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Applied and Environmental Microbiology, March 2000, p. 1084-1092, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization and Determination of Origin of Lactic Acid
Bacteria from a Sorghum-Based Fermented Weaning Food by Analysis of
Soluble Proteins and Amplified Fragment Length Polymorphism
Fingerprinting
Nokuthula F.
Kunene,1
Ifigenia
Geornaras,2
Alexander
von Holy,2 and
John W.
Hastings1,*
School of Molecular and Cellular Biosciences,
University of Natal, Scottsville, 3209,1 and
Department of Molecular and Cell Biology, University of the
Witwatersrand, Wits 2050,2 South Africa
Received 12 July 1999/Accepted 26 November 1999
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ABSTRACT |
The group that includes the lactic acid bacteria is one of the most
diverse groups of bacteria known, and these organisms have been
characterized extensively by using different techniques. In this study,
180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked
fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological
methods, as well as by analyzing the electrophoretic profiles of total
soluble proteins. A total of 58 of the 180 strains were
Lactobacillus plantarum strains, 47 were Leuconostoc
mesenteroides strains, 25 were Lactobacillus
sake-Lactobacillus curvatus strains, 17 were Pediococcus
pentosaceus strains, 13 were Pediococcus acidilactici strains, and 7 were Lactococcus lactis strains. L. plantarum and L. mesenteroides strains were the
dominant strains during the fermentation process and were recovered
from 87 and 73% of the households, respectively. The potential origins
of these groups of lactic acid bacteria were assessed by amplified
fragment length polymorphism fingerprint analysis.
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INTRODUCTION |
The group that includes the lactic
acid bacteria is a heterogeneous group of bacteria that are generally
regarded as safe for use in food and food products (15). The
use of these organisms in food products dates back to ancient times,
and they are used mainly because of their contributions to flavor,
aroma, and increased shelf life of fermented products
(28). Various members of this group are used
commercially as starter cultures in the manufacture of food products,
including dairy products (39), fermented vegetables (25), fermented doughs (45), alcoholic beverages
(33, 34), probiotics in animal feeds (8), and
meat products (44). Lactic acid bacteria have also been used
for lactic acid fermentation of sorghum- or maize-based cereals
used as infant-weaning foods (26, 27, 31, 38).
Various techniques have been used to characterize lactic acid bacteria;
these techniques include whole-cell protein analysis, cell wall
composition analysis, and morphological, physiological, and biochemical
analyses (41). While combined use of these methods is
invaluable for distinguishing lactic acid bacteria at the species level, the methods are not sufficiently discriminatory to differentiate organisms at the subspecies and strain levels (11, 14).
Determining the electrophoretic patterns of total soluble proteins and
computer-assisted analysis of the resulting protein profiles are
well-established procedures in bacterial taxonomy (13, 23,
35). This technique has been used for taxonomic studies of lactic
acid bacteria obtained from meat, dairy products, and other
environments, but it is hampered by the fact that it can yield
discriminatory information only at the species level, which requires
some degree of preidentification. This problem has been overcome by
creating a database of digitized and normalized protein patterns for
most known species of lactic acid bacteria (35).
DNA-based techniques, such as DNA base ratio analysis, DNA-DNA
hybridization analysis, rRNA homology analysis, plasmid profiling, ribotyping, restriction fragment length polymorphism analysis, and
randomly amplified polymorphic DNA analysis, have also been used to
characterize lactic acid bacteria (42). However, some of
these techniques have been hampered by a lack of reproducibility of
results between experiments (randomly amplified polymorphic DNA
analysis) and requirements for large amounts of time and labor (1). Recently, workers developed amplified
fragment length polymorphism (AFLP) analysis, which detects
genetic variation in microorganisms. The variation is assessed at a
large number of independent loci and can be found in any part of the
genome without prior knowledge of the sequence (47). The
results are highly reproducible, as the technique is based on
selective amplification with primers that recognize
double-stranded DNA adapters that are ligated to the ends of DNA
fragments in order to generate template DNA for amplification under
stringent conditions. This technique has been used in taxonomic
studies of Acinetobacter strains (18) and
Aeromonas strains (17), in epidemiological studies of Bacillus anthracis strains (21),
Salmonella strains (1), and
Xanthomonas translucens strains (7), and in
determining Vibrio vulnificus biotypes (2). It
has also been used to determine molecular evolutionary origins and
geographic correlations of Pseudomonas syringae strains
(9).
