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Appl Environ Microbiol, July 1998, p. 2616-2623, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Modeling of Growth of Lactobacillus
sanfranciscensis and Candida milleri in Response to
Process Parameters of Sourdough Fermentation
Michael G.
Gänzle,
Michaela
Ehmann, and
Walter
P.
Hammes*
Institut für Lebensmitteltechnologie,
Universität Hohenheim, D-70599 Stuttgart, Germany
Received 5 February 1998/Accepted 29 April 1998
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ABSTRACT |
We investigated the effect of the ecological factors pH,
temperature, ionic strength, and lactate, acetate, and ethanol levels on Candida milleri and two strains of Lactobacillus
sanfranciscensis, organisms representative of the microflora of
sourdough. A mathematical model describing the single and combined
effects of these factors on the growth of these organisms was
established in accordance with the following criteria: quality of fit,
biological significance of the parameters, and applicability of the in
vitro data to in situ processes. The growth rates of L. sanfranciscensis LTH1729 and LTH2581 were virtually identical
under all conditions tested. These organisms tolerated >160 mmol of
undissociated acetic acid per liter. Growth occurred in the pH range of
3.9 to 6.7 and was completely inhibited by 4% NaCl. C. milleri had a lower optimum temperature for growth (27°C) than
the lactobacilli. The growth of the yeast was not affected by pH in the
range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete
inhibition of growth occurred at 150 mmol of undissociated acetic acid
per liter, but acetate at concentrations of up to 250 mmol/liter
exerted virtually no effect. The model provides insight into factors
contributing to the stability of the sourdough microflora and can
facilitate the design of novel sourdough processes.
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INTRODUCTION |
Sourdough fermentation is a process
to obtain bread from wheat or rye flours by the combined metabolic
activity of lactic acid bacteria and yeasts. Due to the superior
sensory quality and the prolonged shelf life of the resulting baked
goods, sourdough processes have retained their importance in modern
baking technology (11, 20, 34). These processes are employed
in the production of more than 20% of the bread produced in Central
Europe, France, and Italy and are essential for a wide variety of
specialty products.
Sourdough fermentations are characterized by a stable association of
yeasts and lactobacilli. In sourdoughs with a tradition of continuous
propagation, Lactobacillus sanfranciscensis (37) ("Lactobacillus brevis subsp. lindneri"
[30]), Candida milleri, and
Saccharomyces exiguus (anamorphic form: Torulopsis
holmii) are the predominant microorganisms (1, 26, 35).
The process conditions of traditional fermentations ensure a high level
of metabolic activity for these organisms and permit the production of
breads with sensory qualities superior to those prepared from pure
cultures of lactobacilli or yeasts (33). The raw materials of bread are essentially flavorless. Therefore, the formation of flavor
compounds relies on endogenous cereal enzymes, microbial metabolism,
and the baking process. The significant contribution of lactic acid
bacteria as well as yeasts to the formation of aroma volatiles or
precursors available for thermal transformation to aroma compounds is
well established (6, 13, 28).
The adaptation of artisanal processes to new products and technologies
requires profound knowledge of the factors determining microbial
metabolism and the stability of the microflora. In order to achieve a
balanced metabolic activity of lactobacilli and yeasts in sourdough,
interactions between these organisms have to be taken into account.
Recent research has focused mainly on the metabolism of carbohydrates
and amino acids (3, 10, 31). Little information is available
on the response of the sourdough microflora to the ecological factors
temperature, pH, ionic strength, and accumulation of metabolic end
products. A useful tool to assess the effects of environmental factors
on the growth of microorganisms is the development of mathematical
models that meet the criteria proposed by Rosso et al. (24):
simplicity of the model, biological significance of the parameters,
applicability, and quality of fit. Models based on in vitro experiments
permit the evaluation of the single effect of an environmental factor
independent of the choice of raw materials, whereas during in situ
fermentations, usually only the sum of several factors can be
evaluated. Wijtzes et al. (38), Rosso et al.
(24), and Cuppers et al. (5) have validated
models describing the combined effects of temperature and pH, as well
as temperature and NaCl concentration, on microbial growth. It was the
aim of our study to expand the scope of the proposed models to more
than two factors, taking into account the combined effects of pH,
temperature, salt concentration, and accumulation of metabolic end
products, and to use the model to identify the most important factors
contributing to the stable association of lactobacilli and yeasts.
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MATERIALS AND METHODS |
Organisms and media.
