MICROBIOLOGICAL STUDIES ON A NIGERIAN MAIZE PRODUCT, KWOKA, SUPPLEMENTED WITH SOYBEAN

B.J.O. EFIUVWEVWERE’ and 0. AKOMA

Department of Microbiology Food & Industrial Division

P.O. Box 148-Uniport Post Ofrice Port Harcourt, Nigeria

Accepted for Publication August 8, 1997

ABSTRACT

Microbiological studies on a popular maize product (kwoka) with or without a soybean supplement (20 or 30%) were carried out during production and storage at 26-32C. Soybean -supplemented product had greater microbial diversity and higher populations than the unsupplemented control. The divers@ was most evident at the slurry stage (blended mixture before steaming) which contained ten different microbial genera including Aspergillus, Enterobacter, Bacillus, Lactobacillus, Micrococcus and Pediococcus. A dramatic decrease occurred after steaming which killed most of the molds and Gram-negative bacteria. However, within I day of storage, a sharp increase was observed in the microbialpopulation of all samples and maximum load occurred in 20% soybean supplemented “kwoka” at the end of storage. The microbiota became less diverse with storage and was dominated by Bacillus, Lactobacillus and Micrococcus. Packaging of ‘kwoka ’ in traditional leaves was microbiologically inferior to polyethylene

packaging. Changes in the acidity of 30% supplemented ‘kwoka’ were less dramatic compared to the other products. The Iactics were primarily responsible for the spoilage of the products (especially the 20% supplemented) after approxi- mately 3 days of storage at tropical ambient temperature of 26-32C.

INTRODUCTION

Cereal-based products are highly popular foods and are major dietary components in developing countries. They are known by various names in different parts of the world (Odunfa 1985; Tellez-Giron et al. 1988; Efiuvwevwere and Amadi 1992). ‘Ugali’ in Kenya, ‘Tortillas’ in Central America, ‘Chapati’ in

‘Corresponding author and current address: Agrotechnological Research Institute (ATO-DLO), Dept. of Food Safety & Applied Microbiology, P.O. Box 17, NL-6700 AA Wageningen, THE NETHERLANDS, Tel: 3 1-3 17-475048, Fax: 31 -3 17-475347

Journal of Food Safety 17 (1997) 249-259. AII Rights Reserved. ” Copyright I997 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 249

2.50 B.J.O. EFIUVWEVWERE and 0. AKOMA

Pakistan, ‘Koga’ in West Africa and ‘Kwoka’ or ‘Asuruasu-oka’ in Nigeria are consumed as snacks both in urban and rural areas. ‘Kwoka’ is a highly perishable product which is traditionally packaged by wrapping it in leaves of Thaitmatococcus dunieli, but recently, polyethylene bags have been introduced for packaging.

The microbial quality of such packaged local foods is of interest (Steinkraus et uI. 1983). Because of increased tourism and emigration, there is increased international concern for microbiological data and safety of traditional foods (Bryan 1988).

Soybeans are a major source of nutrients used for fortification of less nutritious cereals (Nyotu el ul. 1986; Almeida-Dominguez et ul. 1990). Whereas many studies have investigated nutritional aspects of soybean-supplemented maize products, there is no information on the microbiology of such supplementation. The present work was therefore undertaken to focus attention on the microorganisms and the microbial load associated with maize products supplememented with different levels of soybean, the effects of packaging materials on the microbial quality and acidity changes during tropical ambient storage of these products.

MATERIALS AND METHODS

Raw Materials

Yellow maize (Zea mays), soybean (Glycine m a ) , the other raw materials (red pepper, sugar, onions, palm fruits and salt) as well as the polyethylene bags and wrapping leaves were purchased from local markets in Port Harcourt, Nigeria. The maize and soybeans were sorted and ground respectively, into grits of 4 5 0 - 5 5 0 p as determined by a test sieve shaker (Endocolt Octagun 200, London, England).

Formulation of ‘Kwoka’

Blends of maize-soybean (70 or 80% w/w of maize to soybean) were prepared and mixed with the other ingredients (sugar 16.7%, onions 33.7%, salt 3.3%, red pepper I .3% and palm nut fruit extract3 10.0 g) to form a slurry as traditionally practiced in Southern Nigeria. Figure 1 depicts the flow-diagram for production and packaging of soybean supplemented and unsupplemented kwoka which was stored under tropical ambient conditions (26-32C) for analyses. Production of the unsupplemented kwoka has earlier been described (Efiuvwevwere and Amadi 1992).

