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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2008, p. 5662–5673 Vol. 74, No. 180099-2240/08/$08.00�0 doi:10.1128/AEM.00418-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Polyphasic Approach to Bacterial Dynamics during the Ripening ofSpanish Farmhouse Cheese, Using Culture-Dependent and

-Independent Methods�

Antonio M. Martın-Platero, Eva Valdivia, Mercedes Maqueda,Ines Martın-Sanchez, and Manuel Martınez-Bueno*

Departamento de Microbiologıa, Facultad de Ciencias, Fuentenueva s/n, 18071 Granada, Spain

Received 19 February 2008/Accepted 15 July 2008

We studied the dynamics of the microbial population during ripening of Cueva de la Magaha cheese usinga combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat’smilk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures wereanalyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) andidentified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate thatthe majority of the 225 strains isolated and enumerated on solid media during the ripening period werenonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillusspecies, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning andend of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were alsodetected at the end of the ripening period. Microbial community dynamics determined by temporal temper-ature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabledus to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated.Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcelydetected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolutionby using length heterogeneity PCR.

Lactic acid bacteria (LAB) are functionally related by theirability to produce lactic acid via either homo- or heterofermen-tative metabolism. The acidification and enzymatic processes ac-companying the growth of LAB impart distinctive flavors andtextures to a wide variety of fermented dairy products, meats, andvegetables. In addition, their preservative qualities make thesebacteria useful in the control of undesirable microorganisms (16).For this reason LAB have important applications in the food,agricultural, and medical sectors and have been the subject ofconsiderable research and commercial development over the pastdecade. Lactobacilli are well represented in fermented milk,yogurt, and cheese, as either natural or intentionally added mi-crobiota (starter cultures). Lactobacillus casei, Lactobacillus para-casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactoba-cillus fermentum, Lactobacillus brevis, Lactobacillus buchneri,Lactobacillus curvatus, Lactobacillus acidophilus, and Lactobacil-lus pentosus are common members of the nonstarter LAB(NSLAB) communities in dairy products (11, 32, 35). In cheeseNSLAB are represented mainly by L. casei and L. paracasei (5, 11,20), but L. brevis, L. plantarum and L. curvatus are also important(4, 5, 43). Although these organisms are usually present at lowlevels in curd (102 to 103 CFU/g), their concentrations increaseduring ripening to 107 to 108 CFU/g (15, 21). NSLAB have beenreported to be responsible for many of the flavor qualities ofcheese, and their use as adjuncts to starter cultures might either

improve taste or introduce off-flavors, depending upon the strainsused (4, 38, 43).

Because complex interactions in the microbial communityoccur during milk curdling and cheese ripening, accurate iden-tification of the microorganisms involved provides informationessential for understanding their role in this process (7), whichshould lead to more objective criteria for the selection ofstrains for starter or adjunct cultures. However, LAB identifi-cation on the basis of phenotypic features is very difficult be-cause of the great variation in the fermentation profiles of theorganisms, which differ considerably for strains belonging tothe same species. Identification is now approached by usingDNA-based techniques, which allow workers to distinguishbetween different species with similar phenotypic characteris-tics and also between strains belonging to the same species.Among the various molecular techniques used for identifica-tion of LAB species are species-specific PCR (33), restrictionfragment length polymorphism (8), and 16S rRNA gene se-quencing (41, 42), and the latter technique is the techniquemost extensively used to identify species. For intraspecies char-acterization, pulsed-field gel electrophoresis (28), repetitiveenterobacterial palindromic sequence PCR (9), and randomlyamplified polymorphic DNA (RAPD) analysis (25) are themethods currently used for LAB isolates from foodstuffs anddairy products. Nevertheless, all of these molecular techniquesare culture dependent. It is, however, well known that only asmall proportion of microorganisms are culturable, and there-fore culture-dependent techniques do not reflect real microbialcommunities (24). Consequently, different culture-indepen-dent techniques have been used for fermented foods and dairyecosystems, mainly terminal restriction fragment length poly-

* Corresponding author. Mailing address: Departamento de Microbio-logıa, Facultad de Ciencias, Fuentenueva s/n, 18071 Granada, Spain.Phone: 34 958 243184. Fax: 34 958 249486. E-mail: [email protected].

� Published ahead of print on 25 July 2008.

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morphism analysis (44), length heterogeneity PCR (LH-PCR)(30), single-strand conformation polymorphism analysis (18),denaturing gradient gel electrophoresis (42), temperature gra-dient gel electrophoresis (10, 11), and temporal temperaturegradient gel electrophoresis (TTGE) (3).

Cueva de la Magaha is a cheese made in the south of Spain(Jayena, Granada) from raw goat’s milk inoculated with thestarter cultures Lactococcus lactis and Streptococcus thermophi-lus. The aim of this study was to characterize the microbialcommunities isolated during the ripening process. The dynam-ics of microbial diversity was also studied by using the culture-independent techniques TTGE and LH-PCR; the latter wasused to evaluate the Lactobacillus community, which provideda quick and easy way to fingerprint this bacterial group.