The strains used in this study are
L. sanfranciscensis LTH2581 and LTH1729 and C. milleri LTH H198. These organisms were isolated by Böcker et
al. (2) from a commercial mother sponge. L. sanfranciscensis LTH2581 and LTH1729 were differentiated by their
carbohydrate fermentation pattern, plasmid content, and colony
morphology on mMRS agar (2). The strains have been
maintained in this sourdough starter for at least 20 years, accounting
for more than 90% of the microflora of that product. The lactobacilli
were grown in modified MRS medium (mMRS) containing the following per
liter: 10 g of tryptone, 5 g of meat extract, 5 g of
yeast extract, 2.5 g of maltose, 1.25 g of fructose, 8 g
of KH2PO4, 3 g of diammonium citrate,
3 g of NH4Cl, 0.5 g of cysteine HCl, 1 g of
Tween 80, 6 g of DL-lactic acid (90%), 4.9 g of
sodium acetate · 3H2O, 0.2 mg of
MgSO4 · 7H2O, 0.05 g of
MnSO4 · H2O, 0.5 µg each of cobalamin, folic acid, niacin, pantothenic acid, pyridoxal, and thiamin. The pH
was 5.44 after autoclaving. Sugars were autoclaved separately, and
vitamins were sterilized by filtration. For cultivation of yeast,
maltose and fructose were replaced by 2.5 g of glucose. To
determine the effect of pH, NaCl, ethanol, lactate, and acetate, the
medium composition was changed as follows.
(i) pH.
The pH of mMRS media was adjusted to values ranging
from 3.5 to 7 with 4 N NaOH or HCl.
(ii) NaCl.
mMRS media containing 3.6, 4.8, 7.2, 9.6, 10.8, or 16.2% NaCl (wt/vol) were diluted with mMRS medium (0% NaCl) to
obtain the desired NaCl concentrations in the ranges of 0 to 4% (for
lactobacilli) and 0 to 8% (for yeasts).
(iii) Ethanol.
mMRS media were adjusted to a concentration
of 6, 8, or 18% ethanol (vol/vol) and mixed with mMRS medium to obtain
ethanol concentrations ranging from 0 to 8%. Media containing ethanol were prepared immediately prior to inoculation to minimize evaporation losses.
(iv) Lactate and acetate.
mMRS media containing 360, 240, or
0 mmol of acetate or lactate per liter were mixed with mMRS without
acetate or lactate to obtain the desired concentration of acetate and
lactate in the range of 0 to 240 mmol/liter. The pH of these media was
4.45.
(v) Combination of the effects of pH, acetate, NaCl, and
lactate.
The pH of mMRS media (0 mmol of acetate and lactate per
liter) containing 12 g of K2HPO4 · 3H2O was adjusted to 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, and 3.5 with 0, 22, 49, 78, 91, 140, and 230 mmol of lactic acid/liter,
respectively. For each pH level, media were prepared containing 0, 4, and 8% NaCl (wt/vol). Salt containing mMRS was diluted with mMRS (0%
NaCl) to give NaCl concentrations of 0, 1.8, 2.7, and 4% for
lactobacilli and 0, 3.6, 5.3, and 8% for yeasts. The acetate
concentration was adjusted to 0, 30, 60, 90, 120, or 150 mmol of
acetate/liter. Blanks were prepared for each combination of parameters
(144 for both lactobacilli and yeasts), and the pH was measured.
Determination of the growth rates.
Standardized inocula were
prepared by growing overnight cultures in mMRS broth to exponential
growth phase (optical density at 595 nm [OD595], 0.2 to
0.4). The growth media were inoculated to an OD595 of ca.
0.001 and incubated at 30°C in microtiter plates. The total volume
was 150 µl, and the media were overlaid with 100 µl of paraffin to
achieve anoxic growth conditions and to avoid evaporation losses of
water, ethanol, and acetic acid. The growth of the organisms was
monitored by measuring the OD of the growth media with a Bioscreen C
microbiological analyzer (Labsystems, Frankfurt, Germany) for automated
measuring or a microtiter plate reader, model 450 (Bio-Rad, Munich,
Germany). The effects of temperature and pH were studied by using
15-ml reagent tubes incubated in a water bath at the desired
temperature ± 0.5°C. For OD measurements, the tubes were
vortexed and 150-µl samples were transferred to microtiter plates.
The ODs of the cultures were correlated to cell counts by using the
same shaking regimen as that applied in the measurements. The OD
measurements on microtiter plates were correlated to the cell counts of
C. milleri LTH H198 and L. sanfranciscensis
LTH2581 and LTH1729 with correlation coefficients, r2, of 0.972, 0.977, and 0.950, respectively.
The threshold sensitivity of the OD measurements was about
106 cells/ml for both yeast and lactobacilli. Growth below
this threshold level was not recorded or accounted for as lag-phase
growth. Thus, OD measurements are not the most suitable method to
assess the growth of food pathogens or spoilage organisms but are
adequate for modeling the growth of food-fermenting organisms, where
the emphasis is exclusively on growth conditions that allow growth to a
high population density. From each growth curve, the maximum growth
rate, µmax, the lag phase, 
, and the asymptote,
A, were obtained by fitting the OD readings to the logistic
growth curve (41). SigmaPlot 1.02 software was used for all
curve fit routines.
