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Applied and Environmental Microbiology, August 1999, p. 3418-3426, Vol. 65, No. 8
Dairy Products Research Centre,
Received 11 January 1999/Accepted 25 May 1999
Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar
cheeses, obtained from six manufacturers. From countable plates of
Lactobacillus-selective agar, 20 single isolated colonies
were randomly picked per cheese. All 331 viable isolates were
biochemically characterized as mesophilic (i.e., group II)
Lactobacillus spp. Phenotypically, the isolates comprised
96.4% L. paracasei, 2.1% L. plantarum, 0.3%
L. curvatus, 0.3% L. brevis, and 0.9%
unidentified species. Randomly amplified polymorphic DNA (RAPD)
analysis was used to rapidly identify the dominant strain groups in
nine cheeses from three of the factories, and through clustering by the
unweighted pair group method with arithmetic averages, an average of
seven strains were found per cheese. In general, strains isolated from
cheese produced at the same factory clustered together. The majority of
isolates associated with premium-quality cheese grouped together and
apart from clusters of strains from defective-quality cheese. No
correlation was found between the isomer of lactate produced and RAPD
profiles, although isolates which did not ferment ribose clustered
together. The phenotypic and genotypic methods employed were validated
with a selection of 31 type and reference strains of mesophilic
Lactobacillus spp. commonly found in Cheddar cheese. RAPD
analysis was found to be a useful and rapid method for identifying
isolates to the species level. The low homology exhibited between RAPD
banding profiles for cheese isolates and collection strains
demonstrated the heterogeneity of the L. paracasei complex.
Non-starter lactic acid bacteria
(NSLAB) is a term used to describe the adventitious bacterial flora
capable of growth under the selective conditions (typically 32 to 39%
moisture, 4 to 6% salt in moisture, pH 4.9 to 5.3, and 5 to 13°C) of
ripening cheese (15). During the ripening of Cheddar cheese,
the starter organisms autolyze, releasing growth substrates and
enzymes; concomitantly, an increase in NSLAB levels occurs to
~107 CFU · g Although NSLAB can be isolated from cheese milk, most of them are
inactivated by pasteurization. Their presence in cheese is probably due
to (i) postpasteurization contamination by the airborne flora
(36) or from strains on the cheese-making equipment or in
ingredients (40) or (ii) thermoduric strains surviving pasteurization (33).
The role of NSLAB in the development of Cheddar flavor was reviewed in
1990 by Peterson and Marshall (37), who concluded that it
was, at best, equivocal. Since then, there have been a number of
studies on the effect of adjunct lactobacilli on Cheddar flavor
development, with most authors reporting increased levels of free amino
acids and enhanced flavor intensity in the ripened cheese. High-quality
Cheddar, ripened for 1 year, was produced by certain strains of
Lactobacillus casei subsp. casei and L. casei subsp. pseudoplantarum, while other strains of
these species resulted in cheese with acidic and bitter flavor defects
(28). The addition of homofermentative lactobacilli to
Cheddar during production reduced the frequency of openness, swelling,
and gas production caused by intentionally added or heterofermentative lactobacilli in a study by Laleye et al. (25). Addition of
L. casei subsp. casei L2A as an adjunct was
reported in several investigations (29, 39, 44-46) to
accelerate ripening and consistently yielded well-aged Cheddar without
bitterness. The production of "aseptic" cheese (32) and
the use of antibiotics (47) or starter cultures producing
bacteriocins (42) to control adventitious NSLAB have facilitated research on the effect of adjunct lactobacilli on flavor development.
Evaluation of the relationship between NSLAB and flavor production
requires knowledge of the relative abundance of NSLAB species and
strain heterogeneity in cheese, but very few data on these topics are
available. NSLAB originating from good-quality mature cheese have been
investigated as potential starter adjuncts (26, 32), since
if they are beneficial to flavor development, such strains should be
abundant in mature, premium-quality cheese. Determination of the
diversity of the NSLAB population to strain level necessitates the use
of a rapid method suitable for handling large numbers of isolates.
