of the product after 24 h.

Obiolor is a traditional non-alcoholic beverage brewed by the Igala in Nigeria. It is a light brown, moderately light gruel with a sweet taste and low viscosity. It is prepared from malted sorghum and millet or maize grains, depending on the locality. Similar beverages are prepared from these cereals in the form of Pito and Buru- kutu in the Northern parts of Nigeria (Ekundayo 1969; Faparusi 1970). Obiolor differs from these products because it is non- alcoholic. During the fermentation process, many micro-organisms develop in the mash, producing substances which include acids (Faparusi 1970). When these acids and other microbial by-products are present in moderate quantities they contribute positively towards the flavour and aroma of the beverage. In high con- centrations, however, they cause off-flavours which render the product unacceptable.

Obiolor is an important beverage drink, dis- tinct in microbial quality, and is consumed daily by the people of Igala land who attribute their good health to it. No studies have been made to date on obiolor and its fermentation process and no information is available on the kinds of micro-organisms involved in the fermentation.

This study was therefore directed at under- standing the fermentation process and at deter- mining the kinds of micro-organisms involved in the fermentation as practised by the Igala tribe of Nigeria.

Materials and Methods

The traditional techniques practised by the local inhabitants of Idah town and the adjoining areas were followed in the laboratory prep- aration of obiolor. Sorghum and millet were purchased from the local market, cleaned and soaked in water for 1 d in 1 I beakers. The grains were drained and resoaked for 15 min before they were wrapped in fresh banana leaves. Germination was allowed to proceed for 3 d. The germinated grains were wet-milled and made into a slurry. Obiolor malt was prepared, based on 80% sorghum flour and 20% millet flours. Boiled water was added to the slurry in a ratio of 4 : 1 v/v. The resulting mash was allowed to cool, rough filtered, and the residue discarded. The filtrate was concentrated by boiling for 30 min with continuous mixing and then allowed to cool rapidly. This was left to

322 0. K. undergo natural fermentation for 24 h at room temperature. The time of cooling was regarded as time 0 of the fermentation.

Temperature and pH variations during the fermentation were monitored periodically. Samples (S10 ml) were removed at intervals of 2 h and examined immediately. Moisture content was determined after drying a known quantity of the sample at 100°C for 24 h. The total soluble solids were measured with an Abbe refractometer according to the method of Hart & Fisher (1971). The amount of total sugar was determined by the Nelson-Somogyi method (Somogyi 1952) with glucose as the standard. The titratable acidity was measured by the method of Amerine et al. (1972).

Selective media were used to count and isolate the different microbial groups. Serial dilutions of the extract were plated out on Tryp- ticase Soy agar, Nutrient agar, Madonkey agar, Malt Extract agar and Sabouraud Maltose agar (all from Oxoid). The spreading plate method was used and 0.1 ml volumes of suitable dilutions of samples were spread on the surfaces of agar plates. All counts were deter- mined per gram of material and were made after incubation for 48 h. All cultures, unless other- wise stated, were incubated at room tem- perature (28"-30°C). The pure cultures were preserved on nutrient agar slants at 5°C with periodic transfers. Obiolor samples prepared locally were also obtained from three different market women in Idah for comparison with laboratory-prepared samples.

IDENTIFICATION OF THE ISOLATES

Representative colonies were subcultured irre- spective of their numbers on the primary medium. Biochemical characterization of the

Achi strains was carried out and species identified by standard microbiological procedures (Lodder 1970; Buchanan & Gibbons 1974; Cowan & Steel 1974; Pederson 1979). The identification of yeasts included : morphological characteristic of yeasts using 48 h slide cultures prepared from malt extract agar, colony observation on malt extract agar plates, growth in broth medium and carbohydrate fermentations.

Results

After malting for 3 d, the sorghum and millet seeds had a plumule length of 1 cm and a strong characteristic aroma. The moisture content of the milled mash was 82% with an initial soluble reducing sugar concentration of 1.8%. The pH of the mash was recorded as 6.81 (Fig. 1). Ini- tially a diverse group of micro-organisms was found on the grains. The dominant flora were Bacillus spp. and Lactobacillus spp. Small numbers of Streptococcus faecalis and Entero- bacter spp. were also isolated. The malting stage contained almost the same groups of bacteria as found on the grains. However, Bacillus species were encountered more frequently than the others.

Two species of Bacillus identified as Bacillus stearothermophilus and B. subtilis were isolated from the boiled mash. The other groups of bac- teria in the paste were eliminated by the heat treatment.

