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Food Microbiology 25 (2008) 616625

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Functional properties of selected starter cultures for sour maize bread

Mojisola O. Edemaa,, Abiodun I. Sannib

aDepartment of Microbiology, College of Natural Sciences, University of Agriculture, P.M.B. 2240, Abeokuta, NigeriabDepartment of Botany and Microbiology, University of Ibadan, Nigeria

Received 13 March 2007; received in revised form 17 December 2007; accepted 30 December 2007

Available online 29 January 2008

Abstract

This paper focuses on the functional properties of maize sour-dough microflora selected and tested for their use as starter cultures for

sour maize bread. Lactic acid bacteria and yeasts isolated from spontaneously fermented maize dough were selected based on dominance

during fermentation and presence at the end of fermentation. Functional properties examined included acidification, leavening and

production of some antimicrobial compounds in the fermenting matrix. The organisms previously identified as Lactobacillus plantarum,

Lb. brevis, Lb. fermentum, Lb. acidophilus, Pediococcus acidilactici, Leuconostoc mesenteroides and Leuconostoc dextranicum and

Saccharomyces cerevisiae were used singly and as mixed cultures in the fermentation (fermentation time: 12 h at 2872 1C) of maize meal(particle size 40.2mm). The pH fell from an initial value of 5.623.05 in maize meals fermented with Lb. plantarum; 4.37 inL. dextranicum+S. cerevisiae compared with the value for the control (no starter) of 4.54. Significant differences (Pp0.05) were observedin values obtained for the functional properties tested when starters were inoculated compared with the control (no starter) except for

leavening. Bivariate correlations at 0.01 levels (two-tailed) showed that significant correlations existed among pH and production of

antimicrobial compounds in the fermenting meals, the highest correlation being between production of diacetyl and acid (0.694), a

positive correlation indicating that production of both antimicrobial compounds increase together with time. Antimicrobial activities of

the fermented maize dough were confirmed by their abilities to inhibit the growth of Salmonella typhi, Escherichia coli, Staphylococcus

aureus and Aspergillus flavus from an initial inoculum concentration of 7 log cfuml1) for test bacteria and zone of inhibition of up to

1.33 cm for aflatoxigenic A. flavus. The findings of this study form a database for further studies on the development of starter cultures

for sour maize bread production as an alternative bread specialty.

r 2008 Elsevier Ltd. All rights reserved.

Keywords: Starter culture; Lactic acid bacteria; Yeasts; Fermentation; Maize meal

1. Introduction

Many foods are fermented before consumption andlactic acid bacteria (LAB) are widely used as starterorganisms in these food fermentations because theyconvert sugars into organic acids thus improving theorganoleptic and rheological properties of the products(Konings et al., 2000; Vogel et al., 2002). Lactic and aceticacid concentrations found in many fermented foods couldalso be sufficient to impart the observed shelf stability.Martinez-Anaya et al. (1994) reported that the efficiency ofsourdough as a possible preservative agent of microbial

e front matter r 2008 Elsevier Ltd. All rights reserved.

.2007.12.006

ing author. Tel.: +234 8037119671.

esses: moedemao@yahoo.co.uk (M.O. Edema),

yahoo.co.uk (A.I. Sanni).

spoilage of bread depends on its ability to produce aceticacid. Pepe et al. (2003a) observed increased viscousproperties during fermentation and increased crumbfirmness in baked pizza dough leavened with LAB andyeast. LAB are often inhibitory to other micro-organismsand this is the basis of their ability to improve the keepingquality of many fermented food products (Corsetti et al.,1998a; Corsetti et al., 2000). LAB have been known to takepart in bread fermentations such as in the production ofthe Swedish rye sourdough (Lonner and Preve-Akesson,1988) and the Indian Idli (Mukherjee et al., 1965) whereinthey improve flavor, texture and keeping quality throughthe production of metabolites such as diacetyl, hydro-gen peroxide and bacteriocins (Armero and Collar, 1996;Arendt et al., 2007; Lacaze et al., 2007). For example, Pepeet al. (2003b) observed an inhibition of rope-producing

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ARTICLE IN PRESSM.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625 617

Bacillus subtilis spores for more than 15 days in breadsproduced with strains of LAB isolated from sourdough. Inaddition, results obtained by Budde et al. (2003) indicatedstrong antilisterial activity by bacteriocin-producingLeuconostoc carnosum without any observable undesirableflavor components.

It was customary in the beginning when cereals werefermented by their natural flora, to put aside pieces of thedough called sours or starters for fermenting subsequentbatches in bread making. This results in irregularities andunpredictability that led to the development and use ofdefined starter cultures of micro-organisms in modernsourdough fermentations. To ensure products of consistentflavor, texture and shelf stability, as well as to improveproduct safety, most processors have developed puremicrobial cultures to control the fermentation of theirproducts (Holzapfel, 2002). It is evident that with a starterculture, the pH drops much more rapidly; hence the wholemanufacturing process is accelerated, leading to economic-al gains for the processor. The majority of starter culturesare natural isolates of the desirable micro-organisms foundnormally in the substrates (Holzapfel, 2002; De Vuyst andVancanneyt, 2007).

