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Annals of Microbiology ISSN 1590-4261 Ann MicrobiolDOI 10.1007/s13213-014-0997-6

Zymomonas mobilis: biomass productionand use as a dough leavening agent

Alida Musatti, Manuela Rollini, CeciliaSambusiti & Matilde Manzoni

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ORIGINAL ARTICLE

Zymomonas mobilis: biomass production and use as a doughleavening agent

Alida Musatti & Manuela Rollini & Cecilia Sambusiti &Matilde Manzoni

Received: 10 September 2014 /Accepted: 28 October 2014# Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Zymomonas mobilis ferments only glucose, fruc-tose and sucrose via the Entner-Doudoroff pathway, providingan equimolar mixture of ethanol and CO2 and theoretically, asfor Saccharomyces cerevisiae, the gas evolved can be used toleaven a dough. However, the capability of Z. mobilis toproduce CO2 has rarely been exploited. In the present studywe first evaluated the growing performance of two Z. mobilisstrains (DSMZ 424 and 3580) in a culture medium lackingyeast extract, with added glucose or fructose (20 and 50 g/L)comparatively; the results demonstrated that biomass yield is50% higher with glucose. The best conditions were up-scaled,obtaining a biomass yield of 1.3–1.4 g dcw/L in a 14-Lfermenter. Leavening trials performed in a model system withthe biomass collected from fermenters after 9 or 16 h incuba-tion evidenced that Z. mobilis can leaven a model dough asS. cerevisiae does, and showing a CO2 production rate (9–11 mL g dcw−1 min−1) statistically higher than that ofS. cerevisiae (6–7 mL g dcw−1 min−1), especially when using9-h-grown biomass. Bakery products leavenedwith Z. mobiliscould thus be available to people with adverse responses to theingestion of bakery food, providing innovation in the area ofyeast-free leavened baked goods.

Keywords Zymomonas mobilis . Leavening performance .

Dough-model system . CO2 production rate . Yeastintolerance . Yeast-free products

Introduction

In Western countries, alcoholic beverages such as beer, wineand cider are generally produced by fermentation with yeasts,the most common among them being Saccharomycescerevisiae. In many tropical areas of America, Asia and Afri-ca, other types of alcoholic beverages are very popular andwidely produced: these consist of plant saps undergoing fer-mentation processes by mixed cultures containing bacteriabelonging to the genus Zymomonas. In Europe, bacteria ofthis genus can be found occasionally as contaminants of beer,cider and perry (Swings and De Ley 1977; Sahm et al. 2006;Alcantara-Hernandez et al. 2010).

Zymomonas mobilis is an asporigenous Gram-negativebacterium, able to consume only glucose, fructose and su-crose, via the Entner-Doudoroff (ED) metabolic pathway,giving an equimolar mixture of ethanol and CO2. Zymomonasmobilis produces ethanol faster than Saccharomyces cerevisiae,the most popular ethanol producer, and also has both a highsugar tolerance (up to 400 g/L) and ethanol resistance (up to12 %) (Sprenger 1996). Many studies have investigated theadvantages of the use of Z. mobilis in ethanol productioninstead of S. cerevisiae. This research line is lively, and thedepletion of fossil fuel reserves is pushing research towardsalternative energy sources (Panesar et al. 2006; Schuster andChinn 2013). Regarding ethanol production, several studieshave been trying to widen the range of fermentable substratesand to reduce the formation of by-products through geneticmanipulation of Z. mobilis (Lawford and Rousseau 2003; So

A. Musatti :M. Rollini :M. ManzoniDeFENS, Department of Food, Environmental and NutritionalSciences, Università degli Studi di Milano, Via G. Celoria 2,20133 Milan, Italy

C. SambusitiDICA, Department of Civil and Environmental Engineering,Environmental Section, Politecnico di Milano, Piazza L. da Vinci,32, 20133 Milan, Italy

M. Rollini (*)Via Mangiagalli 25, 20133 Milan, Italye-mail: manuela.rollini@unimi.it

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et al. 2014). Moreover the production of organic acids(Malvessi et al. 2013), sorbitol (Cazetta et al. 2005; De Barrosand Celligoi 2006) or levan (De Oliveira et al. 2007), consid-ered by-products in the ethanol production process, have been afocus of great attention.