The most prevalent species isolated during the fermentation stage and their numbers are given in Table I. Lactobacillus plantarum was the predominant organism followed by Bacillus spp. and Strep. lactis. Lactobacillus plantarum occurred at a total count of 4.8 x 10" organisms/g after 22 h.

T 8 k 1. Vanations in microbial populations of major organisms encountered in obiolor fermentation (per g)

period (h) SPP. lactis SPP. Yeasts Fermentation Bacillus Sireprococcus Lactobacillus

- - - - 0 2 2.0 x 10' 6 1.2 x lo2 5.0 x 10' 1.2 x 10' -

10 1.5 x 10' 3.0 x 10' 5.0 x 104 - 14 2.8 x 10' 3.6 x lo' 4.6 x lo6 18 3.5 106 3.8 x 103 5.3 x 107 - 20 2.4 x 107 2.5 x 103 4.1 x 108 1.0 x 10' 22 3.6 x lo7 2.6 x 10' 4.8 x 10' 3.2 x 10' 24 2.2 x 107 2.2 x 1 0 2 3.2 x lo' 2.8 x 10'

- - -

-

'Obiolor' fermentation 323

8

7

6

z 5

4

3

2

0.8

0.7 30

- 0.6 z

!2 z ??

20 2 ?

0 a n E

F 0.4

- - a c -

0.5 - - e e I-"

0.3 10

0.2

0 0 2 4 6 8 10 12 14 16 18 20 22 24

Fig. 1. Fermentation profile of obiolor beverage. A, temperature; 0, % titratable acidity; 0, pH.

The numbers of Srrep. lactis increased pro- gressively during the first 14 h of the fermenta- tion but generally declined towards the end. Very few yeasts were found.

In comparison, bacterial levels of 4.2- 5.4 x lo9 organisms/g were obtained in locally- prepared samples of obiolor. Lactic acid bacteria were also found as the dominant bac-

Table 2. Chemical analysis of obiolor beverage

Before After fermentation. fermentation

terial population at levels of 5.2 x 10' organisms/g. Appreciable numbers of Acero- bacter strain were found up to 1.2 x lo6 organisms/g at the end of the fermentation.

The chemical analysis of the fermented bever- age before and after fermentation is shown in Table 2. The total soluble solids at the end were 9.2% while the sugar concentration was reduced to 0.5%. The percentage of titratable acidity appeared to increase as fermentation progressed in relation to the fall in pH; the final value was 0.75%.

Specific gravity 1.12 1 a4 PH 6.8 I 4.90 Moisturecontent (%) 82.6 84.0 Titratable acidity 0.58 0.75 Per cent sugar 1.82 0.51 Total soluble solids 10.5 9.2

Values are for reconstituted malt ( 1 part malt: 4 parts water, w/v).

Discussion

Obiolor is produced by the fermentation of cereal or starchy foods in a liquid menstruum. The organisms responsible for the fermentation are mainly amylolytic organisms capable of hydrolysing the starchy constituents of the

3 24 0. K. Achi mash. Several Bacillus strains were found to be present in the samples. Ekundayo (1969) pro- posed that the presence of these micro- organisms in such environments as maize and sorghum grains have an important bearing, and explained that extracellular amylolytic enzymes of these organisms help to break down the starchy substrates into products which are likely to stimulate the growth of other organisms such as the lactic acid bacteria. Bacillus species have been associated with fermenting cereals for Burukutu (Faparusi 1970) and Ogi (Akinrele 1970) fermentations.

The preponderance of Enterobacter spp. and Strep.jaecalis may be due to the fact that they are soil organisms and are found mostly as food contaminants. Their total numbers during fer- mentation suggest an initial diverse contami- nation of the cereals. That they could proliferate may be as a result of the favourable moisture and temperature conditions provided during malting.

The numbers of the Bacillus spp. were very high during the malting stage but remained vir- tually unchanged during the early part of the fermentation. The presence of these organisms in samples taken after heat treatment does not signify that conditions were favourable for their development. Rather, their ability to form spores and survive adverse conditions may explain their dominance over other groups of organisms during the later high temperature treatment.