Starter cultures can come in fresh, frozen or freeze-driedforms, and they can be single or mixed cultures of selectedstrains of micro-organisms with definite characteristics thatare beneficial in the manufacture of the desired product.A wide variety of species of organisms have been used asstarter cultures in the food industry and many are beinginvestigated for their potential use as starter cultures(De Vuyst and Neysens, 2005; Gaggiano et al., 2007). Whatwere probably the first starter cultures for sourdoughs werethose developed by Kline and Sugihara (1971) for the SanFrancisco sourdough. One of them was a pure cultureconsisting of Lactobacillus sanfranciscensis (formerlyLb. san Francisco) that had previously been isolated fromthe San Francisco sourdough.

However, the use of sourdough starter cultures in thebaking industry is only in its infancy in Sub-Saharan Africaand in Nigeria, is almost non-existent. Yet, there are anumber of substrates that can be exploited for use in thedevelopment of new sourdoughs other than those existingin Europe today. Maize is one such promising substratesparticularly as it lacks gluten which is a major source ofconcern in baked goods from wheat and other cereals thathave gluten proteins, in order to avoid coeliac disease(Di Cagno et al., 2002). Fairly successful attempts havebeen made to develop sour maize bread using thesourdough technique (Sanni et al., 1998). The sourdoughsystem is however a very complex one and there is a need tostudy and understand the system in order to effectivelymanage new products developed from novel sourdoughssuch as the sour maize meal. The composition anddynamics of the micro-flora developing in spontaneouslyfermented maize meal has therefore been studied (Edemaand Sanni, 2006). The dominating organisms in the micro-population of the fermented maize meal were Pediococcus

acidilactici, Lactobacillus plantarum, Lactobacillus brevis,Lactobacillus fermentum, Leuconostoc mesenteroides, Leu-conostoc dextranicum, Lactobacillus casei, Candida albi-cans, Schizosaccharomyces pombe and Saccharomycescerevisiae. The aim of the present study was to selectstarter cultures from the dominant microbial flora of sourmaize meal by investigating their functional properties witha view to developing appropriate sour maize meal starterfor bread making.

2. Materials and methods

2.1. Sample collection and processing

A commercial flour variety of white maize (Zea mays) wasobtained from Bodija market in Ibadan, southwesternNigeria. The grains were milled into maize meal with particlesize greater than 0.2mm which is particularly valuable as aningredient for maize bread as well as meal mixes, maizemuffins and some extruded maize snack products comparedto maize flour with less than 0.2mm particle size (Okoruwa,1995). A knife mill (Fritsch Industriestr. 8 0-55743, Idar-oberstein, Germany) was used for milling. The chemicalcharacteristics of the maize meal were as follows: moisturecontent 7.15%, fat 4.09%, protein (N 5.70) 8.96%, fiber1.48%, ash 1.33% and total carbohydrate 77.06% of drymatter (Edema et al., 2005).

2.2. Experimental design

In the preliminary study, 34 LAB belonging to 15 speciesand 13 yeasts belonging to nine species were isolated duringthe spontaneous fermentation of maize meal (fermentationtime 48 h, ambient temperature 28 1C, final pH 3.71)(Edema and Sanni, 2006). Eight test cultures comprisingseven LAB and one yeast were chosen from these isolateson the basis of dominance during fermentation andpresence at the end of fermentation (Holzapfel, 2002).These were Lb. plantarum, Lb. brevis, Lb. fermentum, Lb.acidophilus, P. acidilactici, L. mesenteroides, L. dextranicumand S. cerevisiae.These organisms were tested for those functional proper-

ties that are important in the sourdough technology that isacidification and production of antimicrobial substances.The antimicrobial properties of the test cultures in maizedough were confirmed with inhibition of some selectedpathogens (Kingamkono et al., 1995; Holzapfel, 2002). Toeliminate bias, selected test cultures were used singly and asmixed cultures in the fermentation of maize meal using acompletely randomized block design on a factorial basiswith three replicates. The spontaneously fermented maizemeal without added starter culture served as control.

2.3. Culture conditions and fermentation

Purified isolates of the test LAB that had been keptas stock cultures on Hogness freezing medium were

ARTICLE IN PRESSM.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625618

cultivated by transferring 2ml of each stock culture into8ml of de Man, Rogosa & Sharpe (MRS) broth medium(Oxoid, Hampshire, UK). The tubes were incubated at30 1C for 48 h. The broth cultures were again inoculatedinto fresh MRS broth medium and incubated as above.During incubation, 1ml each of broth culture was platedon MRS agar plates using the pour plate technique withroutine incubation at 37 1C for 24 h. Colonies were countedand broth cultures of corresponding plates containingabout 2 108 cfuml1 were used as inoculums. Brothcultures containing the required concentration of viablecells were centrifuged (Labofuge 200, Kendro LaboratoryProducts, Germany) at 6000 g for 10min, washed insterile distilled water (pH 7.0) and re-centrifuged beforebeing suspended in sterile distilled water. The washedharvested cells were then used as inoculum in thefermentation of maize meals according to the method ofLonner et al. (1986).