The objectives of this study were to characterize lactic acid bacteria
that occur naturally in sorghum powder and corresponding fermented and
cooked fermented porridge samples by using both physiological and
biochemical methods and analyzing total-soluble-protein patterns.
In this study we also investigated the identities and origins of lactic
acid bacteria that are dominant during the fermentation process by
using AFLP fingerprinting.
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MATERIALS AND METHODS |
Culture selection and maintenance.
The 180 lactic acid
bacterial strains used in this study were obtained during a previous
microbiological survey of 45 sorghum samples collected from an informal
settlement in the Gauteng Province of South Africa (24).
These lactic acid bacteria were isolated from plate count preparations
of sorghum powder samples (44 strains) and corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples.
Presumptive lactic acid bacterial colonies were randomly selected from
MRS agar (Oxoid, Hampshire, England) plates supplemented with 0.1%
L-cysteine monohydrochloride (Sigma Chemical Co., St.
Louis, Mo.) (12) and 40 µg of cycloheximide (Merck,
Darmstadt, Germany) per ml (30). The isolates were purified by growing them on MRS agar at 25°C for 48 h. Working cultures were maintained on MRS agar plates and were subcultured every 3 weeks.
Stock cultures were maintained in MRS broth (Oxoid) supplemented with
30% glycerol and were stored at 
70°C. Thirteen reference strains
were included (Table 1).
Analysis of total-soluble-protein profiles.
Protein extracts
were obtained from 180 strains by a previously described method
(13) and were separated on 12% polyacrylamide gels by using
a model SE 600 electrophoresis apparatus (Hoefer Scientific
Instruments, San Francisco, Calif.); the gels were stained with
Coomassie brilliant blue. Midrange molecular weight markers
(Boehringer, Mannheim, Germany) were used as standards. Images were
captured (UVP Image store 5000; Ultra Violet Products Ltd., Cambridge,
United Kingdom), and electrophoretic patterns were analyzed by using
the software package GelManager, version 1.5 (Biosystematica, Devon,
United Kingdom). Levels of similarity between patterns were calculated
by using the Pearson product moment correlation coefficient
(5). Strains were clustered by using the unweighted pair
group method with arithmetic averages (UPGMA) (5), and the
clusters were delineated at an arbitrary r level of 0.60.
Physiological and biochemical tests.
Of the 180 isolates
analyzed in this study, 72 were randomly selected from clusters
obtained from the electrophoretic profiles of total soluble proteins
(Fig. 1), and biochemical and
physiological tests were performed with these strains. Of the 72 isolates, 24 (41%) belonged to cluster I, 13 (46%) belonged to
cluster II, 3 (43%) belonged to cluster III, 5 (29%) belonged to
cluster IV, 5 (38%) belonged to cluster V, 10 (53%) belonged to
cluster VI, and 12 (48%) belonged to cluster VII. Each isolate was
grown on MRS agar for 48 h and Gram stained. Cell morphological
characteristics were examined microscopically by using a Kontron image
analyzer (Vidas, Darmstadt, Germany). Fermentation of carbohydrates was studied as described by Schillinger and Lücke (40).