Obiolor fermentation is primarily a lactic fer- mentation as is indicated by the great increase in the population of the lactic acid bacteria accompanied by a high amount of titratable acidity. Most of these micro-organisms were probably derived from the environment, e.g. air, food utensils, and also from persons involved in obiolor manufacture. According to Frazier (1967) and Pederson (1979) changes brought about in fermenting vegetables are due mainly to the activities of lactic acid bacteria. Lacto- bacillus plantarum was the single most abundant group along with Strep. lactis and their numbers increased progressively until the end of the fer- mentation.

The lactic acid bacteria are able to produce a variety of chemical compounds relative to fer- mentation conditions. These organisms are known to attack sugars to form mixed acids (Brouk 1975). It is quite likely that their meta-

bolic products contribute to the acidity as well as adding a distinctive taste and aroma to fer- menting material.

Through the activities of the lactic acid bac- teria the pH decreased from 6.81 at the begin- ning of the fermentation to 4.9 after 24 h due to substantial acid production during the fermen- tation. The fermentation is characterized by vigorous bubbling. Apparently the temperature of the fermenting mash, which was between 28" and 30°C and the availability of reducing sugars (I.8% at the beginning of the fermentation) allowed the development of this homo- fermentative lactic acid bacterial species.

It is interesting to note that most of the organisms isolated in the laboratory-prepared obiolor were also isolated from the locally- fermented samples. The only difference was perhaps the isolation of Acetobacter spp. Their presence is likely to be as a result of contami- nation from previous ground malt on the milling machines in addition to the dregs of a previous brew added by the local brewers to accelerate the fermentation process. These organisms are common spoilage bacteria associ- ated with cereal products by their production of acetic acid which imparts a vinegary flavour to the food (Hamid 1978). Presumably, spoilage by acetic acid bacteria makes obiolor unacceptable because of the accumulation of volatile acids. On the whole, the microbial content of the locally fermented product was higher than that prepared in the laboratory.

The low count of yeasts shows that obiolor fermentation is not an alcoholic fermentation. Whether they participate in bringing about any desirable flavour changes is not clear. It is of interest that samples held under refrigeration could keep for 14 d after which yeasts prolifer- ated and produced an alcoholic flavour.

The results have demonstrated that the micro-organisms involved in obiolor fermenta- tion resemble those found in the fermentation of other cereals (Hesseltine & Wang 1979). The role of the individual micro-organisms in the fermentation is not quite clear. Thus an under- standing of the metabolism of the individual organisms in relation to the fermentation process in conjunction with analytical studies may lead to a clear understanding of the factors related to the development of an industrially based finished product. This and the spoilage of obiolor will be the subject of further study.

'Obiolor' fermentation 325

References FAPARUSI, S.I. 1970 Sugar changes during production of 'Burukutu' beer. Journal of the Science of Food

AKINRELE, 1.A. 1970 Fermentation studies on maize and Agriculture 21.79-8 I . during the preparation of a traditional African FRAZIER, W.C. 1967 Food Microbiology, 2nd edn. pp. starchcake food. Journal of the Science of Food and 201-251. New York: McGraw-Hill. Agriculture 21,619425. HAMID, A.D. 1978 A Microbiological study of Suda-

AMERINE, M.A., BERG, H.W. & C A ~ , M.V. 1972 The nese Merissa Brewing. Journal of Food Science 43, Technology of Wine Making, 3rd edn. Westport: 16831686. AVI Publishing Co. HART, F.L. & FISHER, H.J. 1971 Modern Food

BROUK, B. 1975 Plants Consumed by Man. pp. 386- Analysis. Berlin: Springer Verlag. 393. London: Academic Press. H s L n m , C.W. & WANG, H.L. 1979 Fermented

BUCHANAN, B.E. & GIBBONS, N.E. (ed.) 1974 Bergey's foods. Chemistry and Industry 12,393399. Manual of Determinative Bacteriology, 8th edn. Bal- IADDER, J. 1971 The Yeasts-Taxonomic Study. pp. timore: Williams and Wilkins. 863-1357. North Holland Publishing Co.

COWAN, S.T. & S ~ L , K.J. 1974 Manualfor the Iden- PEDERSON, C.S. 1979 Microbiology of Food Fermenta- tification of Medical Bacteria. 3rd edn. Cambridge: tions, 2nd edn. pp. 153203. Westport: AVI Publi- Cambridge University Press. shing Co.

EKUNDAYO, J.A. 1969 The production of 'Pito', a SOMOGYI, M. 1952 Notes on sugar determination. Nigerian fermented beverage. Journal of Food Tech- nology 4,2 17-225.

Journal of Biological Chemistry 195, 19-23.