Isolation and Enumeration of Microorganisms

Samples (20 g each) of slurry and finished products were homogenized with 180 mI, of sterile 0.1% peptone water using a stomacher (Stomacher Laboratory,

MICROBIAL COMPOSITION OF ‘SOY-KWOKA’ 25 1

SORTED IMAIZE SORTED SOYBEAV

U U

GROUND MAIZE GROUND SOYBEAN

U .e Palm Nut Fruit Extract * u SLURRY; MAIZE( lOO%,)-MAIZUSOYBEAN- SLURRY; SOYBEAN( 100%)

(20 or 30% soybean)

U U

DISPENSED and PACKAGED DISPENSED and PACKAGED

-. STEAMED FOR 50 MIN (70-75 C) c

11.

CONSUMED or STORED

FIG. 1. FLOW-DIAGRAM FOR THE PRODUCTION OF THE MAIZE PRODIJCT, KWOKA, SUPPLEMENTED AND UNSUPPLEMENTED WITH SOYBEAN (SL= SLURRY)

Seward Medical, London, England). Following preparation of serial dilutions, total viable counts of the samples were

determined by pour-plating in duplicate on Tryptone soya agar (TSA; Unipath Ltd.; formerly Oxoid, Ltd) and incubated at 30C for 24-48 h to obtain 25-250 colonies per plate (Speck 1984). Lactic acid bacteria were enumerated in duplicate on deMan Rogosa Sharpe (MRS) agar following pour-plating and anaerobic incubation (Gas Pak, BBL, Becton Dickinson and Co. USA) at 30C for 5 days. Total coliforms were determined using MacConkey agar incubated at 37C for 24 h (Harrigan and McCance 1976). Other microorganisms isolated involved use of Baird-Parker agar for Staphylococcus spp., acidified Malt extract agar for fungi and Bacillus cereus agar supplememeted with polymyxin-egg yolk-mannitol bromothymol blue agar for Bacillus species. All the culture media were purchased (through their agents) from Unipath (formerly Oxoid).

Identification of the Isolates

Typical colonies picked randomly from countable plates were purified, kept in slants under refrigeration, and subjected to different morphological and biochemical tests. Among the morphological tests were Gram- and spore-staining. Biochemical and physiological characterization included growth at different

252 B.J.O. EFNJVWEVWERE and 0. AKOMA

temperatures (35, 45, 50 and 60C), catalase tests, coagulase production, IMVC tests, oxidatiodfermentation of carbohydrates (glucose, lactose, fructose, mannitol, arabinose, cellobiose, and esculin) and lipolytic activity (tributyrin hydrolysis). The bacterial isolates were identified after these tests by conventional microbiological procedures (Harrigan and McCance 1976; Krieg and Holt 1984; Sneath et al. 1986).

The mold isolates were subjected to morphological identification using scotch tape preparation and lactophenol cotton blue staining (Harrigan and McCance 1976; Samson and van Reenen-Hoekstra 1988) while the yeasts were identified following cultural, morphological and carbohydrate fermentation data (fructose, glucose, maltose and sucrose) (Samson and van Reenen-Hoekstra 1988).

Determination of Acidity Changes

The pH of the samples was measured with a pH meter (Pye Unicam, England) after homogenization ( 1 :2 sample to deionized water). Titratable acidity was determined by titration against 0. IN NaOH to a phenolphthalein end point (pink) and expressed as percentage lactic acid.

Statistical Analysis

The Genstat statistical program 5 release 3.2 (Lawes Agricultural Trust 1995) was used for the analysis of variance for mean comparisons of the microbial. populations during storage of the different products at the probability level of p=0.05.

RESULTS

Microbial Composition and Population of the Samples

The microbial biota of the samples varied widely (Table I ) . The slurry samples showed the greatest diversity with the supplemented material being the most heterogeneous and containing ten microbial genera (Table I). After steaming, fungi and Gram-negative bacteria were virtually eliminated (except Aspergillus) and the samples were dominated by Gram-positive bacteria, particularly Bacillus, Lactobacillus and Micrococcus.

The total viable count decreased dramatically following steaming but during storage increased rapidly in all samples. Significant differences were not observed between the 20% and 30% supplemented products in most cases (Table 2). The changes in lactic acid bacteria and total coliforms are comparable to those of the total viable counts (Table 2). Use of traditional wrapping leaves resulted in higher microbial populations than did the use of polyethylene bags for packaging the samples (Table 3).

MICROBIAL COMPOSITION OF ‘SOY-KWOKA’ 253

TABLE I . VARIATIONS IN THE OCCURRENCE OF THE MICROBIAL GENERA COMMONLY

ISOLATED FROM THE SAMPLES

Storage t i i i ie

NA‘ Ground niaize

NA‘ Ground aoyhean

NA’ S1iirry:Suppleiiieiited

Unsuppleinenred

0 Kwoka:Supplemented

Unrupplemeiired

I Supplemented

Unsupplemented

3 Suppleinented

Unhupplemented

5 Supl~lcineiited

Unsuppleiiiented

I\sp

+

+

+

+

+

+

+

ILIUC. PEN. SAC. --_ + + -

- + +

+ - + + .