Isolates were tested to determine whether they fermented acetate,
adonitol, arabinose, cellobiose, erythritol, fructose, galactose,
glycerol, inositol, lactose, maltose, mannitol, mannose, melibiose,
rhamnose, ribose, saccharose, sorbitol, sucrose, trehalose, and xylose. The terminal pH values of broth cultures were determined after 48 h of incubation in MRS broth at 25°C (15), and growth at pH 3.9 was determined in MRS broth (40). Tolerance to NaCl
was examined by testing for growth in MRS broth containing 7 and 10% NaCl; the ability to grow at different temperatures was determined by
growing the organisms in MRS broth after incubation at 4°C for 7 days
and at 15 and 45°C for 3 days; and slime production was determined by
using MRS agar containing sucrose instead of glucose (40).
The presence of meso-diaminopimelic acid (m-DAP) in cell walls was determined as described by Keddie and Cure
(20) by using paper chromatography (37). The
configurations of lactic acid enantiomers were determined enzymatically
by using D- and L-lactate dehydrogenase
(Boehringer) (14). Production of gas from glucose,
H2S contents, and hydrolysis of arginine were determined as
described by Schillinger and Lücke (40).
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FIG. 1.
Simplified dendrogram derived from whole-cell protein
profiles of 180 lactic acid bacterial isolates from sorghum powder and
corresponding fermented porridge and cooked fermented porridge samples.
Electrophoretic patterns were analyzed by using GelManager software,
version 1.5. Similarity patterns were calculated by using the Pearson
product moment similarity coefficient, and clusters were delineated at
an r value of 0.60.
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AFLP analysis.
Genomic DNAs were extracted from 58 Lactobacillus plantarum strains (Fig. 1, cluster I) and 46 Leuconostoc mesenteroides strains (Fig. 1, clusters II and
VI). Cultures were grown overnight in MRS broth (5 ml), and DNA was
isolated from cells by using the cetyltrimethylammonium bromide (CTAB)
method (4) and was resuspended in 35 µl (final volume) of
TE buffer. RNA was digested by incubating a DNA solution with 1 µl of
DNase-free RNase (Boehringer) at 37°C for 1 h. The purity and
concentration of the DNA were determined on 1% agarose gels by using
lambda DNA (Boehringer) as the concentration standard. Pure DNA was
stored at 20°C until it was used.
The AFLP reactions were performed with an AFLP Ligation and
Pre-selective Amplification kit according to the instructions
of the
manufacturer (Perkin-Elmer, Foster City, Calif.). DNA was
cleaved with
restriction enzymes
EcoRI and
Tru9I
(
MseI isoschizomer)
and was ligated to the adapters by using
T4 DNA ligase (Boehringer);
the preparations were incubated at 37°C
overnight in Eppendorf
tubes containing 1 µl of T4 DNA ligase buffer,
0.5 µl of nuclease-free
NaCl (0.5 M; BDH, Dorset, England), 0.5 µl
of nuclease-free bovine
serum albumin (10 mg/ml; New England Biolabs,
Beverly, Mass.),
and 1.6 µl of a master enzyme mixture. The master
enzyme mixture
was prepared immediately before use and contained 0.5 µl of
EcoRI
(10 U/µl), 0.1 µl of
Tru9I (10 U/µl), and 1 µl of T4 DNA ligase
(1 U/µl). Preselective
amplification was carried out by using
EcoRI and
MseI primers with single 3' A and 3' C selective bases,
respectively. A model 2400 Gene Amp PCR system (Perkin-Elmer)
was used
for PCR. The sequences of primers and adapters were not
supplied by the
manufacturer.
DNA fragments generated by PCR were separated on denaturing 4%
polyacrylamide gels with 8 M ultrapure urea (ICN Biochemicals
Inc.,
Aurora, Ohio). Glass plates were treated as described in
the Promega
manual (
36), except that the short plate was treated
with
Bind Silane (Promega, Madison, Wis.) instead of SigmaCote.
Aliquots (4 µl) of the PCR mixtures were mixed with equal volumes
of loading
buffer (95% formamide, 20 mM EDTA [pH 8], 0.05% bromophenol
blue,
0.05% xylene cyanol), denatured at 95°C for 5 min, and snap
cooled
on ice before loading. Amplified fragments were separated
at 55 W by
using a model S2 sequencing gel electrophoresis apparatus
(Gibco BRL,
Life Technologies, Gaithersburg, Md.) and 1× TBE as
the
electrophoresis buffer. The lower-compartment buffer was supplemented
with 0.5 M sodium acetate to prevent gel "frowning" (
1).