Model development.
The model equations used to describe the
effects of temperature, pH, NaCl, ethanol, lactate, and acetate on the
growth of C. milleri and L. sanfranciscensis are
shown in Table 1. All functions were of
the form µ(x) = µopt 
(x).
The parameter µopt equals µmax at the
growth conditions with the factor x at its optimum value.
The function (x) describes the response of the growth
rate to changes in the factor x, with values ranging from 0 (no growth) to 1 (optimum growth). The models are valid only in the
range of xmin 
x xmax. This scheme allows the description of the
combined effects of the factors x1,
x2, ..., xn by models of the type µ(x1,
x2, ..., xn) = µopt · (x1) · (x2) · ... · (xn).
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TABLE 1.
Model development to describe the effect of temperature,
pH, ionic strength, ethanol, acetate, and lactic and acetic acids
on microbial growth
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RESULTS |
Effect of temperature on growth.
The dough temperature is
affected by the temperatures of the raw materials, water and flour, and
the incubation temperature. In practice, these are not strictly
controlled but are subject to changes on a daily and seasonal basis.
The effect of temperature on the growth rates of L. sanfranciscensis LTH2581 and LTH1729 and C. milleri LTH
H198 is shown in Fig. 1, and the
parameter estimates for models 1a and 1b fitted to the experimental
data are compiled in Table 2. From
parameters a, b, and c of model 1a,
the optimum temperature for growth (Topt),
µopt, and a' were calculated as
(Tmax 
b/c),
µ(Topt), and a/µopt,
respectively (see Appendix), where Tmax is as
defined below. L. sanfranciscensis LTH1729 and LTH2581
have Topt values of 33 and 32°C, respectively, whereas C. milleri grows fastest at 27°C. With model 1a,
the quality of fit was not improved if Tmax was
incorporated as a regression parameter; Tmax was
therefore defined as the lowest temperature above
Topt at which no visible growth occurred after 7 days of incubation. Tmax was 41°C for both
strains of L. sanfranciscensis and 36°C for C. milleri. These values do not differ appreciably from those
obtained with model 1b. Model 1a gave the better curve fit results for
all strains except LTH1729. Compared with model 1b, model 1a gave a
poor estimate around the minimum growth-permitting temperature
(Tmin), whereas the prediction of growth rates
around Topt is more accurate. The growth rates
of L. sanfranciscensis LTH1729 and LTH2581 are almost
identical over the entire temperature range, with a higher
µopt and a slightly lower Topt
determined for strain LTH2581. In comparison to the lactobacilli,
C. milleri has a lower Topt and
Tmax. Sourdough fermentations are commonly performed at temperatures between 20 and 28°C. Remarkably, L. sanfranciscensis and C. milleri exhibit the same
response to changes at temperatures of <26°C.
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FIG. 1.
Effect of temperature on the µmax values
for L. sanfranciscensis LTH2581 (A) and LTH1729 (B) and
C. milleri LTH H198 (C). The solid and dashed lines
represent growth rates predicted with models 1a and 1b, respectively.
Error bars indicate the standard deviations from the means of two
independent experiments. The shaded area represents the range commonly
encountered during sourdough fermentations.
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Effect of pH on growth.
The initial pH of the sourdough
fermentation ranges from 4.5 to 5.5, depending on the size of the
inoculum. Sourdoughs are acidified to a pH of 3.5 to 3.7. The effect of
pH on the growth rates of L. sanfranciscensis LTH2581 and
LTH1729 and C. milleri LTH H198 is shown in Fig.
2A; the parameter estimates for model 2 are given in Table 3. Growth of C. milleri is not affected by the pH in the range tested, i.e., 3.5 to 7. The model describing growth therefore simplifies to µ = µopt. Remarkably, the same values of pHmin,
pHmax, and pHopt (the minimum, maximum, and
optimum pH values for growth, respectively) and thus the same (pH)
were obtained for the two strains of L. sanfranciscensis.
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FIG. 2.
Effect of pH on µmax values for L. sanfranciscensis LTH2581 ( ) and LTH1729 ( ) and C. milleri LTH H198 ( ) (A) and effect of NaCl addition expressed
as ionic strength on the growth of L. sanfranciscensis
LTH2581 and C. milleri LTH H198 (B). The lines represent the
predicted growth rates. Error bars indicate the standard deviations
from the means of three independent experiments. The shaded areas
represent the ranges commonly encountered during sourdough
fermentations.
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Effect of ionic strength on growth.
