antimicrobial effect of fermented ghanaian maize dough

Journal of Applied Bacteriology 1991, 70, 203-210 ADONIS 002108479100031 H

Antimicrobial effect of fermented Ghanaian maize dough

Patience Mensah, A.M. Tomkinsl, B.S. Drasar‘ and T.J. Harr/son2 Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana and ’Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London, UK

Accepted 23 July 1990

Paper number: 3360/04/90

P . MENSAH, A.M. TOMKINS, B.S. D R A S A R AND T.J. HARRISON. 1mi.Unhygienic conditions of a typical rural community in a developing country were simulated in the laboratory by inoculating fermented maize dough porridge with Shigella Jexneri and enterotoxigenic Escherichia coli (ETEC). T h e antimicrobial effects of the different processes involved in the preparation of fermented maize dough porridge were assessed. T h e soaking process reduced the p H but no antimicrobial effect against shigella and ETEC was noted. Unfermented maize dough did not inhibit any of the test strains. When the fermentation process had become established, half of the strains tested were inhibited by the fermented maize dough when examined 8 h after inoculation. Cooking the fermented maize dough into porridge reduced the antimicrobial effect but there was still significant inhibition of pathogens. This suggests that the antimicrobial effect of fermented maize dough is not due to pH per se. Fermentation of maize dough appears to be a useful strategy for reducing contamination of weaning foods by Sh.Jexneri and ETEC. The possible nature of the antimicrobial agent(s) produced during the fermentation of maize dough is discussed.

INTRODUCTION

At some stage in its development the breastfed infant requires nutritionally adequate and hygienically prepared weaning foods (Rowland 1986). A review by Underwood & Hofvander (1 982) emphasized the importance of achieving a balance between adequate nutritional quality and hygiene during the preparation of weaning foods. In view of the interaction between infection and nutrition (Tomkins & Watson 1989), the need for improved hygiene during weaning food preparation has been highlighted (Esrey & Feochem 1989). This need is further emphasized by the high levels of bacteria that have been detected in weaning foods from developing countries (Rowland et al. 1978; Barrel1 & Rowland 1980; Elegbe 8z Ojofeitimi 1984). Indeed, if weaning foods with little or no bacterial contamination could be obtained, the question of the optimal timing of introduction of solid food could become less criti- cal.

Metchnikoff (1907) attributed the longevity of Cauca- sians to the consumption of fermented milk products and

Correspondence to : Prof: A . M . Tomkins, Department of International Child Health, Institute of Chrld Health, Guildford Street, London WCIN IEH, UK. ’ Present address : Academic Department of Medicine, Royal Free School of Medicine, Rowland Hill Street, London NW3 2PF, UK.

postulated that such products could inhibit the growth of diarrhoea1 pathogens. A number of studies on fermented milk showed inhibitory activity against a variety of bacteria including Escherichia coli (Nyaga et al. 1982). Fermentation of fish silage inhibited salmonellas, staphylococci and coliform bacteria (Yeoh 1982). Fermented sausages were inhibitory to Staphylococcus aureus (Raccach 1986). The fermentation of soybean by Rhizopus oligosporous resulted in a product that showed inhibitory activity against Staph. aureus, Bacillus subtilis and Klebsiella pneumoniae (Wang et al. 1969). In 1981, Mbugua studied ‘uji’, a fermented maize product from Kenya and showed complete inhibition of all coliform bacteria which formed about 40% of the resident flora in maize. It appears that fermentation of foods could produce an environment that is inhibitory to a variety of bacteria although the substrate for fermentation may vary. Indeed, if this antimicrobial effect is a property of fermented foods, it could be an important strategy for the reduction of the high levels of faecal bacteria in weaning foods in developing countries.

In Ghana the traditionally recommended weaning food is prepared from fermented maize dough. The preparation of this porridge or pap, known as ‘akasa’ or ‘koko’, is outlined in Fig. 1. The soaking process, which involves the addition of a large volume of water to whole maize grains, allows the selection of desirable organisms, such as lactic acid-

rn PATIENCE MENSAH E r A L .

Maize grain (whole kernels). Winnow, wash and soak (steep1

in water for 1-2 d at ambient temperature

i Dram and mill

i Moisten and form dough

i Ferment for 2-3 d

I Cook porridge

fermented m o w dough- I port water-6 parts

Fig. 1 Preparation of fermented maize dough porridge

producing bacteria, yeasts and moulds (Akinrele & Bassir 1967). The metabolic activities of these organisms reduce the pH and increase the titratable acidity. A number of fatty acids are also produced (Akinrele 1970; Muller 1970). These have been shown in vitro to inhibit a wide variety of bacteria (Hentges 1967; Adams & Hall 1988).