Randomly amplified polymorphic DNA (RAPD) analysis is a PCR-based
method that uses a single primer of arbitrary sequence under
low-stringency conditions to amplify fragments of the test organism's
genome. The amplicons may be species or strain specific. This method
has been used to estimate the diversity among lactobacillus strains of
the Centre National de Recherches Zootechniques (CNRZ) collection
(43), to type strains of L. plantarum
(21), and to establish the correct nomenclature and
classification of strains of L. casei (12).
In this study we report the development and application of the RAPD
technique for the rapid identification of NSLAB to strain level and the
study of this population in mature commercial Cheddar cheese.
Cheese samples and compositional analyses.
Fourteen
premium-quality, 20-kg blocks of mature Cheddar cheese and three
defective-quality cheeses were obtained from six manufacturers.
Standard procedures were used to analyze cheeses for fat
(17), NaCl (18), and moisture (19).
The pH was measured on a cheese slurry prepared from 20 g of
cheese in 12 g of water (2).
Bacteriological analysis of cheese and NSLAB isolation.
Each
cheese was aseptically sampled, emulsified in sterile 2% (wt/vol)
trisodium citrate (pH 8.75), diluted, plated on
Lactobacillus-selective agar (Becton Dickinson and Co.,
Cockeysville, Md.) (41), overlaid, and incubated at 30°C
for 5 days. Twenty isolated colonies were randomly selected, from a
countable Lactobacillus-selective agar plate, for each
cheese. These isolates were propagated in MRS (10) broth
(Difco Laboratories, Detroit, Mich.), and purified. All isolates were
checked for catalase reaction and examined microscopically prior to
being stocked. Cultures were maintained in the Dairy Products Research
Centre collection (Teagasc, Moorepark, Fermoy, County Cork, Ireland) at
Type and reference strains.
A selection of 31 type and
reference strains of mesophilic lactobacilli were obtained from the
Laboratorium voor Microbiologie, Universiteit Gent (LMG) culture
collection, Universiteit Gent, Ghent, Belgium. These strains were used
to corroborate the genotypic and phenotypic methods employed to
characterize cheese isolates. Details of the species and strain numbers
for these cultures are given in Table 1.
Phenotypic characterization of isolates.
Lactobacillus species commonly found in Cheddar cheese
include L. paracasei, L. plantarum
(3), L. curvatus (23), and Lactobacillus brevis (40). These species may be
distinguished on the basis of differential levels of fermentation of
various sugars, growth responses in the absence and presence of Tween 80 (23), isomers of lactate produced, and levels of ethanol production (see Table 2), as reported by Kandler and Weiss
(24). Fermentations of rhamnose, cellobiose, melibiose,
mannitol, ribose, raffinose, mannose, and glucose in MRS broth (made up
without Lab Lemco, glucose, or citrate) were determined. Glucose, which is metabolized by all these species, was included as a positive control. Sterile sugar solutions were aseptically added to broths to a
final sugar concentration of 1% (wt/vol) in a total volume of 5 ml.
Cultures were incubated at 30°C for 7 days; a pH differential of
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Phenotypic and Genotypic Characterization of
Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 of cheese 6 to 8 weeks
postmanufacture (22, 34). The NSLAB community is
predominated by mesophilic (group II) lactobacilli, though pediococci
and micrococci may also be found (1, 8, 16). A recent study
of the NSLAB population in 8-week-old Irish Cheddar revealed that it
was composed of 55% Lactobacillus paracasei, 28%
Lactobacillus plantarum, and 14% Lactobacillus
curvatus (23).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C in a 1:1 glycerol-MRS mixture. Isolates from stock were
subcultured in MRS broth and streaked on MRS plates before use.
TABLE 1.
Differential characteristics of the clusters shown in
Fig. 3
0.7 U relative to the pH of a control (sterile water substituted for
sugar solution) was regarded as fermentation. A comparison of the
growth responses (pH differential) in MRS broth minus acetate, citrate,
and Tween 80 and in MRS broth minus acetate and citrate after 2 days at
30°C was used to determine the requirement of Tween 80 for growth.