The ionic strength of
sourdough is affected by the ash content of the flour, the dough yield
(grams of dough/100 g of flour), and the formula. The effect of ionic
strength on growth was studied by adjusting ionic strength by the
addition of NaCl. The results are shown in Fig. 2B; the models used and
the parameter estimates are shown in Table 3. Model 3 was used to
describe the effect of ionic strength on the growth of L. sanfranciscensis. It is based on model 2 with
Imin = 0, where Imin is
the minimum ionic strength for growth. As a stimulatory effect of the
ionic strength on the growth of C. milleri was not observed
(i.e., Iopt [optimum ionic strength for
growth] and Imin are outside the range of
investigation), the use of model 4 was considered to be more
appropriate. The yeast grows in up to 8% NaCl (I = 3.2), whereas the growth of lactobacilli is inhibited by 4% NaCl
(I = 1.9). An increasing ionic strength will therefore
inhibit the growth of lactobacilli to a much greater degree than it
inhibits the growth of yeasts.
Effects of ethanol, lactate, and acetate.
Lactate, acetate,
ethanol, and CO2 are the major metabolic end products of
cofermentations with L. sanfranciscensis and C. milleri. Their accumulation in the dough may lead to an inhibition of growth of the microflora. The production of lactate and acetate is
influenced by appropriate technological measures, e.g., buffering capacity of the dough, aeration, and the addition of citrate or fructose (12). The effects of ethanol, acetate, and lactate on microbial growth are shown in Fig. 3;
the regression parameters are presented in Table 3. Differences in the
µopt obtained with model 4 for the effects of lactate and
acetate reflect experimental error. The values of maximum lactate
concentration for lactic acid bacteria and yeasts as well as those of
maximum acetate concentration for lactobacilli are not supported by
experimental data. The maximum ethanol concentration is approximately
the same for both organisms. For the yeasts a linear decrease of the
growth rate is evident with increasing ethanol concentration, whereas
the growth of L. sanfranciscensis LTH2581 is affected by
ethanol only at concentrations of >5%. A more pronounced inhibitory
effect of acetate on the growth of yeasts than on the growth of
lactobacilli is evident. The growth of C. milleri was
completely inhibited by 166 mmol of acetate per liter, corresponding to
110 mmol of undissociated acid per liter, while L. sanfranciscensis LTH2581 tolerated more than 240 mmol of acetate
per liter. The experimental design does not allow differentiation
between the inhibitory effects of acetate and undissociated acid. As
opposed to acetate, lactate inhibited the growth of lactobacilli and
yeasts to the same degree. As pointed out above for acetate, the
inhibitory effects of lactate and lactic acid could not be
distinguished.
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FIG. 3.
Effect of ethanol (A), acetate (B), and lactate (C) on
µmax of L. sanfranciscensis LTH2581 () and
C. milleri LTH H198 (). The lines represent the growth
rates predicted with model 4. Error bars indicate the standard
deviations from the means of three independent experiments. The shaded
areas represent the ranges commonly encountered during sourdough
fermentations.
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Combined effects of pH, NaCl, lactic acid, acetic acid, and acetate
on growth.
It was the intent of the study to verify that a model
of the type µ(x1,
x2, ... , xn) = µopt · (x1) · (x2) · ... · (xn) gives an accurate prediction of the
combined effects of the factors of importance in sourdough on the
growth of the fermentation flora. Therefore, the combined effects of
pH, NaCl, lactate, and acetate were evaluated. The models describing
the single effects of these factors on growth were used to predict by
extrapolation the combined effects of two factors on appropriate data
subsets. The predictions of the combined effects of pH and ionic
strength on L. sanfranciscensis LTH2581 and acetic acid and
ionic strength on C. milleri LTH H198 are compared to the
experimental data in Fig. 4A and 4B,
respectively. The value of r2 was 0.829 for
LTH2581 and 0.880 for LTH H198. The experimental design allows
distinction between the inhibitory effects of acetic acid and acetate
as well as between those of lactic acid and lactate, because several
concentrations of undissociated acid-sodium salt were tested at
different pH values. Thus, the data were fitted to an expanded model
taking into account the combined effect of these factors. The models
used and parameters obtained are compiled in Table
4; plots of the observed
growth rates against the predicted growth rates are shown in Fig.
5. The values determined for
pHopt, pHmax, and Iopt
(LTH2581) and Imax (maximum ionic strength for growth; LTH2581 and LTH H198) correspond to those determined for the
single effects of the factors on growth (Table 3). The
pHmin determined for L. sanfranciscensis
LTH2581 was slightly higher (4.16 versus 3.94) than that determined for
the single effect of pH on growth. This deviation can be explained by
the experimental design, as pH values between 3.9 and 4.1 were not
evaluated. The growth of C. milleri is completely inhibited
by 150 mmol of acetic acid per liter, while the inhibitory
concentration of acetate was about 10 times higher. It is of special
interest that growth inhibition of L. sanfranciscensis was
correlated to acetate concentration, with little or no effect of the
undissociated acid.
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FIG. 4.
