Microbial Growth Responses in Fermented Maize Dough Systems

Abstract

Food safety and the occurrence of diarrhoea are a challenge in the management of food systems in Africa. Infections arising from diarrhoea can be very devastating on the population, especially children. The traditional fermented maize dough systems have been identified as being able to reduce considerably the growth of diarrhoeal causing organisms and improve safety The study was set up to investigate: 1. The survival of selected diarrhoeal causing bacteria (Salmonella, Shigella and Escherichia coli) in maize dough fermenting systems (steeping water, maize dough, Ga kenkey water and maize dough porridge koko) to determine the safety of the products; 2. The survival of four Escherichia coli strains in synthetic medium containing lactic and acetic acids; 3. Tolerance of the dominant yeasts (Candida krusei and Saccharomyces cerevisiae) involved in maize fermentation for lactic acid. 4. Changes in short-term intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae in the presence of high and low concentrations of lactic acid to explain their tolerance for lactic acid. Five Salmonella species, three Shigella species, five pathogenic Escherichia coli strains and two non-pathogenic strains were inoculated into fermented maize dough systems at a concentration of 106 - 107 cfu/ml at 28 °C. Viable cells were recovered on selective and non-selective media. Almost all the bacteria survived in maize steeping water for 48 h without reduction in numbers. All the salmonellae and shigellae and two E. coli strains were completely inhibited in fermenting maize dough after 48 hours whilst three E. coli strains (026 (VTEC), 0157 (VTEC II) and 03 EAggEC) survived for 48 h but were significantly reduced by more than 3 log units. None of the Salmonella strains survived in koko for 24 h but Sh. flexneri 2a II and four pathogenic E. coli strains (0157 (VTEC II), 026 (VTEC), 03 (EAggEC), 0111 (EPEC)) and one laboratory strain E. coli K12, survived in koko for 48 h with less than 2 log reductions. All the bacteria were completely inhibited in kenkey water after 24 h. The decrease in populations of the bacteria in the different fermentation systems was observed at pH of < 4. Greater numbers of survivors were recovered with non-selective medium than with a selective medium for Gram-negative bacteria. In Trypticase Soy Broth Yeast Extract (TSBYE) supplemented with lactic and acetic acids in concentrations found in fermented maize dough systems, E. coli strains 0111 (EPEC), 03 (EAggEC), K12 and M23 were inhibited to various extents. Concentrations of 31 - 62 mM, undissociated lactic acid had only a bacteriostatic effect on the four pathogens, while above 62 mM, a bactericidal effect was noted after 24 h. Concentrations of > 17 - 33 mM undissociated acetic acid were required to completely inhibit the four E. coli strains. These results confirm that fermented maize dough systems have antimicrobial properties which may inhibit the survival of some pathogenic and non-pathogenic bacteria. The extent of inhibition varied among the species investigated, namely Salmonella, Shigella and Escherichia coli; and also among the maize dough systems. The presence of lactic acid at low pH was found to be the main anti-microbial property of the fermented maize dough systems. The influence of different lactic acid concentrations (0.2, 0.4, 0.8 and 1.2% V/V), within a pH range of 3.5 to 4.2 on the survival kinetics of E. coli 0111: H2 (EPEC) was determined in TSBYE at 30 °C. Survival data were analysed and fitted with the model of Peleg and Cole (1998). The model parameters b and n were estimated and used to calculate the time to one log decrease in bacterial population. The model of Peleg and Cole gave a good description of the survival of E. coli under the experimental conditions tested. A strong correlation of the time to one log reduction in bacterial numbers with the undissociated lactic acid concentration was demonstrated. Also using the Number Cruncher Statistical Sytems (NCSS), a multiple regression analysis was performed on the data and a model was obtained which relates the death rate (time to one log reduction) to the lactic acid concentration and pH. The proposed model for the death rate of E. coli 0111 (EPEC) in response to pH and total lactic acid concentration in TSBYE provided a good description of the data. Except for the low pH of 3.5 and 1.2% lactic acid, where the model predicted a negative value for the death rate, all other predicted values were in agreement with actual values obtained in the broth studies. Growth responses of two strains each of Candida krusei and Saccharomyces cerevisiae singly and as mixed cultures were determined in MYGP broth, pH 2.5 and pH 3.5 with or without lactic acid at 30 °C. At pH 2.5, in the presence of 106.4 mM undissociated lactic acid, C. krusei strains grew within 48 h from 4.0 log10 cfu/ ml to 7.0 logio cfu/ ml irrespective of whether cultured singly or combined as mixed cultures with either strain of Sacch. cerevisiae. But Sacch. cerevisiae strains did not grow when cultured individually in combination with either strain of the two C. krusei investigated. When the Sacch. cerevisiae strains were cultured individually as single cultures only one grew at pH 2.5 in the presence of 106.4 mM undissociated lactic acid. At pH 3.5, irrespective of the presence or absence of 77 mM lactic acid, C. krusei 29 grew from 4.0 logio cfu/ml to about 8.0 logio cfu/ml whether cultured singly or in combination with either strain of Sacch. cerevisiae. In contrast, both strains of Sacch. cerevisiae showed good growth as single cultures but reduced growth when cultured with C. krusei as mixed cultures and the reduced growth was greater in the presence of lactic acid. These results indicate that C. krusei is more tolerant to lactic acid at low pH than Sacch. cerevisiae. To explain the differences in lactic acid tolerance of the two yeast species, fluorescence-ratio-imaging microscopy and a perfusion system were used to determine the short-term intracellular pH (pH,) changes in single cells of C. krusei and S. cerevisiae. The changes were investigated both in the presence of low (20.7 mM) and high (106.4 mM) concentrations of undissociated lactic acid. For both the investigated species 20.7 mM undissociated lactic acid did not seem to influence the initial pH; which for C. krusei was found to be approx. 8.0 and for S. cerevisiae 6.9- 7.5. For both C. krusei strains, perfusion with 106.4 mM undissociated lactic acid induced only weak short-term pH responses with a decrease in pH of less than one pH unit. Contrary for both strains of Sacch. cerevisiae, perfusion with 106.4 mM undissociated lactic acid resulted in a significant decrease in pH from initially 6.9 – 7.5 to 6.2 - 6.4 after 1 min and further to a pH, of < 5.5 after 3 min after which it remained constant. The results obtained show that C. krusei is more resistant to short-term pHj changes caused by lactic acid than S. cerevisiae, and this, in turn, may explian why C. krusei is more tolerant to lactic acid than S. cerevisiae.