Yeast culture inoculum was prepared using malt extractbroth. Five ml sterile malt extract broth were added to theyeast cultures growing on malt extract agar slants andshaken to make a suspension. Each suspension was pouredinto another 5ml malt extract broth and incubated at 30 1Cfor 24 h. Dilutions of the broth cultures were plated onmalt extract agar and incubated for another 24 h at 30 1C.Plates of broth cultures that gave 2 108 cfuml1 wereused as inoculum. The broth cultures having the requiredcounts were then centrifuged (Labofuge 200) and washedtwice in sterile distilled water before use as inoculum (Halmet al., 1996).

Equal amounts (w/v) of maize meal to tap water wereused in all the trials. Five ml of inoculum containingapproximately the same concentration of cells each(2 108 cfuml1 from pour plates) was used in all caseswhether singly or mixed for 50 g maize meal. Mixing wasdone manually (for up to 5min) in glass bowls using glassrods as stirrers. Fermentation was carried out at ambienttemperature (2872 1C) for up to 24 h. During fermenta-tion, the number of inoculum cells was estimated by plating1 g of fermenting maize meal on MRS agar at 30 1Cfor 48 h.

2.4. Analyses of fermenting maize meal

2.4.1. pH

After fermentation, the pH values of the starter-fermented maize meals were determined with a combinedglass electrode and pH meter (Mettler-Toledo, EssexM3509 Type 340).

2.4.2. Acidity

The amount of acid produced in the fermented mealswas determined by the standard titration procedure fortotal titratable acidity (TTA) according to Lonner et al.(1986). One gram of the fermenting meal was mixed with9ml sterile distilled water and homogenized. The mixture(10ml) was titrated with 1N NaOH using phenolphthalein

as indicator. Acid equivalent is the amount of NaOHconsumed in ml. Each ml of 1N NaOH is equivalent to90.08mg of lactic acid.

2.4.3. Leavening

Leavening in the fermented meal was determined byrecording the level of the fermenting maize meal on thegraduated bottles used for fermentation at mixing and atthe end of fermentation. The difference between the initialand the final readings were taken as the level of leaveningin centimeters.

2.4.4. Diacetyl

Diacetyl production was determined by mixing 10 gfermenting maize meal in 90ml tap water. To 25ml each ofthe homogenized mixture, 7.5ml of hydroxylamine solu-tion (1M) was added in two flasks (one flask was forresidual titration). Both flasks were titrated with 0.1N HClto a greenish yellow end point using bromophenol blue asindicator (Sanni et al., 1995). The equivalence factor ofHCl to diacetyl is 21.52mg. The concentration of diacetylproduced was calculated as follows:

Ak R S100EW

,

where Ak (mg) is the percentage of diacetyl, R the ml of0.1N HCl consumed in residual titration, S the ml of 0.1NHCl consumed in titration of sample, E the equivalencefactor and W is the volume of sample.

2.4.5. Hydrogen peroxide

Hydrogen peroxide production was determined bymeasuring 25ml of homogenized mixture (from the samebatch used for diacetyl) into a 100-ml flask. To this wasadded 25ml of dilute H2SO4 (10%). The preparation wasthen titrated with 0.1N potassium permanganate(KMnO4). The end point was the point at which the palepink color persisted for 15 s before de-colorization. Eachml of 0.1N KmnO4 is equivalent to 1.701mg of H2O2(Sanni et al., 1995). The volume of H2O2 produced wasthen calculated as follows:

H2O2 concentration KMnO4ml KMnO4N M:E: 100

H2SO4ml volume of sample.

2.4.6. Antimicrobial activity

Antimicrobial activities of the fermenting maize mealswere determined by two procedures (Piddock, 1990). Theagar well diffusion method was used to test the anti-microbial activity of fermenting meal against aflatoxigenicmold, Aspergillus flavus previously isolated from grilled,dry meat and confirmed to produce aflatoxin by fluores-cence under ultraviolet radiation in Yeast Extract Sucrosemedium was used (Cotty, 1994; Onilude et al., 2005).Spores were harvested from stock culture maintained onagar slant by adding 10ml sterile distilled water to dislodge

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0123456789

0 12 24 36 48Time (h)

Log

of c

ount

s (c

fu/g

) LAB

Yeasts

PCAcounts

Fig. 1. Plate counts of LAB and yeasts during spontaneous fermentation

of maize meal.