Supernatants from the latter cultures were analyzed for the isomer of
lactate enzymatically (Boehringer, Mannheim, Germany) and for ethanol
production by high-performance liquid chromatography (Waters, Watford,
Hertfordshire, United Kingdom). Similar levels of lactate and ethanol
indicated heterofermentation of sugars. Twenty microliters of
supernatant was injected onto an HPX87H column and guard column
(Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, United Kingdom)
held at 50°C. The mobile phase was 5 mM
H2SO4, and the flow rate was 0.6 ml · min1 (Waters model 510 pump). A refractive-index detector
(Waters model 410) was used. Data were collected and analyzed with a
Minichrom system (VG Data Systems, Cheshire, United Kingdom). Growth at 15 and 45°C was recorded as a pH differential (range as described above), relative to the pH of an uninoculated control, after 7 days of
incubation. Species designations were based on the phenotypic characteristics detailed in Table 2
(24).
TABLE 2.
Differential physiological and biochemical
characteristics of the facultatively and obligately
heterofermentative species of the
genus Lactobacillusa
Genotypic characterization of isolates. (i) Rapid extraction of DNA from lactobacilli. Genomic DNAs were extracted as described previously (4) from 1.5-ml samples of fresh overnight cultures (inocula used for phenotypic characterization) by harvesting by centrifugation for 5 min; 2 µl of each DNA in a subsequent 50-µl PCR mixture was sufficient to give reproducible results. Lambda DNA (Boehringer) standards were used to estimate the level of DNA extracted.
(ii) DNA concentration.
Tenfold serial dilutions from
100 to 106 of template DNA were used in PCR
preparations to determine the influence of DNA concentration on RAPD profiles.
(iii) Random primers. Eight primers, the sequences of which were chosen at random, were tested at final concentrations of 0.1, 0.5, or 1 µM and were as follows: P1 (5' ACGCGCCCT 3') (21), P2 (5' ATGTAACGCC 3') (20), P3 (5' TAGACTAT 3'), P4 (5' GTCAACTA 3'), P5 (5' CAATCTGATC 3'), P6 (5' AGCTATACTA 3'), P7 (5' CTGCGGCAT 3'), and P8 (5' CCGCAGCGTT 3'). P3 to P6 were designed with low G+C contents in order to maximize binding to the Lactobacillus chromosome (33 to 53% G+C). Primers were synthesized at the National Food Biotechnology Centre, University College, Cork, Ireland.
(iv) Amplification of Lactobacillus DNA by PCR. DNA amplification was performed by the method of Coakley and Ross (4). Taq polymerase (1.25 U; Bioline, London, United Kingdom) was added during a 6-min hot start at 94°C, followed by 35 amplification cycles of 94°C for 1 min and the desired annealing temperature for 1 min and an extension step of 72°C for 1 min. Annealing temperatures ranged from 25 to 60°C, and for extended annealing, the following conditions were used: 25°C for 1 min, 30°C for 1 min, 36°C for 2 min, and 40°C for 1 min. In an attempt to increase the proportion of low-molecular-weight fragments, a combined annealing and extension temperature of 36°C for 1 min was used. PCR products (5 to 10 µl) were separated on a 1.5% (wt/vol) gel (Promega Corp., Madison, Wis.) that was run for 1.25 h at 130 V, and the DNA was detected by UV transillumination.
(v) Reading of patterns, numerical analysis, and reproducibility. RAPD patterns on Polaroid photographs were digitized with a Scanjet 4P scanner and Deskscan II version 2.7 software (Hewlett-Packard Co., Dublin, Ireland), normalized, and further processed with the GelCompar version 4.0 program (Applied Maths, Kortrijk, Belgium). Strains were grouped by using the Pearson product moment correlation coefficient (r) and cluster analysis by the unweighted pair group method with arithmetic averages (38). Reproducibility of RAPD banding profiles was determined from triplicate loadings of independent, triplicate RAPD reaction mixtures from three LMG collection strains of L. paracasei subsp. paracasei on three gels, with cluster analysis being performed as described above.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Compositional profiles of cheeses.