Molecular
weight marker X (Boehringer) was included in every fourth
track
as a standard in order to normalize patterns in different gels.
Gels were stained by using a modified silver staining method
(
3).
The modifications included fixing in 12% acetic acid,
staining
with 1 g of silver nitrate per liter, and developing in a
solution
containing 0.5 ml of sodium thiosulfate (10 mg/ml) per liter
and
2.5 ml of formaldehyde (37%) per
liter.
The gels were air dried and scanned with a ScanJet IIcx scanner
(Hewlett-Packard, Santa Clara, Calif.). The electrophoretic
patterns
were analyzed by using the software package GelManager,
version 1.5 (Biosystematica). Levels of similarity between AFLP
fingerprints were
calculated by using the Dice coefficient (
SD),
which equaled the ratio of twice the number of bands shared by
two
patterns that were compared to the total number of bands in
both
patterns (
5). Strains were clustered by using UPGMA
(
5),
and clusters were delineated at
SD levels of 0.70 (for
L. plantarum strains) and 0.74 (for
L. mesenteroides strains).
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RESULTS |
Total-soluble-protein profiles and biochemical and physiological
characteristics.
Results of our analysis are shown in Tables
2 and 3.
All of the isolates utilized fructose and did not utilize adonitol, erythritol, inositol, or rhamnose. On the basis of a computerized numerical analysis of protein electrophoretic patterns, we grouped the isolates into seven major clusters (Fig. 1 and Table 3).
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TABLE 2.
Biochemical and physiological characteristics of 72 lactic acid bacterial isolates selected from clusters based on an
analysis of electrophoretic profiles of total soluble proteins
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TABLE 3.
Distribution of lactic acid bacteria according to sample
type and household, based on an analysis of total-soluble-protein
profiles and biochemical and physiological tests
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The largest cluster (cluster I) (Fig.
1) consisted of 58
L. plantarum strains, which were characterized by their rod-shaped
cells, the presence of
m-DAP in their cell walls, production
of
DL-lactate, fermentation of ribose, and an inability to
utilize
arginine (Table
2). The reference strain of
L. plantarum (DSM
20174
T) also was a member of this
cluster. Of the 58 strains in this
cluster, 20 were isolated from
sorghum powder samples, 27 were
isolated from fermented porridge
samples, and 11 were isolated
from cooked fermented porridge samples
(Table
3). The strains
isolated from sorghum powder and fermented
porridge samples were
recovered most frequently and were obtained from
11 and 13 of
the 15 households, respectively (Table
3). The 11 strains
isolated
from cooked fermented porridge samples were recovered from
four
households.
The second largest cluster (cluster II) (Fig.
1) consisted of 28
L. mesenteroides strains, all of which produced the
D isomer
of lactic acid, utilized acetate, produced dextran
from sucrose,
and were not able to produce ammonia from arginine (Table
2).
A total of 23 of these strains were isolated from fermented
porridge
samples and were obtained from six households, whereas five
strains
were isolated from cooked fermented porridge samples and were
obtained from five households (Table
3). No strain in this cluster
was
isolated from sorghum powder
samples.
Cluster III was the smallest cluster and consisted of seven
Lactococcus lactis strains (Fig.
1), all of which had
coccoid
cells, produced the
L isomer of lactic acid, were
not able to
produce gas from glucose, and could not utilize lactose.
The terminal
pH produced in MRS broth was more than 4.0 (Table
2). The
reference
strains
L. lactis subsp.
lactis DSM
20481 and
L. lactis subsp.
cremoris DSM 20069 were members of this cluster. Of the seven
strains in this cluster,
three were isolated from sorghum powder
samples, two were isolated from
fermented porridge samples, and
two were isolated from cooked fermented
porridge samples (Table
3). The three strains isolated from sorghum
powder samples were
obtained from three households, whereas the four
strains isolated
from fermented and cooked fermented porridge samples
were obtained
from the same household (Table
3).