M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625 619

the spores. Molten Potato Dextrose Agar (Oxoid) inErlenmeyer flask was inoculated with the spore suspensionat a concentration of 5% (v/v). Incubation was 30 1C forup to 72 h on. For test bacteria, Salmonella typhi, E. coliand S. aureus, a modification of the method used by Rusolet al. (1997) was adopted. From stock cultures obtained inthe Department of Microbiology, University of Agricul-ture, Abeokuta, one colony each of test organisms wereinoculated in MacConkey broth for E. coli, tetrathionatebroth for S. typhi and BairdParker medium for Stapho-lyococcus. After incubation at 37 1C for 24 h, the cells wereharvested by centrifugation at 6000 g, 15min and washedwith sterile distilled water. Ten-fold dilution of each samplewas then plated to obtain 7 log cfuml1. Dilutions contain-ing the required number of cells were used as inoculum bymixing 1ml of the cell suspension with 9 g of fermentingmaize meal. The tubes were incubated at 37 1C for 24 hbefore enumeration.

2.4.7. Inoculum growth assessment

The growth of each inoculum was monitored during thefermentations by plating out dilutions using the pour platemethod. Counts were also taken for the un-inoculatedsample before and after fermentation for comparison.Total viable counts were made on MRS of de Man et al.(1960) for the LAB and on malt extract agar for yeasts.MRS was adjusted to pH 5.4 while streptomycin sulfateand penicillin were added to MEA in the ratio 2:1,respectively/liter, to inhibit the growth of unwantedorganisms. As expected, cell concentrations increasedduring fermentation (Muller et al., 2001).

2.4.8. Analysis of data

All experiments were carried out in triplicate trials andthe data generated were subjected to one-way analysis ofvariance (ANOVA) at 5% level of significance andbivariate correlations using SPSS11.0 for windows. Meanswere separated by Duncans multiple range tests.

3. Results

Thirty-four LAB belonging to 15 species and 13 yeastsbelonging to nine species were isolated during thespontaneous fermentation of maize meal (fermentationtime 48 h, ambient temperature 2872 1C, final pH 3.71) ina previous study by the authors (Edema and Sanni, 2006).Counts of LAB increased steadily from 4.62 log at mixing(0 h) to 6.45 log after 48 h fermentation while yeast countsincreased from 4.18 to 6.64 log within the same period offermentation (Fig. 1). Eight test cultures comprising sevenLAB and one yeast were chosen from these isolates on thebasis of dominance during fermentation and presenceat the end of fermentation. These were Lb. plantarum,Lb. brevis, Lb. fermentum, Lb. acidophilus, P. acidilactici,L. mesenteroides, L. dextranicum and S. cerevisiae.

Some functional properties important in soudough wereexamined by inoculating the selected organisms into maize

meal (fermentation conditions, 2872 1C for 12 h; inoculumrate, 108 cfu g1; dough yield, 192). Functional propertiesexamined in this study included acidification and leaveningpatterns as well as the production of some antimicrobialcompounds. The initial pH of the maize meal beforefermentation was 5.62. For single cultures, all LAB specieswere able to lower pH during fermentation more thanyeasts with pH values ranging from 3.05 for Lb. plantarumto 3.37 for L. mesenteroides as against 3.65 for S. cerevisiae(Table 1). Mixed cultures also lowered the pH of thefermenting meal to varying degrees with significantdifferences (Pp0.05) in the values obtained. All starters,whether single or mixed were however able to lower the pHof the fermenting meals significantly more than that of thecontrol, i.e. 4.54 at the same probability level. Conversely,production of acid, diacetyl and hydrogen peroxide in thefermenting meals were significantly higher in mealsfermented with starter cultures than in the spontaneoussour. Leavening in fermented maize meals ranged from1.20 to 1.80 cm. Significant differences (Pp0.05) wereobserved in values obtained for the functional propertiestested when starters were inoculated compared with thecontrol (no starter) except leavening.Bivariate correlations at 0.01 levels (two-tailed) were

used to evaluate correlations among the functional proper-ties examined. Significant negative correlations existedamong pH, acid production and diacetyl production inthe fermenting meals while significant positive correlationswere observed in the production of H2O2 and pH on onehand, and in the production of acid, diacetyl and leaveningon the other hand (Table 2). The highest correlation wasobserved between production of diacetyl and acid (0.694).It was a positive correlation indicating that production ofboth antimicrobial compounds increase together with time.Antimicrobial activities of the fermented maize dough

were confirmed by testing their abilities to inhibit thegrowth of selected indicator organisms: S. typhi, E. coli,S. aureus and A. flavus. Numbers of inoculum weredrastically reduced from about 6 log cfuml1 (approximatecell concentration after inoculating 7 log cfuml1 of

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Table 1

Functional properties of sour maize meals started with single and mixed cultures of LAB and yeasts

Starter pH Acid equivalent

(ml)

Diacetyl

(mg)

Hydrogen

peroxide (mM)

Leavening

(cm)