The 17 cheeses analyzed
were between 9 and 27 months old. The averages ± standard
deviations of the four compositional parameters of pH, percent salt in
moisture, percent moisture in nonfat substance, and percent fat in dry
matter were 5.10 ± 0.15, 5.27% ± 0.75%, 55.46% ± 2.40%, and
52.82% ± 1.64%, respectively (Table
3). NSLAB counts averaged log 7.42 ± 0.43 CFU · g1. No correlation was found between
commercial grading score and compositional parameters, since the latter
could not separate premium-quality cheeses (A to R) from downgraded
cheeses (1 to 3).
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Phenotypic characterization. (i) Type and collection strains. Biochemical characterization, based on sugar fermentation patterns and end products of glucose metabolism, classified the collection strains as expected. However, comparison of phenotypic characteristics with those reported in the literature revealed some anomalies: LMG 6904T (ATCC 393) did not ferment ribose (12), LMG 12586 (ATCC 334) fermented raffinose (9), and LMG 6907T (ATCC 14917) fermented rhamnose (7). Two different colony morphologies (a and b) were observed for the following LMG isolates: 6400T, 14769, 9191T, and 13724.
(ii) Cheese isolates. All 331 NSLAB were found to be catalase-negative, nonmotile rods, indicating that they were members of the genus Lactobacillus. Phenotypic characterization of this population revealed a predominance (96.4%) of the species L. paracasei (Fig. 1). The other species identified were L. plantarum (2.1%), L. curvatus (0.3%), and L. brevis (0.3%). The NSLAB population of 7 of the 17 cheeses analyzed was comprised solely of L. paracasei strains. An additional five cheeses also contained ribose-negative variants of L. paracasei, such that 70% of the sampled cheeses had an NSLAB flora wholly comprised of strains from the L. paracasei complex. L. curvatus was found only in cheese O, which also contained L. brevis and a raffinose-negative variant of L. plantarum (tentatively identified as Lactobacillus paraplantarum). The six L. plantarum strains were all isolated from cheese N, which was obtained from the same factory as cheese O. The fermentation profiles of three strains, which were mutually dissimilar, were such that these isolates could not be phenotypically identified.
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, L. paracasei; 
, ribose-negative L. paracasei; 
, L. plantarum; 
, L. paraplantarum; 
, L. curvatus; 
, L. brevis; 
, unidentified.
DNA amplification with random primers.
Dilution of DNA from
100 to 102 resulted in identical RAPD
patterns for four strains (data not shown); thus, the undiluted
template was subsequently used in all PCR preparations. Primers P3 to
P6 did not give PCR products, despite extended annealing times at low
temperatures, probably due to their low melting temperatures. The
remaining primers, P1, P2, P7, and P8, all gave acceptable PCR
products; P1 gave the largest number of bands, while P2 gave easily
identifiable species-specific profiles (Fig.
2). Optimal primer concentrations for P1
and P2 were 1 and 0.5 µM, respectively, and the optimal annealing
temperature was 44°C, based on the intensities of
low-molecular-weight fragments.
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Reproducibility of RAPD. Triplicate loadings of triplicate PCR products from three individual strains of L. paracasei subsp. paracasei were separable, following cluster analysis, at the 80% similarity level (data not shown); i.e., separation above this level of similarity was insignificant.
Resolving power of RAPD. RAPD profiles exhibiting species-specific bands were generated with the P2 primer for L. paracasei (Fig. 2a); Lactobacillus rhamnosus, L. plantarum, and Lactobacillus pentosus (Fig. 2b); and L. curvatus (Fig. 2c). Differentiation of isolates to the subspecies and strain levels was possible by RAPD analysis.