On the contrary, the ability of Z. mobilis to produce CO2

has rarely been exploited. Like with S. cerevisiae, which isused in bakery products, the gas evolved can be used to leavena dough. Perhaps the fact that Z. mobilis has a low molargrowth yield—among the lowest known for bacteria—mayhave discouraged any research into its biomass production andapplication. Z. mobilis metabolism leads to a lower energyyield compared to S. cerevisiae: only a small percentage(< 3 %) of sugar consumed by Z. mobilis ends up in biomass(Sahm et al. 2006). To date, the only published study relatingto investigation of the leavening ability of five Z. mobilisstrains is insufficient to conclude if these bacteria could beused in bread making (Oda and Tonomura 1994).

In recent years in Western countries the incidence of yeastintolerance, due to an adverse immune response towardsmannans, has been increasing (Annese et al. 2004; Brunneret al. 2006); S. cerevisiae has also been related to fungemia inimmunosuppressed and critically ill patients (Munõz et al.2005). Therefore, these health issues may justify deeper in-vestigation into the use of Z. mobilis in dough production.

In the present study, we investigated the use of Z. mobilis asalternative to the commercial yeast S. cerevisiae. This study isthe first step in research aimed at optimizing Z. mobilis bio-mass growth yield (despite the fact that yields are much lowerthan with S. cerevisiae): we evaluated Z. mobilis growthperformance in media with glucose and fructose as energysource, avoiding the ingredient yeast extract. The best condi-tion was then up-scaled to a pilot plant fermenter, and thebiomass obtained was employed in dough-model systems toevaluate Z. mobilis leavening performance compared withS. cerevisiae.

The potential baked product will be targeted at peoplehaving adverse responses to the ingestion of bakery foodleavened with S. cerevisiae.

Materials and methods

Microorganism

Zymomonas mobilis DSMZ 424 (DSMZ: DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH)and DSMZ 3580 were employed in this research. Thesestrains were chosen on the basis of their fermentative perfor-mance, as reported byOda and Tonomura (1994). A sample ofbaker’s yeast strain in compressed form (Saccharomycescerevisiae, ABMauri, Italy) was employed as reference. Yeastwas stored at 4 °C and used in the 1st week after purchase.

Culture conditions

Strains were maintained weekly in DSM liquid medium of thefollowing composition (g/L): bacto-peptone (Costantino Spa,Turin, Italy) 10, yeast extract (Costantino Spa) 10, glucose 20,pH 6.8, sterilization 118 °C for 20 min. Cultures were inocu-lated (10 % v/v) with a pre-culture in the same medium,incubated at 30 °C for 24 h in stationary conditions andsubsequently stored at 5 °C. Cultures grown in DSM mediumwere used as inoculum of the following biomass productiontrials.

Zymomonas mobilis growth in flasks

Biomass was grown in 300 mL and 1 L flasks containing 200and 600 mL, respectively, of the following IC medium (g /L):bacto-peptone 10, casein enzymatic hydrolysate (CostantinoSpa) 10, pH 6.8. In 300mL flasks, mediumwas supplementedwith 20 or 50 g/L glucose (G20-G50) or fructose (F20-F50),while trials performed with 1 L flasks employed glucose only(G20-G50). Carbon sources were sterilized separately frommedia (112 °C for 30 min) and added before inoculum.

All flasks were inoculated with a 10 % (v/v) amount of a16-h-old culture grown in IC G20 medium for the biomassproduction trials with glucose and IC F20 for those withfructose. Cultures were incubated at 30 °C for 16 or 24 hunder stationary conditions.