Three-dimensional plot of observed growth rates ( )
versus surfaces of predicted growth rates. Observed growth rates are
taken from data subsets for the evaluation of combined effects;
calculated growth rates were extrapolated with the models describing
single effects. (A) L. sanfranciscensis LTH2581; (B)
C. milleri LTH H198.
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FIG. 5.
Plot of observed growth rates versus growth rates
predicted with the model for the combined effects. (A) L. sanfranciscensis LTH2581; (B) C. milleri LTH H198.
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DISCUSSION |
The experimental design used in this study has several limitations
with respect to conditions during sourdough fermentations: (i) in
practice, fermentations are not performed under static conditions for
environmental factors; (ii) the differences in nutrient supply between
mMRS medium and wheat or rye doughs may alter the response of the
microorganisms to these factors; and (iii) the microflora of the dough
may be affected by antagonistic or synergistic interactions such as
competition for nutrients or by release of nutrients by extracellular
enzymes during growth in mixed culture. Therefore, the model
predictions require careful validation by comparison with data from
sourdough fermentations.
The parameter estimates for the effect of temperature on the growth of
L. sanfranciscensis and C. milleri determined in
our work are in good agreement with literature data. Böcker et
al. (1) and Spicher (29) reported that strains of
L. sanfranciscensis have a Topt
in the range between 30 and 37°C. C. milleri and S. exiguus do not grow at temperatures above 35°C (17,
39). The observation that L. sanfranciscensis LTH2581
and LTH1729 and C. milleri LTH H198 exhibit the same
response to temperatures below 26°C provides an explanation for the
stable association of these organisms in a sourdough for more than 20 years (2). Our data are furthermore in agreement with the
"baker's rule" that low temperatures during sourdough
fermentations (20 to 26°C) are better for yeast growth than higher
temperatures (30).
Sourdoughs usually are prepared without the addition of salt;
nevertheless, several processes that make use of the incorporation of 2 to 5% NaCl in the sourdough have been developed (30). Our data are consistent with the observations of Röcken et al.
(22) and Gianotti et al. (9) that increasing
dough yields leads to faster acidification of doughs. The addition of
salt may alter the composition of the microflora, because C. milleri is much less sensitive to salt than the strains of
L. sanfranciscensis. Yeast growth in dough may even be
stimulated by the addition of NaCl. In agreement with this assumption,
it was observed that the addition of NaCl to wheat doughs inhibited the
growth of lactic acid bacteria while it exerted a stimulating effect on
yeast (9). We expressed the salt concentration as ionic
strength rather than water activity. This was considered justified on
the basis of the results of Dossmann (7), who observed a
more pronounced growth inhibition of Lactobacillus sakei if
NaCl replaced glycerol for adjusting the water activity to values
ranging from 0.92 to 0.99.
The response of L. sanfranciscensis to changes in pH
described in this study is in accordance with the original species
description of Kline and Sugihara (15), who reported a
pHopt of about 5 and a pHmin between 3.6 and
4.0. We observed that the growth of lactobacilli is favored over yeast
growth at pH values of >4.5, corresponding to the first stage of dough
fermentation. L. sanfranciscensis does not grow below pH
3.8, indicating that the pH is a decisive growth-limiting factor for
this organism in sourdough. This conclusion is confirmed by the
observation that factors increasing the buffering capacity of the dough
do not alter the final pH of the fermentation but result in a higher
content of lactic acid (23, 25). The parameter estimates
describing the pH effect on the growth of L. sanfranciscensis LTH1729 and LTH2581 are strikingly similar, suggesting that the stable coexistence of these organisms in the same
substrate is explained in part by their identical growth rates. The
finding that the pH has little effect on the growth of C. milleri is in accordance with our earlier observations of a
cereal-based growth medium (8).
During sourdough fermentation, acidification is achieved by the
production of lactate and acetate to levels of 100 to 200 mmol/liter
and 40 to 60 mmol/liter, respectively. The lactate content is
determined by the buffering capacity of the sourdough, and the acetate
content depends on the availability of substrates used as electron
acceptors by the lactobacilli (12). Heterofermentative lactobacilli including L. sanfranciscensis tolerated up to
250 mmol of acetate and lactate per liter in rye and wheat doughs (18, 23). Because lactate and acetate production by
lactobacilli has various effects on microbial growth, i.e., increase of
ionic strength, decrease of pH, and additional effects of undissociated organic acids, it is necessary to distinguish between these effects. Remarkably, the growth of L. sanfranciscensis was not
inhibited by undissociated acetic acid.
The growth of C. milleri was strongly inhibited by acetic
acid, and was inhibited to a much lesser extent by lactic acid. The
inhibitory concentrations of these organic acids are in good agreement
with our earlier studies of a cereal-based medium, rye bran extract
(8). Increased acetate contents of sourdough resulting from
technological measures such as those proposed by Röcken et al.