L. plantarum 3.05a 3.20ijkl 163.55q 4.25ef 1.33bcd

L. brevis 3.16ab 2.90ghij 180.77u 4.22def 1.33bcd

L. fermentum 3.26ab 2.80ghi 172.16s 4.25ef 1.37cde

L. acidophilus 3.09a 3.00hijk 154.94o 4.08bcdef 1.30abc

P. acidilactici 3.36abc 2.60efgh 154.94o 4.25ef 1.20a

Leu. mesenteroides 3.37abc 2.50cdefg 129.12j 4.29ef 1.43def

Leu. dextranicum 3.36abc 2.50cdefg 111.90d 4.20def 1.37cde

S. cerevisiae 3.65abc 1.33b 94.69b 4.08bcdef 1.50efgh

L. plantarum and L. brevis 3.20ab 3.28jkl 176.46t 4.42f 1.57fghij

L. plantarum and L. fermentum 3.12ab 3.36kl 172.20s 3.17abcd 1.63hij

L. plantarum and L. acidophilus 3.03a 3.49l 172.30s 3.07ab 1.63hij

L. plantarum and P. acidilactici 3.04a 2.80ghi 173.07s 3.17abcd 1.57fghij

L. plantarum and Leu. mesenteroides 3.15ab 3.46l 172.77s 3.35abcde 1.67ij

L. plantarum and Leu. dextranicum 3.40abc 2.92ghij 185.07v 3.31abcde 1.53fghi

L. plantarum and S. cerevisiae 3.35abc 3.50l 154.94o 4.25ef 1.80k

L. brevis and L. fermentum 3.41abc 2.15cde 155.07o 3.11abc 1.62hij

L. brevis and L. acidophilus 3.48abc 2.71gh 156.77p 3.17abcd 1.59ghij

L. brevis and P. acidilactici 3.46abc 2.63fgh 154.40o 4.02bcdef 1.58ghij

L. brevis and Leu. mesenteroides 3.39abc 2.16cde 134.70m 3.56abcdef 1.58ghij

L. brevis and Leu. dexranicum 3.92abc 2.16cde 167.88r 4.08bcdef 1.70jk

L. brevis and S. cerevisiae 3.55abc 2.70gh 124.81i 4.17def 1.80k

L. fermentum and L. acidophilus 3.91abc 2.10c 122.17h 4.01bcdef 1.57fghij

L. fermentum and P. acidilactici 3.88abc 2.59defgh 121.20gh 4.05bcdef 1.60ghij

L. fermentum and Leu. mesenteroides 3.85abc 2.15cde 120.33fg 3.99bcdef 1.60ghij

L. fermentum and Leu. dextranicum 3.85abc 2.16cde 121.00gh 3.99bcdef 1.50efgh

L. fermentum and S. cerevisiae 3.95abc 2.53cdefgh 130.37k 4.06bcdef 1.63hij

L. acidophilus and P. acidilactici 3.71abc 2.83ghi 119.67ef 3.94bcdef 1.47defg

L. acidophilus and Leu. mesenteroides 4.19abc 2.15cde 120.13fg 3.96bcdef 1.50efgh

L. acidophilus and Leu. dextranicum 4.08abc 2.12cd 120.03fg 3.98bcdef 1.53fghi

L. acidophilus and S. cerevisiae 4.12abc 2.69gh 151.40n 4.06bcdef 1.67ij

P. acidilactici and Leu. mesenteroides 3.93abc 2.17cdef 118.70e 4.11bcdef 1.47defg

P. acidilactici and Leu. dextranicum 3.78abc 2.48cdefg 118.80e 4.07bcdef 1.43def

P. acidilactici and S. cerevisiae 4.08abc 2.50cdefg 122.13h 4.16cdef 1.63hij

Leu. mesenteroides and Leu. dextranicum 3.75abc 2.17cdef 100.27c 4.44f 1.57fghij

Leu. mesenteroides and S. cerevisiae 4.04abc 2.88ghij 120.17fg 4.22def 1.67ij

Leu. dextranicum and S. cerevisiae 4.37bc 2.50cdefg 133.42l 4.18def 1.70jk

Control (no starter added) 4.54c 0.80a 84.47a 2.89a 1.23ab

Values are means of three replicates. Mean values followed by different letters within columns are significantly different by Duncans multiple range tests

(Pp0.05).

M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625620

washed cells into moistened maize meal). All startersincluding the control were able to inhibit growth of thetest bacteria from an initial inoculum concentration ofapproximately 6 log cfuml1 at 35 1C; 24 h to as low as2 log cfuml1 for S. typhi and E. coli. S. cerevisiae recordedthe least inhibitory activity against the pathogenic bacteriatested (Fig. 2).