Zymomonas mobilis growth in fermenter

Trials were carried out in a 14-L fermenter (10 L workingvolume) (Omnitec, Sedriano, Italy) employing the IC G50medium, sterilized directly in the fermenter; glucose wassterilized separately and added before the inoculum. Growthconditions were set as follows: temperature 30 °C, pH stat 5.5with 1 M NH4OH, 200 rpm, no aeration (nitrogen was bub-bled for 10 min before inoculum), overpressure 0.15 bar,incubation time 9 or 16 h, inoculum 10 % (v/v) of a 16-h-old culture grown in IC G20 medium as reported previously.

Biomass growth determination

An aliquot (200 mL) of culture broth obtained at the end ofincubation was centrifuged at 8,600 g for 20 min at 5 °C in aBeckman J2-21 centrifuge. The recovered cell pellet waswashed with deionized water and centrifuged again underthe same conditions. Dry cell weight (dcw) was determinedafter drying the cell pellet at 105 °C for 24 h.

Evaluation of leavening performance in a model dough

Gas production ability (mL/g dcw) was tested employing aprocedure adapted from Burrows and Harrison (1959). Cell

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sample (160 mg dcw) was suspended in 15 mL distilled waterand then added to 20 g commercial Type 0 wheat flour(Manitoba), suitable for bread making. In the case ofZymomonas, glucose (2 g) was also added. The dough wasmade bymixing all ingredients (kneading done by handwith aspatula for 45 s) in a double chamber glass flask, connected toa water bath at 29±0.5 °C. The flask was connected with agraduated burette, filled with saturated NaCl solution (tominimize CO2 dissolution), equilibrated with atmosphericpressure. The kinetics of gas production (mL) was followedby measuring the liquid level at appropriate intervals. Datawere normalized per gram dcw.

In each trial, the time course of gas production was fitted byemploying DMFit 3.0 software, to estimate gas productionrate (mL/g dcw min), lag leavening phase duration (min) andtotal amount of gas evolved (mL/g dcw), according to Baranyiand Roberts (1994) (Eq. 1):

y tð Þ ¼ y0 þ μmaxt þ1

μmaxln e−vt þ e−h0− e−vt−h0� �

−1

mln 1þ e

m μmaxtþ1=μmaxln e−vtþe−h0− e−vt−h0ð Þ − 1

em ymax−y0ð Þ

0

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ð1Þ

In the present paper, the equation was applied to model gasproduction, where y(t)=mL gas/g dcw; y0=mL gas/g dcwpresent at t0; ymax=(mL gas/g dcw)max; μmax is the maximumgas production rate (mL gas/g dcw min); m is a curvatureparameter to characterize the transition from the exponentialphase; v is a curvature parameter to characterize the transitionto exponential phase; h0=v, as suggested by the authors whenfitting situations other than bacterial viable counts.

Determination of gas composition

The volume of gas evolved was also monitored by using amanometric device (LEO 2, Keller, Winterthur, Switzerland).Gas composition was determined using a gas chromatograph(Master GC Analyzer DGA52, Dani, Cologno Monzese, Ita-ly) equipped with two different capillary columns: (1)Hayesep Q column (3 m, 1 mm ID, 1/16″ OD, 80/100 mesh)that enables the separation of CO2 from the other gases, whichwere separated with: (2) Molesieve 5 Å column (3 m, 1 mmID, 1/16″ OD, 80/100 mesh) in H2, O2, N2, CH4. The carriergas was helium. The temperatures were 40 °C for the oven,250 °C for the injector and 200 °C for the detector. Detectionwas done using a thermal conductivity detector (TCDA). Thevolume of injected biogas was 250 μL. The calibration wasperformed with a standard gas composed of 24.79 % CO2,4.98 % H2, 0.976 % O2, 10 % N2 and 59.254 % CH4.