Cluster IV was designated a
Pediococcus pentosaceus cluster
and consisted of 17 strains (Fig.
1). The cells of these strains
were
coccoid and were arranged in pairs or tetrads. These strains
utilized
cellobiose, galactose, melibiose, and sorbitol and produced
the
DL isomer of lactic acid. The terminal pH produced in MRS
broth after 48 h of incubation was less than 4.0 (Table
2). The
cluster IV isolates grouped with the reference strain
P. pentosaceus DSM 20366. Of the 17
P. pentosaceus strains
in this cluster, 2
were isolated from sorghum powder and were obtained
from one household,
11 were isolated from fermented porridge samples
and were obtained
from three households, and 4 were isolated from
cooked fermented
porridge samples and were obtained from three
households (Table
3).
Cluster V was designated a
Pediococcus acidilactici cluster
and consisted of 13 strains, including reference strain
P. acidilactici DSM 20284 (Fig.
1). The cells of these isolates were
coccoid and
occurred in tetrads or pairs. The cluster V strains
differed from
the
P. pentosaceus strains in that they
hydrolyzed arginine and
fermented xylose and arabinose but did not
utilize sorbitol, melibiose,
galactose, or cellobiose (Table
2). Eight
of these strains were
isolated from sorghum powder samples and were
obtained from three
households, whereas four strains were isolated from
fermented
porridge samples and were obtained from two households. One
strain
was isolated from a cooked fermented porridge sample (Table
3).
Cluster VI was another
L. mesenteroides cluster and
consisted of 19 strains (Fig.
1). These strains produced the
D isomer
of lactic acid and dextran from sucrose but
no ammonia from arginine.
They differed from the members of the
first
Leuconostoc cluster
(cluster II) (Fig.
1) in
that they utilized arabinose, lactose,
ribose, and trehalose (Table
2).
The cluster VI isolates grouped
with reference strain
L. mesenteroides DSM 20343
T. Of the 19 strains in this
cluster, 13 were isolated from fermented
porridge samples and were
obtained from 11 households, whereas
3 were isolated from sorghum
powder and were obtained from three
households. The remaining three
strains were isolated from cooked
fermented porridge samples and were
obtained from the same household
(Table
3).
Cluster VII consisted of 25
Lactobacillus sake-Lactobacillus
curvatus strains (Fig.
1). Cells of these strains were typically
rod shaped and produced gas from glucose and the
DL isomer
of
lactic acid. These strains did not contain
m-DAP in their
cell
walls (Table
2). Eleven of these strains were isolated from
fermented
porridge samples and were obtained from four
households, while
seven strains were isolated from sorghum powder
samples and were
obtained from two households; the remaining seven
strains were
isolated from cooked fermented porridge samples and were
obtained
from three households (Table
3).
There were 13 isolates that did not belong to any major cluster (Fig.
1). Although two of these isolates were very similar,
the other
isolates constituted a heterogeneous group along with
seven of the
reference strains, which were randomly distributed
among them. No
isolate grouped with
Lactobacillus alimentarius DSM
20249
T,
Lactobacillus brevis DSM
20054
T,
Lactobacillus casei DSM
20011
T,
Lactobacillus coryniformis DSM
20001
T,
L. curvatus DSM 20019
T,
Lactobacillus confusus (
Weissella confusa) DSM
20196
T or
Leuconostoc paramesenteroides
(
Weissella paramesenteroides)
DSM 20288
T
(results not
shown).
AFLP analysis.
A representative silver-stained AFLP gel is
shown in Fig. 2. Our analysis of the DNA
electrophoretic patterns of the 58 L. plantarum strains
resulted in five major clusters (Fig. 3
and Table 4). Cluster A consisted of six
strains; three of these strains were isolated from sorghum powder
samples and were obtained from three households (households 1, 4, and
12), and the other three strains were isolated from fermented
porridge samples and were obtained from two households (households 1 and 7). Cluster B was the largest cluster, comprising 38 strains.