In an earlier investigation we showed that fermented maize dough was inhibitory to four strains of Shigellajlex- neri (Mensah et al. 1988). We therefore examined the antimicrobial effect of the different processes involved in the preparation of fermented maize dough porridge on 20 strains of Sh. flexneri and enterotoxigenic E. coli (ETEC). Shigellu jlexneri was chosen because it is the commonest species of Shigella in Ghana (Afoakwa 1973). Shigella infection may lead to severe dehydration and malnutrition and usually requires treatment with antibiotics. Prevention of infection by shigellas is therefore of great importance. Strains from the UK were included for comparison and to determine if there was any emergence of resistance in the Ghanaian strains after years of using fermented maize dough foods in Ghana. Enterotoxigenic E. coli were also studied because these are important pathogens in children and adults (Echeverria et ul. 1987).

MATERIALS AND METHODS

Experiment8 with Shlgell8 flexnerl

Twenty strains of Sh. jlexneri, isolated from patients with dysentery attending the University College Hospital, London and University of Ghana Medical School Teaching Hospital, Ghana, were tested. Ten strains isolated in each country were studied.

These organisms were characterized by the API 20E (API System S.A., La Balme les Grottes, Montelieu Vercieu, France) and specific shigella antisera (Difco). One

loopful (0401 ml) of a pure culture of Sh. jlexneri was inoculated on nutrient agar. After overnight incubation at 37°C the entire growth was washed off into phosphate-buffered saline (PBS, Oxoid) to give approximately 10' cfu/ml as determined by the spread plate method (Thatcher & Clark 1968). For each experiment, 1 ml of freshly prepared bacterial suspension was added to several samples of food at different stages of preparation to give approximately lo7 cfu per g of food. Two samples of food were studied each time. Each experiment was performed twice and the loglo mean value of the number of cfu from the two experiments was used for constructing Figs 2, 3 and 4.

Soaked maize grain

Sterile distilled water (1 1) was added to 1 kg portions of winnowed maize grains obtained from a market in Accra, Ghana.

I0.C

8.C

6.C

c

1c

0

8 4.c 3

2.c

C

Aseptic precautions were maintained during these

Time ( h )

4 8 ,, 24 48 I I I l

0 -0 .. ..

Fig. 2 Changes in the numbers of Shigellaflesner: after inoculation into 0, unfermented and 0, fermented maize dough

Time ( h 1

4 8 ,, 24 48 I I " I I

10.0

8' 4.0 A A

A M

A '5

2.01

I 4 1- ~ A A A A A 0'

Fig. 3 Changes in the numbers of Shigellaflexneri after inoculation into A, unfermented and A, fermented porridge

FERMENTED MAIZE DOUGH 205

Time ( h )

4 8 , 24 48 I I 1 I

10-0

2.01

OOAAA Q A A M A

*.

Fig. 4 Changes in the numbers of Shigellapexneri after inoculation into fermented maize dough 0, before and A, after cooking

experiments to reduce external contamination to a minimum. The mixture of maize in water was immediately inoculated with Sh. Jlexneri. Samples of soaking liquor were examined for shigellas at 0, 4, 8, 24 and 48 h. For the next stage of the study, maize meal and flour were prepared by grinding soaked maize grains and dry maize grains respec- tively.

Maize dough

Maize dough was prepared by adding 25 ml of sterile distilled water to 25 g portions of maize meal. This model system was used to facilitate even distribution of the inoculated bacteria. T o obtain fermented maize dough, the mixture was allowed to ferment for 72 h at 30°C. The shigellas were added and mixed with the food. Samples were cultured for shigellas at 0, 4, 8, 24 and 48 h. For the unfermented maize dough, a mixture of 25 g dry maize flour and 25 ml of sterile distilled water was prepared and immediately inoculated with shigellas. Samples were again cultured for shigellas after 0, 4, 8, 24 and 48 h.

Porridge

Fermented and unfermented maize doughs were prepared as above. A mixture of the maize dough and water in the ratio of 1 : 6 was brought to a rolling boil for about 10 min to produce a porridge of normal viscosity. The highest temperature during the cooking process was 91°C. The porridge was then allowed to cool to 30°C and inoculated with shigellas. Samples of porridge were examined for shigellas after 0, 4, 8, 24 and 48 h.