Analytical determinations

Sugar (maltose and glucose) consumption, as well as ethanoland other metabolites produced during Z. mobilis growth orleavening trials were determined through an HPLC system (L7000, Merck Hitachi, Tokyo, Japan) equipped with RI andUV (210 nm) serially connected detectors, using a (300–7.8 mm) Polispher OA KC (Merck, Darmstadt, Germany)column, maintained at 30 °C and eluted with 5 mM H2SO4

at 0.4 mL/min. Culture broth (2 mL) or dough (1–2 g) sam-ples, the last previously suspended in distilled water (about10 mL), were centrifuged (Eppendorf 5804, 10,600 g, 10 min)and the supernatants obtained analyzed after appropriate dilu-tion. Data related to molecules detected in the culture broth areexpressed in terms of grams per litre (g/L), while those foundin leavening trials are expressed as grams per 100 g flour.

Statistical analysis

Statistical analysis of the results was performed with SPSSsoftware, version 21.0 (SPSS, Chicago, IL). T-test and one-way analysis of variance (ANOVA) were applied to the data.Differences between means were assessed on the basis ofconfidence intervals using the Tukey-b test at a level ofsignificance of P<0.05. Data are represented as means±stan-dard deviation.

Results

Biomass production in flasks

The two Z. mobilis strains were initially grown in 300 mLflasks, employing a culture medium (IC) formulation avoidingany yeast-derived compound, in order to preserve the yeast-free nature of the final biomass. Fructose or glucose were usedat 20 or 50 g/L as carbon and energy source and cultures wereincubated for 16 and 24 h, comparatively.

No significant differences (P<0.05) were found betweenbiomass growth at 16 and 24 h (data not shown) in all thetested media and for both strains. The lowest biomass yieldswere obtained with fructose as energy source, in particularwhen set at 20 g/L (Fig. 1). Upon increasing fructose to 50 g/Lthe two strains performed differently: Z. mobilis 3580 in-creased biomass yield while Z. mobilis 424 did not.

In contrast, glucose gave the highest results in terms ofbiomass growth, and increasing glucose from 20 to 50 g/Lresulted in a statistically significant growth increase (about32 %) for both bacteria tested.

Trials performed in 1 L flasks (Fig. 2) with glucose (20 or50 g/L) confirm the results obtained with 300 mL flasks: again,no difference in terms of biomass growth was observed for

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cultivation times of 16 and 24 h (data not shown). Z. mobilis3580 and 424 grew similarly at the two glucose concentrationstested, with higher yields (22 % and 28 %, respectively) whenglucose was set at 50 g/L as compared to 20 g/L.

Comparing results obtained in 300 mL and 1 L flasks, wecan in general observe a positive and significant (t-test,P<0.05) up-scaling effect, with a growth increase between11 % (Z. mobilis 3580, 20 g/L glucose) and 16 % (Z. mobilis424, 20 g/L glucose).

Zymomonas growth in fermenter

The next step comprised biomass cultivation of Z. mobilis(3580 and 424) in IC G50 medium in a 14-L fermenter. Theincubation time was shortened, and culture samples analyzed at9 and 16 h incubation. When downstream analysis was per-formed at 9 h, HPLC analysis evidenced for both strains aquantity of residual glucose (about 7 g/L) in the cultures, whichdisappeared at 16 h (Table 1). When prolonging the incubationtime, biomass conversion yield (YX/S) decreased from about3.5–4.0 % at 9 h to 3.0–3.1 %, while ethanol conversion yield(YP/S) increased from 42–46 % to nearly 50 %.

The obtained data indicate a biomass production increaseof about 60 % up-scaling the process from 1 L flask tofermenter.

Leavening performance in model system: S. cerevisiae

Dough leavening trials were first performed employingS. cerevisiae (control sample). Leavening ability was testedboth in the absence of glucose (according to the proceduredescribed by Burrows and Harrison 1959) and with glucoseadded in the dough, as performed for Z. mobilis. Resultsobtained employing the water displacement method werecompared with those recorded with the manometric procedurecoupled with gas analysis, to assess whether the gas produc-tion measured with the first method was equivalent to the CO2

determined by the second procedure. The two methods pro-vided similar results (Fig. 3) and, as expected, S. cerevisiae inpresence of glucose produced a total amount of gas evolvedhigher than in its absence, about 1,000 vs 600 mL/g dcw in180 min, respectively.