Thirteen of these strains were isolated from sorghum powder samples and
were obtained from nine households (households 2, 3, 5 through 9, 13, and 15), while nineteen strains were isolated from fermented porridge samples and were obtained from eleven households (households 1 through
3, 5, through 9, 11, 12, and 15) and six strains were isolated from
cooked fermented porridge samples and were obtained from six households
(households 2 through 5, 8, and 10). Cluster C contained
four strains, three of which were obtained from the same
household (household 9) but were isolated from three different types of samples; the remaining strain was isolated from a cooked fermented porridge sample obtained from household 8. Cluster D was composed of six strains, one of which was isolated from a sorghum
powder sample and was obtained from household 10; three strains were
isolated from fermented porridge samples and were obtained from three
households (households 8, 10, and 11), and two strains were isolated
from cooked fermented porridge samples obtained from households 3 and
5. Cluster E contained four strains, two of which were isolated from
sorghum powder samples and were obtained from two households
(households 10 and 11); one strain was isolated from a fermented
porridge sample obtained from household 7, and one strain was isolated
from a cooked fermented porridge sample obtained from household 2.
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FIG. 2.
AFLP patterns of L. plantarum and L. mesenteroides strains isolated from fermented porridge. Lanes 1, 7, and 8, L. mesenteroides strains; lanes 2 through 4, 6, and 9, L. plantarum strains; lane 5, molecular weight marker
X.
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FIG. 3.
Dendrogram of 58 L. plantarum strains derived
from an UPGMA of AFLP patterns. Levels of similarity between AFLP
fingerprints were calculated by using SD, and
clusters were delineated at an arbitrary SD
level of 0.70. The sources of isolates are indicated as follows: SP,
sorghum powder; FP, fermented porridge; and CP, cooked fermented
porridge.
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TABLE 4.
Distribution of 58 L. plantarum strains
according to sample type and household, based on an AFLP analysis
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The 46
L. mesenteroides strains analyzed by using AFLP
profiles grouped into five minor clusters and two major clusters (Fig.
4 and Table
5) that were similar to the two
Leuconostoc clusters
obtained after total-soluble-protein
patterns were analyzed (Fig.
1). Cluster F was the largest cluster and
contained 15 strains.
Only one strain was isolated from sorghum powder
(household 11),
while 14 strains were isolated from fermented porridge
samples
and were obtained from seven households (households 4, 7, 9, 11,
12, 14, and 15). The second cluster (cluster G) comprised nine
strains; five of these strains were isolated from fermented porridge
samples and were obtained from three households (households 7,
8, and
12), and four were isolated from cooked fermented porridge
samples and
were obtained from two households (households 8 and
10). The third
cluster (cluster H) was composed of four strains,
three of which were
isolated from fermented porridge samples and
were obtained from two
households (households 12 and 13); the
other strain was isolated from a
sorghum powder sample obtained
from household 10. The fourth cluster
(cluster I) was composed
of three strains, one from each type of
sample. These strains
were obtained from different households
(households 1, 10, and
11). The fifth cluster (cluster J) comprised
five strains; four
of these strains were isolated from fermented
porridge samples
and were obtained from four households (households 3 and 5 through
7), and the remaining strain was isolated from a cooked
fermented
porridge sample obtained from household 5. The sixth cluster
(cluster
K) was composed of eight strains; six of these strains were
isolated
from fermented porridge samples and were obtained from six
households
(households 4, 5, 7, and 9 through 11), and the remaining
two
strains were isolated from cooked fermented porridge samples
obtained
from households 5 and 7. The last cluster (cluster L)
consisted
of two strains, both of which originated from fermented
porridge
samples obtained from households 2 and 3.
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FIG. 4.