For A. flavus, the agar well diffusion method was usedto determine antimicrobial activity of starter fermentedmaize meals. The zone of inhibition ranged from 0.60 cmfor dough fermented with S. cerevisiae to 1.33 cm fordough fermented with Lb. plantarum and Lb. brevis(Fig. 3). The pattern of zone of inhibition of A. flavusobserved for maize meal fermented with mixed culture ofLb. plantarum and Lb. brevis is shown in Fig. 4. The reasonfor the central zone of inhibition is not clear but could haveresulted from production of bacteriocin-like substances

which moved in the direction of observed zone ofinhibition.

4. Discussion

The selection of starter cultures in this study involved theinvestigation of some important functional properties ofstrains of LAB and yeasts isolated from spontaneouslyfermented maize meal. Cultures for food fermentations areselected primarily on the basis of their stability and theirability to produce the desired products or changesefficiently (Gobbetti, 1998; Leroy and De Vuyst, 2004).These cultures may be established ones obtained fromother laboratories or they may be selected after testingmany numerous strains (Sanni et al., 2002; Gaggiano et al.,2007). The latter alternative was employed in this work.This is because organisms develop niches where they thrive

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Table 2

Correlations among functional properties of starter-generated sour maize

meals

pH Acid Diacetyl Peroxide Leavening

pH

Pearson correlation 1 .288 .426 .384 .132Sig. (two-tailed) .002 .000 .000 .167

N 111 111 111 111 111

Acid

Pearson correlation .288 1 .694 .025 .256Sig. (two-tailed) .002 .000 .797 .007

N 111 111 111 111 111

Diacetyl

Pearson correlation .426 .694 1 .158 .097Sig. (two-tailed) .000 .000 .099 .310

N 111 111 111 111 111

Peroxide

Pearson correlation .384 .025 .158 1 .071Sig. (two-tailed) .000 .797 .099 .462

N 111 111 111 111 111

Leavening

Pearson correlation .132 .256 .097 .071 1Sig. (two-tailed) .167 .007 .310 .462

N 111 111 111 111 111

Correlation is significant at the 0.01 level (two-tailed).

M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625 621

and to transplant an organism from one natural environ-ment to another is not a good formula for success,particularly in sourdough fermentations. The species ofLAB and yeasts isolated from fermenting maize meal andtested for their functional properties in this work were thedominant micro-organisms at the end of 48 h spontaneousfermentation of maize meal (Edema and Sanni, 2006).Previous studies on the natural micro-biological flora ofsourdough made from different cereals report similarorganisms (Spicher, 1987; Gobbetti et al., 1995; Merothet al., 2004; De Vuyst and Neysens, 2005; Ehrmann andVogel, 2005).

Micro-organisms necessary in food fermentations maybe added as pure single or mixed cultures. Although insome instances, no cultures may be added if the desiredmicro-organisms are known to be present in sufficientnumbers in the original raw material (Lonner et al., 1986;Randazzo et al., 2005). In the case of sour maize meal, thepresence of the desired organisms, that is LAB and yeasts,in the raw material whether sufficient or not, is notadequate since all the meals fermented with starter cultureswere better in acidification and other properties than thespontaneously fermented meal used as control. This couldbe because there are usually other numerous and undesir-able competing micro-organisms as earlier observed(Edema and Sanni, 2006). Although given favorableenvironmental conditions, the desired organisms willeventually act in proper succession (Wiese et al., 1996;Gatto and Torriani, 2004), it is advantageous to usecontrolled starter cultures as these will drastically reduce

the period of fermentation, sometimes more than halfdepending on the amount of starter used (Oyewole, 1990;Sanni et al., 1999; De Vuyst et al., 2002).As the preparation of sourdough is very time consuming

if full leavening by LAB is to be obtained, few processeshave been developed in Germany, which ensure that thedough is acidified by the sourdough bacteria whileleavening is achieved by bakers yeast (Ganzle, 2002).However, bakers yeast was not used in this study becauseof the short shelf life recorded in bakers yeastleavenedsour maize bread prepared in a preliminary study (Sanniet al., 1998). Rather, a S. cerevisiae strain isolated from thespontaneously fermented maize meal was used. It was usedsingly and in combination with the selected LAB culturesand was observed to produce inhibition of test pathogensin addition to leavening. In sourdough, yeast is importantfor good batter leavening and bread viscosity while theLAB produce acids and other metabolites which inhibit thegrowth of spoilage organisms such as molds or rope-causing bacilli (Amoa-Awua et al., 1997; Corsetti et al.,2000). Yeasts do not produce appreciable amounts oforganic acids, their main metabolites being ethanol andcarbon dioxide (Campbell-Platt, 1987; Sanni and Lonner,1993). The co-interaction between LAB and yeasts ishowever notably common in many food and beveragefermentations with the LAB providing the acid environ-ment for yeast growth, while the yeasts provide vitaminsand other growth factors for the LAB (Odunfa andAdeyele, 1985; Lonner and Preve-Akesson, 1988; Corsettiet al., 2000). The observed antimicrobial property in maizemeal starter on yeast could therefore have been as a resultof the presence and growth of endogenous LAB in thesubstrate since the flour was not sterile.Maize meals started with mixed cultures containing