(22) and Martinez Anaya et al. (18) are therefore
likely to result in the inhibition of yeast growth. The acetate
tolerances of other sourdough yeasts such as T. holmii were
reported to be in the same range as that of C. milleri LTH
H198 (36).
Certain strains of heterofermentative lactobacilli are known to grow at
ethanol concentrations as high as 18% (4). Because the
ethanol concentration in doughs does not exceed 1%, it is unlikely to
exert an inhibitory effect on lactobacilli but may contribute to the
inhibition of yeast growth. Unlike the metabolic end products lactate,
acetate, and ethanol, CO2 does not accumulate in the dough
during fermentation but rather is dissolved in equilibrium to a 100%
CO2 athmosphere. As the liquid media used in our work were
overlaid with paraffin, the conditions matched those of sourdough fermentation, and a possible effect of CO2 on the growth of
C. milleri and L. sanfranciscensis was not
further considered. L. sanfranciscensis is known to grow
optimally at a CO2 atmosphere of 25 to >90%
(15).
The model used in this study to describe the combined effects of
several factors on growth is based on the hypothesis that the cardinal
parameters of growth for a given factor are independent of the values
of any other factor and of the medium composition. Therefore, all
parameters with the exception of µopt, and thus (x), should be independent of the medium composition.
This hypothesis is supported by the satisfactory quality of fit
obtained by extrapolation of single effects to the combined effect of
different factors. Wijtzes et al. (38) demonstrated that the
cardinal parameters of temperature and pH for Lactobacillus
curvatus are independent of each other. A possible influence of
salt concentration on the Topt values for five
fungi was suggested by Cuppers et al. (5). These authors
nevertheless preferred a model that neglected this effect of NaCl on
Topt. However, there are limitations to the use
of functions to describe the combined effects of environmental factors on microbial growth. For example, the addition of cationic compatible solutes, e.g., betaine and choline, decreased the inhibitory effect of NaCl on the growth of Lactobacillus plantarum
(14); thus, (NaCl) and not µopt has been
modified by the medium composition.
The importance of antagonistic and synergistic interactions between
yeasts and lactobacilli based on the metabolism of carbohydrates and
amino acids was emphasized by Gobbetti and Corsetti (10). However, sourdough yeasts do not affect the cell yield of L. sanfranciscensis in sourdough (8, 27, 32). This is
consistent with the conclusion that under practical conditions the pH
is the limiting factor for growth of lactobacilli. The maltose, amino
acid, and peptide concentrations are not depleted during sourdough
fermentations in wheat or rye doughs (16, 19, 31). Maltose
is the preferred carbon source for L. sanfranciscensis but
is not utilized by either C. milleri or S. exiguus (12). The cell yield of C. milleri and S. exiguus is greatly reduced in the presence of
lactobacilli both in wheat and in rye doughs (8, 27). The
accumulation of metabolic end products of the heterolactic fermentation
inhibits the growth of these yeasts in sourdough. The glucose
concentration in rye flours and whole-wheat flours remains high enough
to support yeast growth throughout the fermentation (19,
23). Fermentations that employ white wheat flours as the raw
materials are characterized by low concentrations of glucose, and small
amounts of lactic acid are produced because of the low buffering
capacity. In these doughs, depletion of glucose and fructose may occur
and limit the growth of yeasts (19, 27).
Generally, the predictions made with the model are in good agreement
with the literature data on dough available, indicating that the most
important factors contributing to the microbial stability of sourdough
fermentations have been taken into account. A more detailed
verification of the model in situ is in progress. The model allows the
assessment of factors contributing to the stable association of
lactobacilli and yeasts in traditional sourdough fermentations, and it
can therefore provide important information for the design of novel
sourdough processes.
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APPENDIX |
With µ(x) = axbe
cx, differentiation yields
= axbecx(b/x c). With ecx 
0 for any
x, the function has a maximum at x = b/c, corresponding to (Tmax Topt). Substitution of µ(b/c) = µopt and a'µopt = a into model 1a gives µ(x) = µopt
a'xbecx.
| |
ACKNOWLEDGMENT |
This work was supported by a grant of the European Union (FAIR
project no. CT96-1126).
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Universität Hohenheim, Institut für
Lebensmitteltechnologie, Fachbereich Allgemeine Lebensmitteltechnologie, Garbenstraße 28, D-70599 Stuttgart,
Germany. Phone: 49 711 459 2305. Fax: 49 711 459 4199. E-mail:
hammeswp{at}uni-hohenheim.de.
| |
REFERENCES |
| 1.
|
Böcker, G.,
P. Stolz, and W. P. Hammes.
1995.
Neue Erkenntnisse zum Ökosystem Sauerteig und zur Physiologie der sauerteigtypischen Stämme Lactobacillus sanfrancisco und Lactobacillus pontis.