Genotypic characterization of cheese and collection strains. The dendrogram in Fig. 3 is based on the combined RAPD profiles generated by P1 and P2 for nine cheeses from three factories (including three poor-quality cheeses from factory I) and was abridged at the 80% similarity level to indicate individually separated strains only. Details of each cluster are given in Table 1. To test the discriminating power of RAPD analysis at the species or subspecies and strain levels, 31 collection isolates of mesophilic lactobacilli, previously typed as 25 individual strains by protein profiling, were analyzed (6). Typed strains of each species clustered together. Cluster analysis of RAPD profiles for collection strains resulted in distinct species clusters (Fig. 3). Fingerprints, generated with the P2 primer for multiple strains of the same species demonstrated the presence of a number of species-specific bands. There were three common bands for all L. paracasei strains (Fig. 2a, lanes 2 to 9 and 11 to 16) at 0.4, at 0.9, and at 1.2 and 1.3 kb (the last being a doublet). Fingerprint bands for L. rhamnosus strains (Fig. 2b, lanes 2 to 7) occurred at 0.47, 1.05, and 1.55 kb, while those for L. plantarum strains (lanes 9 to 16) were at 0.52 (faint band for LMG 9205 [lane 15]), 0.85, and 2 kb. All five L. curvatus strains had a faint band at 0.44 kb, with more intense bands appearing at 0.77, 1.1, and 1.4 kb (Fig. 2c, lanes 2 to 6). Since only one strain of L. pentosus (Fig. 2b, lane 17) was subjected to RAPD analysis, identification of species-specific bands was not possible. However, there were two intense bands at 1.23 and 1.7 kb. This result implies that RAPD analysis has potential application in the rapid classification of mesophilic lactobacilli to the species level. A band at 0.8 kb, present in all L. paracasei subsp. paracasei profiles (Fig. 2a, lanes 2 to 9 and 11 to 13), was not found for the L. paracasei subsp. tolerans isolate (lanes 15 and 16; two different colony morphologies), which displayed a faint doublet at 1.2 and 1.3 kb, relative to that of the L. paracasei subsp. paracasei strains. These data suggest that RAPD analysis has the capacity to distinguish between subspecies of lactobacilli. Although the type strain of L. casei, LMG 6904T (Fig. 2a, lane 17), reclassified as Lactobacillus zeae (12), displayed the intense doublet at 1.2 and 1.3 kb common to L. paracasei strains, a distinctly different fingerprint was obtained with bands at 0.49, 0.56, 0.98 kb.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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NSLAB were isolated from 14 mature, premium-quality and from three defective-quality Cheddar cheeses, obtained from six different commercial manufacturers. Each cheese was graded by the in-house sensory analyst of the factory of origin. Comparison of compositional data (Table 3) with recommended ranges for percent salt in moisture, percent moisture in nofat substance, percent fat in dry matter, and pH for first- and second-grade Cheddar cheese, as devised by Lawrence and Gilles (27), revealed that no cheese, including one awarded a prestigious prize, fulfilled all four premium-grade criteria; only cheeses G and H satisfied three of these criteria. Five cheeses, namely, A, G, H, N, and O, complied with second-grade parameters. However, compositional analysis of cheeses was carried out at least one year postmanufacture, while suggested ranges for compositional parameters are based on analyzes at 14 days postmanufacture. The composition ranges across cheeses A to R were quite wide: the range for moisture was 35.3 to 41.3%, that for salt was 1.69 to 2.55%, and that for pH was 4.86 to 5.38. Similar results were reported by Fox (14) in a study of 27 mature Cheddar samples.
The mean NSLAB level in the cheeses, which were all at least 9 months
old, was log 7.42 ± 0.43 CFU · g1. All
isolates were found to be Lactobacillus species. This was in
agreement with the results of a previous study of commercial Cheddar of
Irish origin (23), indicating that the absence of other
genera of NSLAB may be a feature of Irish Cheddar cheese. Pediococci,
which can survive for 12 months in cheese (24), were found
to be the dominant non-starter flora in a series of English Cheddar
cheeses (16) but constituted far smaller proportions of New
Zealand (8) and American (31) Cheddar cheeses.