- - - - -__ ENr. KLE. BAC. LAC. MIC. STA. PED.

+ - + + + -

- + + - + +

+ + + + + + +

+ + + + + + -

- + + + + +

- + + + + -

- + + - + +

+ + + - - + _ _ _ + -

+ - - + + + + + - +

+ - _ . _ + + + + + -

+ - . . - + + + -

+ - - + + + -

* Not applicable ( i s . before blending/production or storage). + = Isolated; -=Not isolated. ASP. = Aspergillus; MUC. = Mucor; PEN. = Penicillium; SAC. = Saccharomyces; ENT. = Enterobacter; KLE. = Klebsiella; BAC. = Bacillus; LAC. = Lactobacillus; MIC. = Micrococcus; STA. = Staphylococcus.

Acidity Changes

The changes in pH and titratable acidity during the production and storage of kwoka are shown in Fig. 2 . The pH of the samples varied as storage progressed with 30% soybean supplemented product having the highest values and the unsupplemented, the lowest (Fig. 2 ) . The pH of all samples decreased towards the end of storage (Fig. 2) . The percentage titratable acidity increased with storage and was highest at the end of storage in 20% soybean supplemented kwoka (Fig. 2 ) .

254 B.J.O. EFIUVWEVWERE and 0. AKOMA

I

2

1

'I

5

TABLE 2. CHANGES IN MICROBIAL POPULATIONS (LOG,,, CFU G-I) OF THE DIFFERENT

SAMPLES DURING AMBIENT TROPICAL STORAGE

T-

4.30h

4.751

4.h0a.h

2.YRa

I .7Yc

2.30h

5.97a

5.Ylh

4.3Yh

6.Y6h

7.47n

7.22a

7.892

7.908

7.42h

8.59u

8.200.1l

7.90b

8.25b

R.70a

8.3 I b

Lnclic acid haclcriu

3.8Ba.h

3.75h

4.Wa

I .Mia

1.10a

I .70a

5.080

4.97a

4.52h

6.930

6.4Yh

6.760

7.1Ga

1.541

6.8lh

7.4Ro

7.71n

7.69~

7.50~

8.38a

7.Ylb

Tuhl cu l i lh is

3.6611

4.lJRa

3.76h

1.11;1

I .SHh

I .4%1

4.5681

4 zxo

3.86h

5. I8a

4.2611

5.04a

S.YRh

6.4Ga

5.2Rc

6.2611

6.2Rn

5.Y4h

6.26u,h

6.48a

6. I8h

*Not applicable (i.e. before storage). Each value represents the mean of 4 determinations (i.e. duplicates of two replicates). Means within the same columns for respective storage intervals followed by similar letters are not significantly different at p = 0.05.

MICROBIAL COMPOSITION OF ‘SOY-KWOKA’ 255

‘TABLE 3.

UNSUPPLEMENTED KWOKA AS AFFECTED BY PACKAGING MATERIAL MICROBIAL POPULATIONS (CFU G-’) OF SOYBEAN-SUPPLEMENTED AND

-ping leaves’

0 IJiisuppleiiiciited 1.7 (k l .1) x 10’ 1 .2 (*0 . l )x 10’

Supplemented (20%) 7.4 (1t1.0) x 10’ 1.5 (+0.001) x 10’

I I Iiisiip[,IeiiieiitetI I .8 (rtO. I ) x 10’ 7.2 (M.2) x lon

Su~’plclllclltccl (20‘70) 2.1 (M.7) x 10’ 2 . 8 ( r t 0 . 1 ) ~ 109

Eiicli voluc icpicsciils llic mean and the standard deviation of 4 deteiiniiiatioiis.

‘OCtcii rclcirccl (o 3 s wiappiiig leaves Tor “Agidi “ (a popular Nigeriaii maize product).

DISCUSSION

Cereals are exposed to many microbial contaminants in the field and after harvest (Anon. 1980b). In the present work, the microbial profile of kwoka reflects the microbial quality of its raw materials (Table 1). Thus, combining the maize and soybean led to greater microbial diversity in the supplemented product.

It has been reported that the slurry stage contains the most diverse microbial biota (Efiuvwevwere and Amadi 1992). This diversity was accentuated in the soybean-supplemented kwoka (Table 1 ), suggesting that soybean supplementation has adverse microbial implications. In addition, the consistent isolation of Aspergillus indicates its abundance in cereal products. This is of concern since some Aspergillus strains are toxigenic (Speck 1984).