Dendrogram of 46 L. mesenteroides
strains derived from an UPGMA of AFLP patterns. Levels of similarity
between AFLP fingerprints were calculated by using
SD, and clusters were delineated at an arbitrary
SD level of 0.74. The sources of isolates are
indicated as follows: SP, sorghum powder; FP, fermented porridge; and
CP, cooked fermented porridge.
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TABLE 5.
Distribution of 46 L. mesenteroides strains
according to sample type and household, based on an AFLP analysis
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DISCUSSION |
Characterization of lactic acid bacteria.
The bacterial
populations in sorghum powder samples and corresponding fermented
and cooked fermented porridge samples consisted of
Lactobacillus, Lactococcus,
Leuconostoc, and Pediococcus strains. Previous studies showed that naturally fermented cereal-based African
foods are dominated by L. plantarum, Lactobacillus
fermentum, Lactobacillus reuteri, L. mesenteroides, P. pentosaceus, and L. lactis strains (32, 38). The majority of the isolates
characterized in this study belonged to the genera
Lactobacillus (83 of the 180 isolates examined) and
Leuconostoc (47 of the 180 isolates examined). The largest
cluster obtained in the protein profile analysis (cluster I) (Fig. 1)
consisted of L. plantarum strains, while L. mesenteroides strains grouped into two clusters (clusters II and
VI) (Fig. 1), which may represent different subspecies of L. mesenteroides. These two species dominated the fermentation processes in this study. They have been isolated previously from a
variety of food products and environmental sources and reportedly are
two of the most dominant species during fermentation of sorghum-based infant-weaning foods (29, 31).
The third largest cluster identified in the protein profile analysis
(cluster VII) (Fig.
1) was an
L. curvatus-L. sake cluster.
Members of this group belonged to the group of atypical streptobacteria
which are phenotypically diverse and are usually distinguished
from
each other on the basis of fermentation of melibiose and
arginine
hydrolysis.
L. curvatus is negative for both of these
characteristics (
23). Phenotypic characteristics of strains
belonging to this cluster suggested that a mixture of the two
species
was present. In previous studies workers have described
the phenotypic
similarities of
L. sake and
L. curvatus
(
19)
and the inability of biochemical tests and
total-soluble-protein
profiles to consistently differentiate between
strains of these
two species (
13,
16).
Clusters IV and V (Fig.
1) consisted of strains belonging to the genus
Pediococcus. Pediococci have often been found at low
frequencies together with leuconostocs and lactobacilli on plant
material and in various foods. They are also widely used as starter
cultures in the fermentation of sausages and have been used to
control
food pathogens in vegetables (
43). Pediococci have
previously
been isolated from fermented cereal gruels (
22).
The smallest cluster (cluster III) (Fig.
1), which consisted of only
seven strains, was an
L. lactis cluster. The natural
habitat
of lactococci is milk, but
L. lactis subsp.
lactis has
been isolated previously from plants, vegetables,
and cereals
(
10,
39).
Household recovery frequencies.
The greater frequencies of
recovery of L. plantarum, L. sake-L. curvatus,
L. mesenteroides, and P. pentosaceus strains from fermented porridge samples than from sorghum powder samples
suggested that these strains participated in the fermentation
processes. By contrast, the frequencies of recovery of P. acidilactici and L. lactis strains from fermented
porridge samples were lower than the frequencies of recovery from
sorghum powder samples. This was probably due to the inability of the
organisms to compete with other lactic acid bacterial species and to
the inability of L. lactis strains to grow at pH values of
less than 4.
The fact that
L. plantarum strains were present in sorghum
powder samples obtained from 73% of the households suggested that
these strains were present in sorghum powder samples prior to
household
handling and processing and thus that sorghum powder
is a natural
habitat of
L. plantarum. The low frequencies of recovery
of
L. lactis,
L. sake-L. curvatus,
L. mesenteroides,
P. acidilactici,
and
P. pentosaceus strains from sorghum powder in households
(Table
3) indicated that these organisms either were present at low
levels in the powder and thus were not detected or were introduced
through handling and processing at the household level. Members
of these groups have typically been associated with foods,
including
vacuum-packaged meat and meat products and vacuum-packaged
smoked
and salted fish (
6,
15). Members of the
L. sake-L. curvatus group have also been reported to be
responsible for spoilage of
chill-stored vacuum-packaged meat products
(
46).