Detection of Shlgeila fiexneri In food samples

Tenfold dilutions of foods were made in PBS for the detection of shigellas by the spread plate method. The bacteria

were grown on MacConkey (CMllS), Xylose Lysine Deoxycholate and Nutrient Agar (Oxoid). Buffered Peptone Water (Difco) was used as a pre-enrichment medium for the resuscitation of metabolically injured bacteria.

Experiments with enterotoxigenic EsCherlChi8 coll

The strains of ETEC were obtained from patients with diarrhoea1 diseases in Bangladesh and were kindly provided by the International Centre for Diarrhoea] Diseases Research. These bacteria carried plasmids that encoded the production of heat-stable enterotoxin, heat-labile enterotoxin or both enterotoxins. They were characterized by the API 20E system as well as DNA-DNA hybridization assay (Grunstein & Hogness 1975; Moseley e t al. 1980; Vadivelu e t al. 1989). Methods adopted were similar to those for experiments with Sh. Jlexneri. Each experiment was repeat- ed at least once. The log,, of the mean of the changes in numbers of ETEC was used to plot Figs 5, 6 and 7.

Detectlon of enterotoxlgenic Escherlchla coll In food samples

MacConkey Agar (Oxoid) (CM7) was used for the detection of ETEC. Buffered peptone water was employed as a pre-enrichment medium.

Data analysis

The number of cfu/g of food was expressed as a logarithm to the base ten (log,, cfu/g). The Wilcoxon Ranked Sign test was used to determine if any differences in numbers of bacteria observed between fermented and unfermented

Time ( h )

24 48 I

lO .0C

0 1 Fig. 5 Changes in the numbers of enterotoxigenic Escherichia coli after inoculation into 0, unfermented and 0, fermented maize dough

206 PATIENCE MENSAH ET A L . ~-

2-0

Time ( h )

M A M A M A AAAAA

-

M An A M

0 4 8 ,, 24 40 I I I " I I 1

io.oc Y w AA A A

AAA M 3:A A%& Ay~w~B U@AM

O M AAA MM A A

A M AA A 6-0

d M AAA 4.0 A

A A A A A I Fig. 6 Changes in the numbers of enterotoxigenic Escherichia coli after inoculation into A, unfermented and A, fermented porridge

POI-

0 Fig. 7 Changes in the numbers of enterotoxigenic Escherichia coli after inoculation into fermented maize dough 0, before and A, after cooking

foods were due to the fermentation process or chance. To determine how to interpret negative growth on MacConkey agar the spread plate method was compared with the mem- brane filtration method as well as growth after resuscitation with buffered peptone water during some spiking experiments. The results showed that when there was no growth in the spread plate method there were less than 10 organisms in the food sample. Both the filtration technique and buffered peptone water resuscitation showed positive growth except on those occasions when all the bacteria had been completely inhibited. A plate with no visible growth was given a value of &2 loglo cfu/g of food. The pH results were analysed by Student's t-test.

RESULTS

Shloella flexnerl

There were no detectable differences in behaviour between the shigellas isolated in the UK and Ghana. The two sets

Table 1 Changes in the numbers of Shigellajexneri after inoculation into soaked maize

Log,, cfu S h . Time after exposure (h) PH Jexnerilg food

0 4 8

24 48

5.4 7.3 5.1 7.2 4-9 7.2 & 0.1 5.0 7.2 * 0.1 3.8 7.0 0.1

of organisms were considered together during the data analysis.

Soaked maize grain

There was a reduction in pH when maize was soaked in water such that by 48 h the pH was 3.8 f 0.1. However, there was no significant reduction in the number of shigellas at 4, 8, 24 and 48 h (Table 1).

Maize dough

The maize dough which had been fermented for 3 d before the beginning of the experiments had a lower pH than the freshly prepared maize dough (3.3 0.1 against 6.1 & 0.1, P < 0.001). The freshly prepared maize dough started to ferment soon after the addition of water and a significant reduction in p H to 3.7 f 0-3 (P < 0.001) had occurred by 24 h. Thereafter, the pH of the fermented maize dough and the dough that had been freshly prepared remained at similar low levels.

Figure 2 shows that all the 20 strains of Sh.Jexneri survived in the unfermented maize dough until 24 h after inoculation when six started to show evidence of growth inhibition. By 48 h a significant reduction had occurred (P < 0.001). The fermentation process had become established and there was a marked inhibition of all the strains by this time. There were some differences between strains tested; 14 showed complete inhibition whereas six showed partial inhibition. ~

By contrast, fewer shigellas survived in the fermented dough and numbers were significantly lower at 4, 8, 24 and 48 h (P < 0.001 at each sampling time).