Feagan and Dawson (13) noted that micrococci, which are not
lactic acid bacteria and thus not normally considered part of the NSLAB flora of Cheddar cheese, comprised from 16 to 68% of the non-starter flora of 6-month-old Australian Cheddar.
Phenotypic characterization to the species level of the 331 isolates from 17 cheeses indicated that 319 (96.4%) were L. paracasei (including 23 ribose-negative variants), 7 (2.1%) were L. plantarum, including a raffinose-negative variant which is possibly L. paraplantarum (7), 1 (0.3%) was L. curvatus, 1 (0.3%) was L. brevis, and 3 could not be identified, either from Bergey's manual (24) or from information provided with API 50CH kits for identification of lactobacilli (bioMérieux s.a., Marcy-l'Etoile, France). Cheese O from factory II displayed the greatest species diversity and contained five different species. It is interesting that the population of 8-week-old Cheddar cheeses studied by Jordan and Cogan (23), comprising 160 isolates, had a smaller proportion of L. paracasei (55%) and larger proportions of L. plantarum and L. curvatus (28 and 14%, respectively) strains than the cheeses investigated in this study. These data imply that the composition of the NSLAB population of Cheddar changes with age, as was reported from an investigation by Naylor and Sharpe (35), who found differences in the distributions of serological types of lactobacilli, both within a block and within a pair of duplicate cheeses, during ripening.
A study of the changes in the microbial ecosystem during the ripening of raw milk Swiss-type minicheeses (11) revealed that the species identified evolved from a great diversity at the start of ripening to two dominating subgroups of L. paracasei at 24 weeks postmanufacture. A comparison of the relative occurrence of L. plantarum with that of L. casei during the ripening of Cheddar (13) indicated that similar proportions of L. plantarum and L. casei were found in both good- and defective-quality cheeses and that variations in the population of lactobacilli in cheese of comparable ages could not be correlated with particular flavor changes.
In an effort to validate the biochemical classifications to species level, a selection of 31 collection isolates of mesophilic lactobacilli were subjected to the tests outlined in Materials and Methods and Table 2. All were designated to the correct species on the basis of these biochemical tests. Fermentation patterns for the majority of these were as reported in the literature. However, a few anomalies were noted, highlighting the main disadvantage of phenotyping: the designation of isolates to one species or another on the basis of a selection of phenotypic markers, which may not be stably expressed under certain environmental or culture conditions. The difficulty associated with using changes in pH as indicators of growth in the presence of different sugars is determining the cutoff point for a positive or negative reaction. For example, LMG 10755T (ATCC 8041), reported as fermenting rhamnose, gave a weakly positive reaction for this carbon source that was indicated by a change in pH of 0.69 after 7 days of incubation at 30°C, compared to mean changes in pH of 1.43 and 0.27 for positive and negative reactions, respectively, for collection strains.
Although phenotypic tests provide evidence of the metabolic capabilities of strains, they have problems such as nonreproducibility and lack of discriminatory power. The advantages of genotyping include the stability of genomic DNA, its composition being independent of cultural conditions or preparation methods, and amenability to automation and statistical data analysis. The designation of certain neotype strains on the basis of phenotypic characteristics alone has resulted in confusion in the nomenclature of strains of L. casei, which is being resolved with genotypic data (12). The most accurate results are obtained when both phenotypic and genotypic attributes are used to make the correct appellation.
RAPD analysis has previously been reported for rapid typing of L. plantarum strains (21), but this procedure has not been used to study lactobacilli in cheese. To complete the present study, it was necessary to determine if this technique could be extended to other species of mesophilic lactobacilli. Distinct species clusters were obtained following cluster analysis of RAPD patterns for 31 collection isolates (Fig. 3). The P2 primer generated species-specific bands for multiple strains of the same species (Fig. 2).