The isolation of lactic acid bacterial strains from cooked fermented
porridge samples suggested that recontamination occurred,
probably due to use of the same utensils, such as spoons, during
the
preparation and storage of the cooked fermented porridge samples.
Recontamination of cooked samples, however, may also have resulted
from
strains present in households before the study was conducted,
particularly since the storage containers used for sorghum powder
samples were seldom washed before new sorghum powder was added.
It is
very unlikely that contaminating strains were strains that
survived the
cooking process since the cooking process was deemed
sufficient to kill
vegetative cells (
24).
None of the households which we studied yielded a single-species
population of lactic acid bacteria associated with sorghum
samples
after fermentation. It should be mentioned, however, that
the processes
from which the lactic acid bacteria were obtained
were not studied over
a sufficiently long period for natural selection
to result in a stable
microbiological population of lactic acid
bacteria. Usually,
single-species populations are established
only after several cycles of
the fermentation process (
29),
while in our case the samples
were collected after the first cycle,
which lasted 36 to 72
h.
AFLP analysis.
As a result of the highly selective and
stringent PCR protocol used in the AFLP procedure, the discriminatory
power of this technique is such that it is able to detect single point
mutations in strains. However, slight variations in band width and
mobility, as well as background intensities, may result in similarity
levels that are less than 100% for strains that appear to be identical after AFLP patterns are analyzed visually. Thus, the similarity levels
obtained in the present study are not absolute; nevertheless, they are
useful for determining relationships among L. plantarum and
L. mesenteroides strains, as shown in Fig. 3 and 4,
respectively. The presence of a majority (66%) of the L. plantarum strains obtained from sorghum powder in one cluster
(cluster B) (Fig. 4) suggested that this group was the dominant group
in sorghum powder. High household recovery frequencies for strains
belonging to this cluster (Table 4) suggested that the source of these
strains was sorghum powder samples. By contrast, the remaining L. plantarum strains (34%) grouped in four small clusters, and their
household recovery frequencies were low. This indicated that these
organisms were not part of the normal flora of sorghum powder but may
have been introduced at the household level.
One-third of the
L. mesenteroides strains which we studied
grouped in one cluster (cluster F) (Fig.
4), as determined by the
AFLP
analysis. All but one of the strains in this cluster were
isolated from
fermented porridge samples. These strains were recovered
from 47% of
the households, which suggests that they had a common
origin, perhaps
sorghum powder; however, because they were present
in low numbers in
the powder, they were not detected. The presence
of the rest of the
L. mesenteroides strains (two-thirds of the
strains) in six
small clusters and the fact these strains were
recovered at low
household frequencies could suggest that they
were not part of the
normal flora of sorghum powder but rather
were introduced at the
household
level.
Conclusion.
In this study, members of several genera of lactic
acid bacteria were recovered from sorghum powder and corresponding
fermented and cooked fermented porridge. The dominant groups found in
the fermented porridge samples were L. plantarum and
L. mesenteroides. High household recovery frequencies and
the results of an AFLP analysis of the majority of the L. plantarum strains suggested that these organisms originated from
the sorghum powder. The AFLP analysis results also indicated that the
majority of the L. mesenteroides strains did not have a
common origin and thus were likely to have been introduced at the
household level. The remainder of the L. mesenteroides
strains, however, appeared to have had a common source, possibly
sorghum powder, but they may not have been detected due to the low
numbers present.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Molecular and Cellular Biosciences, University of Natal, Private Bag
X01, Scottsville 3209, South Africa. Phone: 27 331 260 5434. Fax: 27 331 260 5435. E-mail: hastings{at}gene.unp.ac.za.
| |
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Abstract
Introduction