The decrease in total viable counts (Table 2) caused by steaming has safety implications. However, the remarkable increase after only one day of ambient tropical storage (Table 2) is beyond the recommended 10’ cfu g-’ for ready-to-eat foods (Jay 1986). This increase may be partly attributed to the survival and growth of many heat resistant spore-forming organisms (particularly Bacillus spp.). There are similar sharp increases in the microbial populations of tortilla or soybean unsupplemented kwoka within 24 h of production (Capparelli and Mata 1975; Efiuvwevwere and Amadi 1992).

256

5.a

5,:

5.6

B 5.5

5 . 4

5.3

B.J.O. EFIUVWEVWERE and 0. AKOMA

J I 1 t I I I

0.03

0.025

0.02 B 0

P .v - z

0.015

0.01

0.005 1 4 51 0 2 3 5

Storage tim (days)

FIG. 2. CHANGES IN pH (OPEN SYMBOLS) FOR UNSUPPLEMENTED 0; 20% SUPPLEMENTED, 0; 30% SUPPLEMENTED, A AND TITRATABLE ACIDITY (TA, CLOSED SYMBOLS) 0; W; A, RESPECTIVELY) DURING PRODUCTION (SL=SLURRY) AND

STORAGE OF THE MAIZE-SOY PRODUCTS

Lactic acid bacteria (LAB) are known for production or spoilage of various foods (Anon. 1980; Steinkraus 1983). This may be due to their ability to exert a wide spectrum of antagonistic effects against both Gram-negative and Gram- positive microorganisms which is attributed to the production of bacteriocins and simultaneously increasing the acidity (DeVuyst and Vandamme 1995). Their dominance as storage progressed after day 3 (Table 2) therefore strongly suggests their involvement in the spoilage of kwoka, which was accompanied by the increase in acidity (Fig. 2).

Soybean supplementation enhanced microbial growth and population diversity. The 20%-soybean supplementation in general exhibited higher microbial load than the 30%-supplemented product (though not significantly different in many occasions) (Table 2). This, therefore showed that microbial population did not increase concomitantly with increase in soybean supplementation. The present findings contrast with those reported by Jay ( 1 986) which indicated an increase in aerobic plate counts of beef-soy blend as concentration was increased from 10,20 to 30% soy-supplementation. The disparity in these findings may be partly due to the differences in the chemical attributes and other interacting intrinsic factors of beef-soy versus maize-soy. For instance, the former is less acidic than the latter and

MICROBIAL COMPOSITION OF ‘SOY-KWOKA’ 257

could influence the microbial ecology, behavior and growth. In addition, these interacting factors (especially acidity) have profound and complex effects on mixed bacterial populations (Anon. 1980a; Padan 1984). Since each food has its characteristic microbial profile which is influenced by the composition and the interacting factors, the qualitative and quantitative microbial composition is in a constant dynamic state until equilibrium is attained. It is very likely that such interplay and microbial succession are at work in kwoka since the optimal pH range for LAB is about 4.7 to 5.4 (Pederson 1979; Steinkraus et al. 1983; Holt et at. 1994). Thus, while the growth of some microorganisms was stimulated, others were inhibited. Evidently, LAB constituted a major component of the microbiota of kwoka thereby creating sub-optimal acidic conditions for many microorganisms such as Micrococcus spp, Staphylococcus spp and many Gram-negative bacteria. Furthermore, since LAB population influenced the total viable counts (TVC), conceivably, the lower TVC observed in 30%-supplemented product especially on day 4 (Table 2) reflects the impact of interactive microbial activities. Although higher bacterial levels are expected in the 30%-supplemented kwoka, the comparable populations that occurred between the 20%- and the 30%- supplemented products could be due to the overriding influence exerted by the LAB; consequently negating the expected effects on microbial growth from the slightly higher pH of the 30%-supplemented kwoka (Fig. 2).

Packaging contributes immensely to the overall quality of food products. The traditional wrapping in leaves resulted in higher microbial loads and greater microbial diversity. Furthermore, samples wrapped with traditional leaves had high number of Bacillus cereus and the associated risk is more apparent since consumers do not often reheat these products before consumption. In general, plastic films are inherently sterile as the microorganisms are destroyed during the plastic film manufacturing/production. But these materials become contaminated due to physical damage or abusive storage (Hayes 1985). Unfortunately, these polymeric materials are usually heaped and retailedistored in dusty areas or humid rooms in Nigeria. These abusive handling and storage conditions may have resulted in contamination of the bags and consequently influenced the microbial population of the products.

Clearly, microbial diversity of kwoka is largely dependent on the raw materials, with the soybean supplemented product being more diverse than the unsupplemented. The shelf-life and potential safety do not exceed 24 h after production. Control measures to minimize health risks include consumption of the product soon after production and avoidance of left-overs since heat-stable microbial toxins occur in comparable cereal products (Bryan 1988). The hazards analysis critical control points concept should be applied in order to achieve a greater safety margin in these products.

2S8 B.J.O. EFIUVWEVWERE and 0. AKOMA

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