Comparision of the Native Microflora of Portuguese “BROA”
and that of Similar Sourdough Breads
J. Miguel Rocha and F. Xavier Malcata
Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200 Porto, Portugal
INTRODUCTION
EXPERIMENTAL RESULTS AND CONCLUSIONS
Ground cereals mixed with water produce a dough which, after some time and owing to
microorganisms present therein, may eventually become a sourdough which is characterized by a
typical acid taste and an increased volume due to formation of gas. Both characteristics result
from fermentation by complex endogenous microflora, and this type of fermentation was
probably one of the first microbial processes employed by Man for food preservations purposes.
According to some authors, the microflora in sourdough is a set of compatible strains of yeasts
and lactococci (or cocci), synergistic interaction of which is likely to be an important factor
towards production of unique flavours and textures. One bread that is still produced following
such an ancient manufacturing procedure, at the farm level, in the Minho Region is (widely)
known as BROA.
8%
1% 2% 2%
3%
3%
1%
2%
2%
1%
1%
2%
1%
1%
1%
3%
1%
1%
1%
1%
5%
7%
1%
1%
38%
2%
8%
1%
2%
8%
2%
1%
2%
2%
2%
1%
2%
3%
1%
7%
1%
1%
1%
1%
Manufacture of BROA plays important roles, from both economic and social standpoints, but a
long way is yet to be tracked before such food specialty can be officially certified. To this goal,
sequential steps of microbial quantification and identification have been taken, namely using
samples from said maize bread manufactured in Cabeceiras de Basto.
Almost 420 isolates were tested, via appropriate BioMérieux API™ kits; most of them had been
already recorded in traditional sourdough breads from other countries, although their profiles
were somewhat unique in our case.
MATERIAL AND METHODS
Flour of maize and rye, as well as sourdough samples (10 g) from Cabeceiras de Basto, were suspended
in 90 ml of sterile 2% (w/v) sodium citrate, homogeneized in sterile beakers for 12 min and kept without
agitation for an extra 8 min. Serial decimal dilutions were then made on 0.1% (w/v) sterile peptone
water. The samples were plated in duplicate.
Total viable counts were performed after inoculation and incubation on such media. Purified strains were
first subject to several tests (Gram and spore staining, motility, catalase, oxidase,
homo/heterofermentative and aerobic/anaerobic fermentation tests). Strains were further characterized
via appropriate API galleries (see Figure 1).
Table 1 - Experimental Conditions
Microorganisms
VRBDA
Enterobacteriaceae
YEDCA
2 vials/l X209
BCM
100 ml/l X073
2 vials/l X074
MRS
Yeasts and Molds
Bacillus cereus
1%
1%
Aerococcus viridians
Bacillus badius
B. circulans
B. macerans
B. pumilus
C. guilliermondii
Chryseomonas luteola
L. curvatus
L. plantarum
Leuconoctoc spp.
Micrococcus kristinae
Pichia ohmeri
P. stutzeri
Staphylococcus aureus
S. schleiferi
Streptococcus agalactiae
Xanthomonas maltophilia
Aeromonas hydrophila/caviae
B. brevis
B. laterosporus
B. mycoides
B. stearothermophilus
C. pelliculosa
Gardnerella vaginalis
L. delbrueckii ssp. delbrueckii
Lactococcus lactis ssp. cremoris
L. mesenteroides ssp. cremoris
Micrococcus spp.
Pseudomonas cepacia
Saccharomyces cerevisiae
S. chromogenes
S. sciuri
S. constellatus
Agrobacterium radiobacter
B. cereus
B. licheniformis
B. polymyxa
Candida colliculosa
C. sphaerica
Lactobacillus brevis
L. lactis spp. lactis
L. lactis lactis
Listeria spp.
Pediococcus spp.
Pseudomonas spp.
Sphingobacterium paucimobilis
S. lentus
S. xylosus
S. equinus
1% 4%
7%
According to these authors [1 12], the microflora in sourdough is
a set of compatible strains of
yeasts and LAB, synergistic
interaction of which is likely to be
an important factor towards
production of unique flavours and
textures.