Getreide Mehl Brot
49:370-374.
|
| 2.
|
Böcker, G.,
R. F. Vogel, and W. P. Hammes.
1990.
Lactobacillus sanfrancisco als stabiles Element in einem Reinzucht-Sauerteig-Präparat.
Getreide Mehl Brot
44:269-274.
|
| 3.
|
Collar, C.,
A. R. Mascaros, and C. Benedito de Barber.
1992.
Amino acid metabolism by yeasts and lactic acid bacteria during bread dough fermentation.
J. Food Sci.
57:1423-1427.
|
| 4.
|
Couto, J. A.,
N. Rozes, and T. Hogg.
1996.
Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance.
J. Appl. Bacteriol.
81:126-132.
|
| 5.
|
Cuppers, H. G.,
S. Oomes, and S. Brul.
1997.
A model for the combined effects of temperature and salt concentration on growth rate of food spoilage molds.
Appl. Environ. Microbiol.
63:3764-3769[Abstract].
|
| 6.
|
Damiani, P.,
M. Gobbetti,
L. Cossignani,
A. Corsetti,
M. S. Simonetti, and J. Rossi.
1996.
The sourdough microflora. Characterization of hetero- and homofermentative lactic acid bacteria, yeasts and their interactions on the basis of the volatile compounds produced.
Lebensm.-Wiss. Technol.
29:63-70.
|
| 7.
|
Dossmann, M. U.
1995.
Einfluß von ökologischen Parametern auf die physiologischen Leistungen von Laktobazillen in Rohwurst. Dissertation.
Universität Hohenheim, Stuttgart, Germany.
|
| 8.
|
Gänzle, M. G.,
S. Häusle, and W. P. Hammes.
1997.
Wechselwirkungen zwischen Laktobazillen und Hefen des Sauerteiges.
Getreide Mehl Brot
51:209-215.
|
| 9.
|
Gianotti, A.,
L. Vannini,
M. Gobbetti,
A. Corsetti,
F. Gardini, and M. E. Guerzoni.
1997.
Modelling of the activity of selected starters during sourdough fermentation.
Food Microbiol.
14:327-337.
|
| 10.
|
Gobbetti, M., and A. Corsetti.
1997.
Lactobacillus sanfrancisco, a key sourdough lactic acid bacterium: a review.
Food Microbiol.
14:175-187.
|
| 11.
|
Hammes, W. P., and M. G. Gänzle.
1997.
Sourdough breads and related products, p. 199-216.
In
B. J. B. Wood (ed.), Microbiology of fermented foods. Chapman and Hall, London, United Kingdom.
|
| 12.
|
Hammes, W. P.,
P. Stolz, and M. Gänzle.
1996.
Metabolism of lactobacilli in traditional sourdoughs.
Adv. Food Sci.
18:176-184.
|
| 13.
|
Hansen, B., and A. Hansen.
1994.
Volatile compounds in wheat sourdoughs produced by lactic acid bacteria and sourdough yeasts.
Z. Lebensm.-Unters.-Forsch.
198:202-209.
|
| 14.
|
Kets, E. P. W.,
M. Nierop Groot,
E. A. Galinski, and J. A. M. de Bont.
1997.
Choline and acetylcholine: novel cationic osmolytes in Lactobacillus plantarum.
Appl. Microbiol. Biotechnol.
48:94-98.
|
| 15.
|
Kline, L., and T. F. Sugihara.
1971.
Microorganisms of the San Francisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity.
Appl. Microbiol.
21:459-465[Medline].
|
| 16.
|
Kratochvil, J., and J. Holas.
1984.
Untersuchungen über proteolytische Vorgänge im Roggensauerteig.
Getreide Mehl Brot
38:330-332.
|
| 17.
|
Kreger van Rij, N. J. W.
1984.
The yeasts, a taxonomic study, 3rd ed.
Elsevier Science Publisher, Amsterdam, The Netherlands.
|
| 18.
|
Martinez Anaya, M. A.,
M. L. Llin,
M. P. Macias, and C. Collar.
1994.
Regulation of acetic acid production by homo- and heterofermentative lactobacilli in whole wheat sourdoughs.
Z. Lebensm.-Unters.-Forsch.
199:186-190.
|
| 19.
|
Martinez Anaya, M. A., and O. Rouzaud.
1997.
Influence of wheat flour and Lactobacillus strains on the dynamics of by-products from amylolytic activities.
Food Sci. Technol. Int.
3:123-136.
|
| 20.
|
Ottogalli, G.,
A. Galli, and R. Foschino.
1996.
Italian bakery products obtained with sourdough: characterization of the typical microflora.
Adv. Food Sci.
18:131-144.
|
| 21.
|
Passos, F. V.,
H. P. Fleming,
D. F. Ollis,
H. P. Hassan, and R. M. Felder.
1993.
Modeling the specific growth rate of Lactobacillus plantarum in cucumber extract.