Time course of CO2 production in each trial was fittedemploying the DMFit 3.0 Excel add-in shareware package,useful for fitting sigmoid curves, to obtain CO2 productionrate (mL/g dcw min), lag leavening phase duration (min) andtotal amount of CO2 evolved (mL/g dcw) as predicted by theBaranyi and Roberts model equation (1994). The resultsshowed that glucose did not affect either the lag leavening

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Fig. 1 Biomass growth (g dcw/L) of Zymomonas mobilis 424 (left) and3580 (right) in IC medium with 20 or 50 g/L glucose (G20-G50) orfructose (F20-F50) in 300 mL flasks. Data were obtained by averaging

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Fig. 2 Biomass growth (g dcw/L) of Z. mobilis 424 and 3580 in ICmedium with 20 (G20) or 50 (G50) g/L glucose in 1 L flasks. Data wereobtained by averaging Z. mobilis growth after 16 and 24 h incubation(means±standard deviation, 6<n<8). Different superscript letters indi-cate significant differences (Tukey-b test, P<0.05)

Table 1 Residual glucose (g/L) and biomass production of Zymomonasmobilis strains DSMZ 424 and 3580 (in terms of g dcw/L and Yx/s %)and ethanol production (g/L and Yp/s %) at 9 and 16 h of incubation.Trials were performed in 14-L fermenter. Data are reported as mean (n=3,CV in the range 5–8 %)

Strain Glucose(g/L)

Biomass(g dcw/L)

Yx/s(%)

Ethanol(g/L)

Yp/s(%)

Z. mobilis 424 9 h 7.7 1.49 4.0 17.3 45.8

16 h 0 1.42 3.1 22.6 49.7

Z. mobilis 3580 9 h 7.2 1.32 3.5 16.2 42.2

16 h 0 1.36 3.0 20.1 44.2

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phase duration (7.8 min) or the gas production rate (6.4 mL/gdcw min), but only the final gas production levels (comparedto line 1, Table 2). When employing glucose, the difference inCO2 levels detected by the two procedures at longer leaveningtimes (936 and 1,090 mL/g dcw) may be related to a com-pression effect created by the dough, which was not consid-ered when applying the water displacement method. Due to itseasier detection, we decided to use the volumetric procedureto monitor Z. mobilis leavening trials, aware of the possibilityof underestimating gas production levels only at long incuba-tion times.

HPLC analysis (Table 3) performed on dough samplescollected at the beginning and at the end (180min) of leaveningtrials evidenced that when glucose was added in the dough,S. cerevisiae used it up (almost 78 %), leaving maltose presentin flour; instead, without glucose addition, S. cerevisiae used allmaltose present into the flour. At 180 min, the dough samplesprepared with and without glucose evidenced similar ethanollevels, at 1.2–1.3 g/100 g flour, respectively.

Leavening performance in model system: Z. mobilis

Figure 4 represents gas evolution during 180 min doughleavening trials with the Z. mobilis biomass samples produced

in the fermenter (two strains, each of them collected after 9 or16 h incubation time). Both strains evidenced better perfor-mance when biomass production step lasted for 9 instead of16 h (Table 2), with a gas production rate statistically higherthan S. cerevisiae. Z. mobilis cells grown for 16 h have a gasproduction rate not statistically different from those obtainedby S. cerevisiae. Lag leavening phasewas absent for Z.mobiliswhile present for S. cerevisiae (11 min).

HPLC analysis (Table 3) performed on dough samplescollected at the end (180 min) of Zymomonas leavening trialsevidenced that, as expected, maltose remained unconsumed.Moreover, biomass grown for 9 h was able to consume allglucose present in the dough, while dough leavened with 16 h-grown biomass evidenced a residual 2 % glucose. Also ethanoldetected in dough leavenedwith 9 h-grown biomass was higher(4.3 and 1.5 g/100 g flour, respectively). HPLC analysis evi-denced the presence of lactic and acetic acid at 180 min and, asfor ethanol, in higher concentration in dough leavened byZ. mobilis grown for 9 h (0.26 and 0.06 g/100 g flour, respec-tively) than for 16 h (0.09 and 0.02 g/100 g flour respectively).