4%
2%
1%
1%
4%
9%
1%
1%
1%
1%
1%
1%
1%
2%
1%
3%
41%
8%
5%
1%
3%
Lactobacillus
(Pediococcus and Leuconostoc)
Staphylococcus aureus
(Micrococcus)
RCM
Clostridium
M17 agar
Streptococcus
(Lactococcus)
Streptococcus
(Enterococcus)
Leuconostoc
KF Streptococcus agar
2%
5%
1%
1%
4%
1%
2%
1%
1%
4%
1%
2%
1%
2%
Aerococcus viridians
Bacillus badius
B. firmus
B. megaterium
B polymyxa
Candida dattila
Enterobacter agglomerans
Lactobacillus brevis
L. lactis ssp. Lactis
Leuconoctoc spp.
Micrococcus spp.
Pseudomonas cepacia
Staphylococcus capitis
S. lentus
S. xylosus
S. equinus
Aeromonas hydrophila/caviae
B. cereus
B. licheniformis
B mycoides
B. pumilus
C. pelliculosa
Enterococcus casseliflavus
L. curvatus
L. plantarum
L. mesenteroides ssp. cremoris
Pediococcus damnosus
Saccharomyces cerevisiae
S. cohnii
S. sciuri
Streptococcus adjacens
Agrobacterium radiobacter
B. circulans
B. macerans
B pantothenticus
B. subtilis
Chryseomonas luteola
Gemella morbillorum
L. delbrueckii ssp. Delbrueckii
Lactococcus lactis ssp. lactis
Listeria spp.
P. pentosaceus
Sphingobacterium paucimobilis
S. hominis
S. warneri
S. constellatus
Figure 3 - Species and corresponding frequency of appearance in MAIZE.
1%
2%
3%
1%
2%
3%
1%
1% 4%
2%
1%
4%
1%
1%
T = 30 ºC; 48 h
Spread plate under anaerobic conditions
T = 30 ºC; 24 - 48 h
T = 37 ºC; 48 h
Spread plate under aerobic conditions
T = 30 ºC; 48 h
Spread plate under anaerobic conditions
T = 30 ºC; 48 h
Spread plate under anaerobic conditions
T = 37 ºC; 24 h
Spread plate under anaerobic conditions
T = 30 ºC; 24 h
Spread plate under aerobic conditions
2%
5%
1%
4%
1%
2%
1%
7%
1%
2%
32%
4%
13%
1%
3%
1%
1%
1%
1%
YEASTS
1%
1%
1%
T = 30 and 55 ºC; 24 h
Spread plate under aerobic conditions
T = 30 and 55 ºC; 24 h
Spread plate under aerobic conditions
T = 30 ºC; 24 h
T = 37 ºC; 24 h
Pour-plate method
T = 30 ºC; 24 - 48 h
Spread plate under aerobic conditions
T = 37 ºC; 24 –48 h
Spread plate under aerobic conditions
1%
1%
5%
A major consideration concerning
sourdough fermentation is its
effect upon texture of the final
bread, as a consequence of the
carbohydrate metabolism of LAB,
which is affected by flour
composition or interactions with
yeasts [7].
1%
2%
5%
4%
MSA
MSE
2%
Conditions
Total viable counts
(general and thermophilic viable counts)
Spore counts
(mesophilic and thermophilic spore counts)
PCA
1%
2%
Figure 2 - Species and corresponding frequency of appearance in SOURDOUGH.
Total viable counts in samples of sourdough, maize and rye flour were obtained after inoculation on 10
different solid selective media poured onto Petri dishes. The identification of 419 isolates was via
appropriate BioMérieux API™ galleries, after several preliminary biochemical and morphological tests;
such identification has shown that the total counts on each medium do not correspond exactly to the
expected group of microorganisms for that culture medium. In order to check for the existence of a wide
diversity of microorganisms, a large number of culture media and incubation conditions were selected
(see Table 1).