Porridge

The pH of the fermented porridge was 3.6 and it remained at this low level throughout the experiment. The unfermented porridge had a pH of 6.1 at the beginning of the experiment, Although more acidic at 48 h (5.2) it was less


acidic than the fermented porridge (pH 4.0) tested at the same time.

The inhibition of shigellas by these two porridges is shown in Fig. 3. In the unfermented porridge there was no significant inhibition of bacteria at 4, 8, 24 and 48 h. In the fermented porridge, however, there was a significant reduction in the number of shigellas at 4 (P < 0-Ol), 8, 24 and 48 h (P < 0.001 at each time point).

Maize dough and porridge

The pH of the fermented porridge was 3.3 and this did not change significantly after cooking (3.6 & 0.1). Both maize dough and porridge remained at a low pH throughout the study. Figure 4 shows that the inhibition of growth of shigellas by fermented maize dough was reduced considerably after cooking. These differences were significant at 4, 8, 24 and 48 h (P < 0.05,0*05, <0*02 and <0-005).

Enterotoxlgenlc Escherlchia coll

Soaked maize grain

As in the shigella studies there was a reduction in pH in maize steeping liquor but there was no inhibition of ETEC by steeped maize grains (Table 2).

Maize dough

The pH changes during experiments on the effect of fermented maize dough on ETEC were similar to the changes that occurred during experiments on shigellas.

In the maize dough that was fermented at the beginning of the experiment, a reduction in numbers of ETEC had occurred after exposure for 4 h. Three of the strains, ET4, ET8 and ET9, were not detectable by this time. The reduction in numbers was significant at 4, 8, 24 and 48 h

Table 2 Changes in the numbers of enterotoxigenic Escherichia coli (ETEC) after inoculation into soaked maize

Log,, cfu Time after exposure (h) PH ETEC/g food

0 4 8

24 48

6.3 7.7 5.9 7.7 5.7 7.6 f 0.1 5.6 8.1 f 0.1 4.1 8.1 f 0.1

(P < 0.001 at each time point). Differences in the suscep- tibilities of the ETEC to fermentation of maize dough were observed as in the investigations that tested Sh. Jexneri. Fifteen strains of ETEC were completely inhibited while five were partially inhibited.

All the unfermented food samples contained high levels of ETEC after 4, 8 and 12 h (Fig. 5). By 48 h, however, eight of the food samples showed partial inhibition of growth of ETEC, presumably as a result of the start of fermentation.

Porridge

Figure 6 shows the changes in the numbers of ETEC after inoculation of the two types of porridge. The porridge prepared from the maize dough that was unfermented did not show any inhibitory effect on ETEC at 4, 8, 24 and 48 h. In the fermented maize dough porridge there was a significant reduction in numbers at 8, 24 and 48 h (P < 0.001 at each sampling time).

Maize dough and porridge

The behaviour of ETEC in fermented maize dough and porridge showed that, despite the low pH of both foods, the maize dough was more effective at inhibiting ETEC at 4, 8, 24 and 48 h than the porridge (Fig. 7). These differences were significant at 4 (P < 0*001), 8 (P < 0*004), 24 (P < 0.002) and 48 h (P < 0.001).

DISCUSSION

Our investigations were conducted to assess the effect of the different processes in the preparation of fermented maize dough porridge on bacterial contaminants such as Sh. fiexneri and ETEC. During the preparation of fermented maize dough porridge, there is a series of microbial processes. These can be considered in two stages; the soaking of whole grain for 24-48 h and fermentation after addition of water to the meal to form a dough (Fig. 1).

The pH of the liquor from soaked whole grain was lower by 24 h but this was not accompanied by any antimicrobial action against S h . fiexneri or ETEC. Soaking is a useful first step in the preparation of maize dough foods as it allows limited fermentation of the grain. The natural selection of desirable organisms including Leuconostoc mesen- teroides probably mcurs at this stage (Akinrele 8z Bassir 1967). The metabolic activities of these fermentation organisms could account for the reduction in pH but it is interesting that the inoculated pathogens continued to grow in this acid milieu.

208 P A T I E N C E M E N S A H ET A L .

The inhibition of the inoculated strains of S h . jlexneri (17/20) and ETEC (18/20) after 48 h by fermented maize dough was striking. Both shigellas and ETEC were suscep- tible to the inhibitory effect of fermented maize dough but ETEC survived somewhat longer in the unfermented maize dough.