Discussion

The ability of Zymomonas mobilis to produce CO2 has rarelybeen investigated, perhaps because this bacterium has a lowmolar growth yield that may increase biomass productioncosts compared with the more consolidated S. cerevisiae.Another technological limit of Z. mobilis vs S. cerevisiae isits ability to produce CO2 only from glucose, fructose andsucrose; for this reason, a baked product leavened byZ. mobilis has to contain at least one of these sugars to allowthe dough to rise significantly. These matters may haveinhibited research into Zymomonas biomass application. Todate, the only data reported in the literature regarding the useof Z. mobilis in bread making focused on the effects of NaCland sucrose on the leavening ability of five Z. mobilis strains:the results showed that Z. mobilis strains are more sensitive

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Fig. 3 Time course of CO2 production (mL/g dcw) (means±standarddeviation, n=3) in dough leavening trials performed with Saccharomycescerevisiae with (gray circles) and without (black diamonds) glucose

addition, evaluated through a water displacement method (a) and amanometric procedure coupled with gas analysis (b)

Table 2 CO2 production rate (mL/g dcw min), lag leavening phaseduration (min) and total amount of CO2 evolved (mL/g dcw) (means±standard deviation, n=3), obtained fitting time courses of CO2 productionreported in Fig. 4 employing the DMFit 3.0 software (different super-script letters indicate significant differences, Tukey-b test, P<0.05)

Strain CO2 rate(mL/g dcw min)

Lag leavening(min)

CO2 evolved(mL/g dcw)

Saccharomycescerevisiae

6.4±0.3a 10.9±2.4 646±15a

Z. mobilis 424 9 h 8.9±0.6b 0 1,497±358c

16 h 7.3±0.5a 0 964±212b

Z. mobilis 3580 9 h 10.9±1.1c 0 1,368±253c

16 h 6.1±0.5a 0 831±91b

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than S. cerevisiae to the presence of NaCl in a dough (Oda andTonomura 1994).

In the present study, we evaluated Z. mobilis growth per-formance in a medium formulated avoiding any yeast-derivedcompound, and adding glucose or fructose as energy source.The best condition was then up-scaled and the collectedbiomass was employed to test Z. mobilis leavening ability ina dough model system. We chose not to consider sucrose inthe initial screening, in order to avoid levan formation, as itssynthesis would have further reduced biomass yield. Levan isan exopolysaccharide synthetized by Zymomonas on sucrose-based substrates (Lee and Huang 2000), and has applicationsin the food industry (De Oliveira et al. 2007) and in bakedgoods (Arendt et al. 2007) where it contributes to softeningand antistaling of wheat breads (Jacob et al. 2012). Of coursethis interesting aspect will be investigated in future trials.

Biomass yields obtained in our yeast-free medium contain-ing 20 g/L glucose (about 0.70 g dcw/L for both strains) aresimilar to those reported by other authors: Oda and Tonomura(1994) obtained 0.59 g dcw/ L with Z. mobilis 3580 (Z6) and0.50 g dcw/L with Z. mobilis 424 (Z8); Zikmanis et al. (1997)obtained about 8 g/mol with Z. mobilis 3580, which corre-sponds to about 0.88 g dcw/L. Nevertheless, all these reportedformulations contain yeast extract.

Results obtained in flasks demonstrate that biomass yield isabout 50% higher with glucose than with fructose, and confirmdata reported by Lee and Huang (2000) that the more glucose

present, the higher the biomass yield. However, biomass con-version yield (YX/S)—which represents how efficientlyZ. mobilis converts substrate into biomass—decreases. Anotherinteresting result is the positive up-scaling effect on biomassproduction, from around 0.85 g dcw/L in flasks to 1.40 g dcw/Lin a fermenter (for both Z. mobilis strains), where incubationtime was also shortened to 9 h, with positive implications bothfor biomass yield (YX/S) and dough leavening performance.