Lb. plantarum produced more acid and diacetyl than theothers. Generally, it was observed that mixed culturesappeared to produce more antimicrobial compounds thanthe single cultures. Indeed maize meals started with mixedcultures were preferred to those started with single culturesin taste and aroma (result not shown). Lb. plantarum hasbeen shown to posses the ability for rapid acidification,production of antimicrobial compounds and the mosteffective antifungal effect against toxigenic strains ofPenicillium and Aspergillus (Niku-Paavola et al., 1999) aswell as the production of exopolysaccharide (Figueroaet al., 1997).Antimicrobial activities of the fermented maize dough

were confirmed by the inhibition of the growth of S. typhi,E. coli, S. aureus and A. flavus. E. coli occurs normally inhuman intestines and can get into foods handled withoutwashing hands after changing diapers or using toilets.S. typhi is transmitted through contaminated poultry, eggsand other foods when they are handled unhygienically.Staphylococcus occurs almost everywhere and grows read-ily in foods stored at room temperature. S. aureus is foundon the skin and in the nostrils of many healthy individualswhere it causes some skin infections, but it is also an agent

ARTICLE IN PRESS

0

1

2

3

4

5

6

7

L. pla

ntarum

L. bre

vis

L. fer

mentu

m

L. ac

idoph

ilus

P. ac

idilac

tici

Leu.

mese

nteroi

des

Leu.

dextr

anicu

m

S. ce

revisi

ae

L. pla

ntarum

and L

. brev

is

L. pla

ntarum

and L

. ferm

entum

L. pla

ntarum

and L

. acid

ophil

us

L. pla

ntarum

and P

. acid

ilacti

ci

L. pla

ntarum

and L

eu. m

esen

teroid

es

L. pla

ntarum

and L

eu. d

extra

nicum

L. pla

ntarum

and S

. cere

visiae

L. bre

visan

d L. fe

rmen

tum

L. bre

visan

d L. a

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L. bre

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d P. a

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visan

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. mes

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vis an

d Leu

. dex

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mentu

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d L. a

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L.fer

mentu

m an

d P. a

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mentu

m an

d Leu

. mes

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mentu

m an

d Leu

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L.fer

mentu

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d S. c

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L.ac

idoph

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nd P.

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i

L. ac

idoph

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L.ac

idoph

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u. de

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L. ac

idoph

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P.ac

idilac

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ndLe

u. me

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s

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idilac

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u. de

xtran

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u. de

xtran

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Leu.

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nteroi

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. cere

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Leu.

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d S. c

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iae

Contr

ol(no

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r add

ed)

Starter

Log

cfu

/g S. typhi

E. coli

S. aureus

Fig. 2. Inhibition of three pathogenic bacteria by sour maize meals started with single and mixed cultures of LAB and yeasts (initial inoculum

concentration 7 log cfuml1, incubation 15 1C for 24 h).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

L. pl

antar

um

L. b

revis

L. fe

rmen

tum

L. a

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P. a

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s

Leu.

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S. c

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L. pl

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and

L. br

evis

L. pl

anta

rum

and

L. fe

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L. p

lanta

rum

and

L. a

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L. pl

anta

rum

and

P. a

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L. p

lant

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and

Leu

. mes

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L. pl

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L. b

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L. b

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and

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evis

and

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iae

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and L

. acid

ophi

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L. fe

rmen

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nd P

. acid

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ci

L. fe

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and

Leu.

mes

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L. fe

rmen

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and

Leu

. dex

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L. fe

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and

S. c

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L. ac

idoph

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nd P

. acid

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L. ac

idoph

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nd L

eu. m

esen

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ides

L. ac

idoph

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eu. d

extra

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m

L. ac

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. cer

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ae

P. a

cidila

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and

Leu.

mes

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P. a

cidila

ctici

and L

eu. d

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nicu

m

P. a

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and

S. c

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Leu.

mes

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s an

d Leu

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Leu.

mes

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s an

d S.

cere

visiae

Leu.

dex

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cum

and

S. c

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Cont

rol (

no st

arte

r add

ed)

Starter

Zone

of i

nhib

ition

(cm

)

Fig. 3. Inhibition of Aspergillus flavus by sour maize meals started with single and mixed cultures of LAB and yeasts.

M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625622

of food poisoning when it gets in contact with food where itmultiplies and produces its toxin (Frazier and Westhoff,1986). A. flavus is a ubiquitous spore-former and its sporesare found everywhere in nature food surfaces inclusive. Thespores germinate in foods such as cereals whenever

prevailing environmental conditions permit. All starterswere able to inhibit growth of the tested pathogens tovarying degrees, including the maize dough started withyeast and the spontaneously fermented maize meal. It isbelieved that the growth of endogenous LAB in the maize

ARTICLE IN PRESS

Fig. 4. Inhibition of Aspergillus flavus by sour maize meal started on a

mixed culture of L. plantarum and L. brevis.