The cooking process appeared to reduce the antimicrobial effect of fermented maize dough on both ETEC and Sh. JIexneri despite the acid pH. Previous studies have shown that pH values below 4.0 are inhibitory to shigellas and ETEC in bacteriological media (Jay 1986). The survival of these organisms in the fermented porridge suggests that the antimicrobial effect of fermented maize dough is not due to low pH per se.

Nout et al. (1987) described the inhibition of a single strain of’ Salmonella typhimurium by fermented sorghum but showed no change in inhibitory activity after cooking. Mbugua (1981) showed a reduction in the level of coliform bacteria present in the maize meal during uji fermentation. The inhibitory effect of the cooked product was, however, not investigated. Wang et al. (1969) studied ‘tempeh’, a fermented soy bean product from the Far East, and reported a loss in antimicrobial activity against Staph. aureus, B. subtilis and KI. pneumoniae after heating. We suggest that the fermented maize dough food has properties similar to ‘tempeh’.

During the fermentation of maize dough a range of fatty acids (lactic, acetic, butyric, propionic and formic acids) is produced and these have been shown to inhibit a variety of organisms (Hentges 1967; Baskett & Hentges 1973; Gal- braith et al. 1971; Silva et al. 1987). Volatile fatty acids such as acetic acid could be lost during cooking and hence reduce the antimicrobial effect.

A number of studies have investigated the production of antimicrobials by various micro-organisms, especially members of the Lactobacillaceae. Mattick & Hirsch (1944) reported the production of a small molecular weight antibiotic by Group N Streptococci. I t inhibited all coliforms tested, Bacillus spp., Clostridium spp. and some Lacto- bacillus spp. Other investigators described lactobacillin as a powerful antibiotic produced by Lactobacillus lactis (Wheater et al. 1951). This substance was found to be heat- labile and was described as hydrogen peroxide by the same authors (Wheater et al. 1952). The antimicrobial activity of lactocidin was described by Vincent et al. (1959) who showed that it inhibited a variety of organisms.

Lactobacillus brevis was shown to have antimicrobial properties against E. coli, Staph. aureus, Sh. Jexneri and Sh. sonnei (Kvasnikov & Sudenko 1967). This antimicrobial was named lactobrevin and was ‘very heat stable’. Branen et al. (1975) found that Streptococcus diacetalactis and Lact. citrovarum produced substances that were inhibitory to Pseudomonas sp. A heat-stable antimicrobial substance was

partially characterized by Pulusani et al. (1979). I t was produced by Strep. thermophilus during the fermentation of milk and inhibited both Gram-positive and Gram-negative organisms. Acidophilin, produced by Lact. acidophilus, inhibited Salm. typhimurium, Sh . dysenteriae and E. coli (Shahani et al . 1977).

The ‘bacteriocins’ also exhibit antimicrobial activity against many bacteria of the same and other species (Reeves 1965; Nomura 1967; Barefoot & Klaenhammer 1984). More recently, Schillinger & Lucke (1989) reported the production of a bacteriocin, sakacin, by Lact. sake. This was found to be inhibitory to various lactic bacteria and Listeria rnonocytogenes. The production of this antimicrobial was found to be encoded by an 18 MDa plasmid. This plasmid was absent in lactobacilli that were sensitive to this antimicrobial substance.

These studies emphasize that there is a wide variety of antimicrobial substances in fermented foods. Such substances appear to have special properties which could depend on the substrate that is fermented. We suggest that the fermented maize dough products that we investigated could also contain some unique antimicrobial substances. We have shown that fermented maize dough loses its antimicrobial effect after cooking. This could be due to the destruction of a heat labile antimicrobial substance, protec- tion of bacteria from antimicrobial activity by the cooked gelatinous porridge or a dilution of the antimicrobial substance through the addition of water to produce porridge of acceptable viscosity for infant feeding. We conclude that studies on the metabolic nature of these antimicrobial factors are indicated.

ACKNOWLEDGEMENTS

We are grateful to the Medical Research Council, UK, the United Nations University and the Noguchi Memorial Institute for financial support. Our sincere thanks also go to Miss Opokua Ofori-Anyinam and Mr Fred Taylor for technical assistance. We are greatly indebted to Mr David Felmingham, D r Asamoah-Adu and Mr Yeboah for sup- plying the shigella strains. The ETEC strains were kindly supplied by the International Centre for Diarrhoea1 Dis- eases Research and Dr B. S. Rowe of Public Health Labor- atory Services, UK. Mr David Alnwick, Nutrition Adviser, UNICEF, Nairobi, gave useful advice.

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antimicrobial effect of fermented ghanaian maize dough

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