Taking into account the low molar growth yield ofZ. mobilis, future investigation will address increased biomassyield, perhaps applying a fed-batch fermentation processwhile maintaining dough leavening ability.

As regards leavening trials, our results demonstrate thatZ. mobilis, when glucose is present, is able to rise a dough,with similar or higher gas production rates (mL/g dcw min)and total amount of gas evolved (mL/g dcw) to S. cerevisiae.Glucose addition in leavening trials with S. cerevisiae pro-duced only higher amount of CO2 evolved but did not affecteither the gas production rate or the lag leavening phaseduration. These data were similar to those reported by Odaand Tonomura (1994), even if the dough composition wasdifferent: the latter authors reported leavening ability in termsof milliliters gas developed in 2 h per 10 g flour on a dry cellweight basis, and stated that Z. mobilis performed about 50 %better than S. cerevisiae. We obtained the following ratios:49 % for Z. mobilis 424 and 70 % for Z. mobilis 3580 (9 h-grown biomass, fermenter).

Table 3 Maltose, glucose, ethanol, lactic and acetic acids (g/100 g flour) present in dough samples at the beginning and at the end (0 and 180 min) ofleavening trials performed with S. cerevisiae and Z. mobilis (means±standard deviation, n=3)

Strain Time(min)

Maltose(g/100 g flour)

Glucose(g/100 g flour)

Ethanol(g/100 g flour)

Lactic acid(g/100 g flour)

Acetic acid(g/100 g flour)

S. cerevisiae −glucose 0 0.97±0.21 0.11±0.04 0 0 0

180 0 0 1.18±0.12 0 0

S. cerevisiae +glucose 0 0.74±0.18 10.49±0.20 0 0 0

180 0.70±0.05 2.35±0.15 1.32±0.20 0 0

Z. mobilis 9 h 0 0.81±0.12 10.29±0.21 0 0 0

180 0.85±0.10 0 4.30±1.41 0.26±0.07 0.06±0.01

Z. mobilis 16 h 0 1.08±0.19 10.07±0.19 0 0 0

180 0.82±0.13 0.2±0.05 1.52±0.50 0.09±0.06 0.02±0.01

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Z. mobilis 424 Z. mobilis 3580Fig. 4 Time course of CO2

production (mL/g dcw) (means±standard deviation, n=3) in doughleavening trials performed withZ. mobilis DSMZ 424 (left) and3580 (right). Biomass sampleswere collected after 9 h (blackdiamonds) or 16 h (gray circles)incubation in 14 L fermenter

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Acetic and lactic acids found in Z. mobilis dough samplesmay give a slight acidic flavor to the final product as com-pared to a yeast-based dough (Oda and Tonomura 1994);nevertheless these compounds are also present in sourdoughfermentations (Gobbetti et al. 2005). Of course, deeper studieshave to be planned to investigate the sensorial and technolog-ical properties of doughs leavened by Z. mobilis, to assess theacceptability of the final product.

On the basis of these considerations, the study of newmicrobial populations in the area of leavened goods may beconsidered of actual relevance. Z. mobilis may provide asolution to the increasing incidence of food hypersensitivitywhere S. cerevisiae is involved. It should be noted that, in thisresearch, yeast-derived compounds (i.e., yeast extract), com-monly present in culture media, were avoided to preserve theyeast-free characteristic of the final leavened product. In sum-mary, our results confirm the possibility of replacingS. cerevisiae, and the present research provides an innovativeapproach in the area of yeast-free leavened baked goods.

Acknowledgment The authors are thankful to Fabbrica dellaBioenergia (Bioenergy Factory) Technical University of Milan, wherethe leavening trials monitored with the manometric device as well as thegas analysis were performed.

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