M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625 623

substrate was responsible for the inhibitory ability of thespontaneously fermented maize meal. Previous studieshave also shown inhibition of similar pathogens by LABin related spontaneous fermentations (Svanberg et al.,1992; Kingamkono et al., 1995; Olasupo et al., 1997; Sanniet al., 1999).

At different pH ranges, the minimum inhibitory con-centration (MIC) of the undissociated lactic acid wasdifferent against pathogens such as Clostridium andEnterobacter has been shown to differ (Lindgren andDobrogosz, 1990). In addition, the stereoisomers of lacticacid also differ in antimicrobial activity, L-lactic acid beingmore inhibitory than the D-isomer (Benthin and Villadsen,1995). Acetic and propionic acids produced by LAB strainsthrough heterofermentative pathways may interact withcell membranes, and cause intracellular acidification andprotein denaturation (Huang et al., 1986). They are usuallymore antimicrobially effective than lactic acid due to theirhigher pKa values (lactic acid 3.08, acetic acid 4.75, andpropionic acid 4.87), and higher percentage of undisso-ciated acids than lactic acid at a given pH (Earnshaw,1992). Acetic acid has been shown to be more inhibitorythan lactic acid toward Listeria monocytogenes (Ahmadand Marth, 1989; Richards et al., 1995), and toward thegrowth and germination of Bacillus cereus (Wong andChen, 1988). Acetic acid also acted synergistically withlactic acid with lactic acid decreasing the pH of themedium, thereby increasing the toxicity of acetic acid(Adams and Hall, 1988). In addition, hydrogen peroxideproduced by LAB has been shown to produce antimicro-bial effect from the oxidation of sulfhydryl groups causingdenaturing of a number of enzymes, and from the

peroxidation of membrane lipids thereby increasing mem-brane permeability. Hydrogen peroxide may also be as aprecursor for the production of bactericidal free radicalssuch as superoxide and hydroxyl radicals which candamage DNA (Yang et al., 1997). It has been reportedthat the production of H2O2 by Lactobacillus andLactococcus strains inhibited S. aureus, Pseudomonas sp.and various psychotropic micro-organisms in foods (Cordsand Dychdala, 1993).Inhibition of A. flavus by sour maize meal started on a

mixed culture of Lb. plantarum and Lb. brevis revealed acentral pattern which could be as a result of production ofbacteriocin-like substance (Corsetti et al., 1998b, 2004;Vanne et al., 2001) that moved in the direction of observedinhibition. The inhibition is not likely to have been entirelya result of acid production by the starters, since it haspreviously been shown that Aspergillus parasiticus NRRL2999 grew in a medium containing up to 0.75% lactic acidat pH 3.5 (El-Gazzar et al., 1987). It can be observedhowever that there is some yeast growth on the plate mostlikely from endogenous culture of the maize meal. Thathowever did not prevent the inhibitory action of the starter,as the starter culture was in a large quantity enough to startoff the fermentation and the action before the growth ofthe endogenous organisms.Studies on sourdough have been carried out especially in

Germany, France and Sweden where the sourdoughfermentation process has been used traditionally for rye,rye-mixes and other flours which are difficult to bakewithout souring. (Lonner et al., 1986; De Vuyst et al., 2002;Catzeddu et al., 2006; Valcheva et al., 2006). Also, in theUnited States, the San Francisco sourdough bread processhas been carried out in the San Francisco bay area for over130 years (Sugihara et al., 1970). The use of this techniqueis relatively new to Nigeria but is desirable as a way ofutilizing local substrates such as maize and cassava fordevelopment of new food varieties. LAB are able toproduce a wide variety of compounds which give fermentedfoods such as sourdough their characteristic flavor and alsoimpart improved safety and rheology to the foods (Arendtet al., 2007). However, the properties of the sourdoughsystem depend on many different concurrent factors as wellas external process conditions all of which influence theproperties of the products. Some of these factors influencethe properties of the system directly, while others have anindirect influence by affecting the activity and themetabolism of the micro-organisms (Lacaze et al., 2007).Consistent quality, safety and acceptability of a newproduct such as the sour maize bread will be significantlyimproved by the use of well-defined starter cultures selectedon the basis of multifunctional considerations. It istherefore imperative to properly study and regulate thefermentation process in order to obtain a desirableproduct, as in the focus on ongoing research by theauthors. Further studies are focused on the development offast starters with emphasis on rate of leavening, ultimatelevels of acidity produced and flavor. Molecular studies,

ARTICLE IN PRESSM.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625624

baking trials and a standardized procedure for pilot-scaleproduction are also in view.

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Functional properties of selected starter cultures for sour maize breadIntroductionMaterials and methodsSample collection and processingExperimental designCulture conditions and fermentationAnalyses of fermenting maize mealpHAcidityLeaveningDiacetylHydrogen peroxideAntimicrobial activityInoculum growth assessmentAnalysis of dataResultsDiscussionReferences