Cluster analysis of RAPD fingerprints of cheese isolates demonstrated that there was an average of seven strains per cheese (based on the number of individually separated strains at the 80% similarity level for combined gels of P1 and P2 profiles). The dendrogram in Fig. 3 illustrates the relationships between collection strains and isolates from cheeses, obtained from three different commercial producers. The majority of strains isolated from cheeses made in the same factory clustered together: isolates from factory I cheeses fell in clusters 32 to 54 and 58 to 61, isolates from factory II cheeses fell in clusters 16 to 30 and 73 to 80, and isolates from factory III cheeses fell in clusters 62 to 64 (Fig. 3 and Table 1). For the most part, isolates from premium-quality cheeses clustered together (87 of 103, in clusters 11 to 54), as did isolates from defective-quality cheeses (clusters 58, 59, and 67). Clustering of biochemically characterized isolates from cheeses of adequate and defective quality within the L. paracasei complex was reported by Lindberg et al. (30) in a study of mature Norwegian and Swedish semihard cheese.
The most striking feature of the cluster structure in Fig. 3 is the heterogeneity of the L. paracasei supercluster (clusters 11 to 71) (Table 1). The type strains of L. paracasei subsp. paracasei (LMG 13087T, cluster 30) and L. casei subsp. casei (LMG 6904T [ATCC 393, reclassified as L. zeae], cluster 69) and the proposed neotype strain of L. casei subsp. casei ATCC 334 (LMG 12586, cluster 57), in place of ATCC 393 (12), were all well separated (Fig. 3). Six collection strains of L. paracasei subsp. paracasei (cluster 58) had a very high homology with poor cheese isolates from factory I (clusters 58 and 59). The type strain (LMG 13087T) was most similar to isolates from factory II (clusters 16 to 28), while the type strain of L. casei subsp. casei (LMG 6904T) was only 32% similar to the majority of isolates in the L. paracasei supercluster, a fact which supports its revised taxonomic status (12).
Cluster analysis of RAPD profiles of cheese isolates from three factories provided evidence for the recurrence of strains in cheeses K and 1 to 3, manufactured 1 year apart at factory I (clusters 51, 53, 58, and 59). Visual observation of fingerprints for the remaining isolates revealed that similar banding profiles were found for isolates from cheeses Q and R, produced by factory VI 1 year apart; however, no common strain was isolated from cheeses P and Q, manufactured 1 month apart at factory VI (data not shown). Although L. plantarum strains were found only in cheeses N and O, produced at factory II in consecutive years, the latter exhibited greater species diversity. This reappearance of certain strains of NSLAB in cheese over time has previously been reported (35), prior to the advent of RAPD analysis.
The majority (~79%) of cheese isolates were L-lactate producers. No correlation could be found between the RAPD profile and isomer of lactate produced; the DL-producing type strain of the former subspecies L. casei subsp. pseudoplantarum LMG 9192T (DSM 20008T, cluster 66) grouped together with isolates in clusters 55 to 66, all of which were L-lactate producers. Furthermore, strains of the ribose-negative phenotype divided into two distinct groups (clusters 17 to 19 and 25 to 26). These findings are in contrast to those of Lindberg et al. (30), who reported that strains of the same phenotype clustered together on the basis of biochemical parameters.
In conclusion, RAPD analysis proved to be a very rapid, effective, and reproducible method of differentiating the NSLAB floras of mature Irish Cheddar cheeses to the strain level. Application of this technique to isolates from mature Cheddar demonstrated that the NSLAB flora was dominated by L. paracasei and that major strain heterogeneity existed among isolates.
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ACKNOWLEDGMENTS |
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This work was supported by European Regional Development Fund, Measure 3(ii), of the Food Sub-Programme. N. Fitzsimons was in receipt of a Walsh Fellowship.
The assistance of the staff of the Department of Food Technology, Chemical Centre, University of Lund, Lund, Sweden, is gratefully acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: Dairy Products Research Centre, Moorepark, Fermoy, County Cork, Republic of Ireland. Phone: 353 25 42304. Fax: 353 25 42340. E-mail: tberesford{at}moorepark.teagasc.ie.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 1999, p. 3418-3426, Vol. 65, No. 8
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