Appl. Microbiol. Biotechnol.
40:143-150.
|
| 22.
|
Röcken, W.,
M. Rick, and M. Reinkemeier.
1992.
Controlled production of acetic acid in wheat sour doughs.
Z. Lebensm.-Unters.-Forsch.
195:259-263.
|
| 23.
|
Röcken, W., and P. A. Voysey.
1992.
Sour-dough fermentation in bread making.
J. Appl. Bacteriol.
79:38S-48S.
|
| 24.
|
Rosso, L.,
J. R. Lobry,
S. Bajard, and J. P. Flandrois.
1995.
Convenient model to describe the combined effects of temperature and pH on microbial growth.
Appl. Environ. Microbiol.
61:610-616[Abstract].
|
| 25.
|
Salovaara, H., and T. Valjakka.
1987.
The effect of fermentation temperature, flour type, and starter on the properties of sour wheat bread.
Int. J. Food Sci. Technol.
22:591-597.
|
| 26.
|
Salovaara, H., and J. Savolainen.
1984.
Yeast type isolated from Finnish sour rye dough starters.
Acta Aliment. Pol.
10:241-246.
|
| 27.
|
Saunders, R. M.,
H. Ng, and L. Kline.
1972.
The sugars of flour and their involvement in the San Francisco sour dough French bread process.
Cereal Chem.
49:86-91.
|
| 28.
|
Schieberle, P.
1996.
Intense aroma compounds useful tools to monitor the influence of processing and storage on bread aroma.
Adv. Food Sci.
18:237-244.
|
| 29.
|
Spicher, G.
1982.
Einige neue Aspekte der Biologie der Sauerteiggärung.
Getreide Mehl Brot
36:12-16.
|
| 30.
|
Spicher, G., and H. Stephan.
1993.
Handbuch Sauerteig: Biologie, Biochemie, Technologie, 4th ed.
B. Behr's Verlag, Hamburg, Germany.
|
| 31.
|
Spicher, G., and W. Nierle.
1988.
Proteolytic activity of sourdough bacteria.
Appl. Microbiol. Biotechnol.
28:487-492.
|
| 32.
|
Spicher, G.,
E. Rabe,
R. Sommer, and H. Stephan.
1982.
Die Mikroflora des Sauerteiges. XV. Mitteilung: über das Verhalten heterofermentativer Sauerteigbakterien und Hefen bei gemeinsamer Kultur.
Z. Lebensm.-Unters.-Forsch.
174:222-227.
|
| 33.
|
Spicher, G.,
R. Schröder, and H. Stephan.
1980.
Die Mikroflora des Sauerteiges. X. Mitteilung: die backtechnische Wirkung der in Reinzuchtsauern" auftretenden Milchsäurebakterien (Genus Lactobacillus Beijerinck).
Z. Lebensm.-Unters.-Forsch.
171:119-124.
|
| 34.
|
Stolz, P., and G. Böcker.
1996.
Technology, properties and applications of sourdough products.
Adv. Food Sci.
18:234-236.
|
| 35.
|
Sugihara, T. F.,
L. Kline, and L. B. McCready.
1970.
Nature of the San Francisco sour dough French bread process.
Baker's Dig.
44:50-52.
|
| 36.
|
Suhiko, M.-L., and V. Mäkinen.
1984.
Tolerance of acetate, propionate and sorbate by Saccharomyces cerevisiae and Torulopsis holmii.
Food Microbiol.
1:105-110.
|
| 37.
|
Trüper, H. G., and L. De' Clari.
1997.
Taxonomic note: necessary correction of specific epithets formed as substantives (nouns) "in apposition."
Int. J. Syst. Bacteriol.
47:908-909[Abstract/Free Full Text].
|
| 38.
|
Wijtzes, T.,
J. C. de Wit,
J. H. J. Huis in 't Veld,
K. van 't Riet, and M. H. Zwietering.
1995.
Modelling bacterial growth of Lactobacillus curvatus as a function of acidity and temperature.
Appl. Environ. Microbiol.
61:2533-2539[Abstract].
|
| 39.
|
Yarrow, D.
1978.
Candida milleri sp. nov.
Int. J. Syst. Bacteriol.
28:608-610[Abstract/Free Full Text].
|
| 40.
|
Zwietering, M. H.,
J. C. de Wit,
H. G. A. M. Cuppers, and K. van 't Riet.
1994.
Modeling of bacterial growth with shifts in temperature.
Appl. Environ. Microbiol.
60:204-213[Abstract/Free Full Text].
|
| 41.
|
Zwietering, M. H.,
I. Jongenburger,
F. M. Rombouts, and K. van 't Riet.
1990.
Modeling of the bacterial growth curve.
Appl. Environ. Microbiol.
56:1875-1881[Abstract/Free Full Text].
|
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Abstract
Introduction