Culture media and
antibiotics
Sourdoughs of similar breads
originating in other countries
contain complex microflora,
where yeasts and lactic acid
bacteria predominate, and which,
owing to synergistic interactions,
produce distinct acidic tastes and
unique flavors [1 - 12]; these
microorganisms and LAB are
favored by the environmental
conditions prevailing during
storage, i.e. low temperatures and
high relative humidities.
1%
8%
3%
1%
4%
1%
1%
1%
ID 32C
LAB and yeasts contributed by
sourdough account for several
volatile compounds produced
during sourdough fermentation
[5]: homofermentative LAB are
responsible for development of a
final bread with good grain and
elastic
crumb,
whereas
heterofermentative LAB improve
taste and contribute to the
leavening process.
4%
2%
COCCUS Gram +
Catalase +
API STAPH
COCCOBACILLUS Gram +
Catalase -
API 20 STREP
Catalase +
Catalase -
API STAPH
API 20 STREP
BACILLUS
GRAM +
Catalase +
B . c e re us
B . s te aro the rm o philus
C . m e m branae fac ie ns
De baryo m yc e s m aram a
E. durans
Lac to bac illus ac ido philus
L. lac tis s s p. lac tis
Lac to c o c c us lac tis s s p. lac tis
M . s pp.
P e dio c o c c us pe nto s ac e us
S ac c haro m yc e s c e re v is iae
S . c o hnii
S . s c iuri
Xantho m o nas m alto philia
Figure 4 - Species and corresponding frequency of appearance in RYE.
It should be emphasized that
sourdough leavening is mostly
determined by CO2 produced as a
result of the fermentative activity
of yeasts present: although the
gas developed in the sourdough
contributes to open up texture,
the lactic and acetic acids
produced by LAB play a
determinant role in taste [2, 8].
GRAM -
REFERENCES
Long Bacilli
Obligate Anaerobes
Obligate Aerobes
Oxidase Catalase -
Aero-Anaerobes
API 20 NE
API 50 CHB
B ac illuc c irc ulans
B . pum ilus
C andida dattila
C hrys e o m o nas lute o la
E. c as s e liflav us
E. fae c ium
L. c urv atus
L. plantarum
M ic ro c o c c us k ris tinae
P as te ure lla ae ro ge ne s
P . ps e udo m alle i
S taphylo c o c c us c hro m o ge ne s
S . le ntus
S . xylo s us
Although this work has attempted to characterize the microecology prevailing in the main precursors of Broa
(maize and rye flours, and sourdough), the characteristics of this matrix create a unique environment in which
specialized, wild strains of microorganisms can grow.
Aero - Anaerobies
Longs rods sporuled
A e ro m o nas hydro phila/c av iae
B . lic he nifo rm is
B . s ubtilis
C . pe llic ulo s a
Ente ro c o c c us av ium
E. fae c alis
L. c o pro philus
L. parac as e i s s p. parac as e i
Lis te ria s pp.
M . v arians /ro s e us
P s e udo m o nas c e pac ia
S phingo bac te rium pauc im o bilis
S . e pide rm idis
S . warne ri
API 50 CHL
API 20 A
Oxidase +
Oxidase -
API 20 E
API 20 E
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ACKNOWLEDGMENTS
API 20 NE
Figure 1 - Procedure used for identification of the isolates strains.
The authors are grateful to several members of the Regional Directorate of Agriculture of Entre Douro e Minho (DRAEDM) and several local farmers for cooperation within the experimental program described.
Financial support for author J. M. R. was provided by a Ph.D. fellowship within the program for the Creation of National Infrastructures in Applied Science and Investigation (CIÊNCIA), administered by the
National Board for Scientific and Technological Investigation (JNICT, Portugal). Partial financial support was received from PAMAF – IED (Ministry of Agriculture, Portugal) through a research grant entitled
“Pão de milho: caracterização do processo tradicional de produção e melhoramento tecnológico”.