MATERIALS AND METHODS

Sample preparation and microbiological analysis. The samples analyzed inthis study were obtained from three different cheeses from the same batch ofCueva de la Magaha cheese, a hard farmhouse cheese made in Jayena in theprovince of Granada (Spain) from raw goat’s milk inoculated with starter cul-tures (EZAL MA-400; Rhodia Food, Sassenage, France). The curd of thischeese is processed by heating it to 37°C, cutting and molding, and then saltingby immersion for 24 h in natural brine (20.4 to 22.2° Baume; 25.6 to 28.3% NaCl)before it is ripened for 8 months by storage under controlled humidity (relativityhumidity, 80 to 90%) and temperature (12 to 13°C) conditions. According to thestarter manufacturer, EZAL MA-400 is composed of a mixture of L. lactis subsp.lactis, L. lactis subsp. cremoris, L. lactis subsp. lactis bv. diacetylactis, and S.thermophilus.

Before sampling, the cheeses were washed with water, and their rinds wereremoved. Ten-gram samples were collected aseptically from three cheeses fromthe same batch after 1, 17, and 34 weeks of ripening, and each sample washomogenized for 2 min in 90 ml of a prewarmed (37°C), sterile, 2% sodiumcitrate solution in a sterile plastic bag with a lateral filter, using a Stomacher labblender (IUL Instruments, Barcelona, Spain). One milliliter of the resultingmixture was taken from the filtrate side, and 10-fold serial dilutions up to 10�7

were prepared using a sterile saline solution (0.8% NaCl). A 100-�l aliquot wasspread in triplicate on agar plates for bacterial enumeration. The plates wereincubated at 28°C for 4 to 7 days, and the bacteria were enumerated by countingcolonies on different media, including Tween APT agar, M-17 agar, and brainheart infusion agar (BHA) (all obtained from Scharlab Barcelona, Spain) toestimate the total number of aerobic, mesophilic bacteria, Man-Rogosa-Sharpe(MRS) agar to estimate the number of LAB, Kenner fecal agar (KFA) toestimate the number of enterococci, Vogel-Johnson agar to estimate the numberof staphylococci, and MacConkey agar to estimate the number of enterobacteria.The average counts for the replicate cheese samples and the standard deviationswere calculated after log transformation. Additionally, between 10 and 20 LABisolates (corresponding to the square roots of the total numbers of colonies) wererandomly picked from general and selective medium plates and cultured on thecorresponding media. The isolates were purified, examined to study their mor-phology, and Gram stained and tested for catalase activity before they werestored for further analyses at �70°C in the corresponding broth media supple-mented with 35% glycerol.

Another strategy used in this study was based on the quantitative PCR(QPCR) most-probable-number assay for detection of LAB growth in enrich-ment cultures. For this, 10-g replicates of 34-week-old cheese were homogenizedas described above, and the suspensions were serially diluted 10-fold in triplicatein tubes containing MRS broth and Rothe azide broth (Scharlau, Barcelona,Spain) and incubated without agitation at 28°C for 48 h. Rothe azide broth wasused for primary enrichment of fecal streptococci due to the presence of sodiumazide, which is both selective for enterococci and an inhibitor of the accompa-nying flora through interference with the electron transport chain. The mostprobable number was determined from the more diluted tubes showing turbidity.Tubes showing turbidity were selected for TTGE analysis or multiple specificPCR.

Genomic DNA isolation. A suspension corresponding to 1 g of cheese wascentrifuged for 5 min at 13,000 � g in a microcentrifuge, and the sediment waseither stored at �70°C or used for extraction and isolation of genomic DNA asdescribed by Martın-Platero et al. (34). The same method was used to extractDNA from pure cultures (34).

RAPD analysis. The genotypes of all strains were determined using a RAPD-PCR procedure (40). The PCR was carried out using a 50-�l (total volume)mixture containing 5 �l of 10� Taq reaction buffer, 3 mM MgCl2, each de-oxynucleoside triphosphate (dNTP) at a concentration of 400 �M, 1 �M primerM13 (5�-GAGGGTGGCGGTTCT-3�), 1 U of Taq DNA polymerase (MBL,Cordoba, Spain), and 5 �l of template DNA. Amplification was performed withan iCycler 170-8720 (Bio-Rad, Hercules, CA) using a program consisting of aninitial denaturing step of 94°C for 60 s, followed by 35 cycles of 60 s at 94°C, 20 sat 40°C (with a 0.6°C/s ramp), and 80 s at 72°C (with a 0.6°C/s ramp) and thena final extension at 72°C for 5 min. The PCR products were analyzed by elec-trophoresis in 1.5% agarose gels at 30 V for 16 h in 1� TAE buffer (40 mMTris-acetate, 2.5 mM EDTA; pH 8) and were revealed by staining with ethidiumbromide (0.5 �g/ml). Gels were photographed under UV light and analyzed byusing Fingerprinting II Informatrix software (Bio-Rad, Hercules, CA). A simi-larity matrix was constructed on the basis of the Pearson product momentcorrelation coefficient, and the corresponding dendrogram was deduced usingthe unweighted-pair group method with arithmetic averages.

16S rRNA gene sequencing. A 700-bp fragment of the 16S rRNA gene, whichincluded variable regions V1 to V4, was amplified for representative strains ofeach RAPD genotype for subsequent sequencing. The PCR was carried out usinga 50-�l (total volume) mixture containing 5 �l of 10� Taq reaction buffer, 10 �lof 5� Taq Enhancer, 1.5 mM magnesium diacetate, each dNTP at a concentra-tion of 400 �M, 0.4 �M primer WO1 (5�-AGAGTTTGATC[AC]TGGCTC-3�),0.4 �M primer WO12 (5�-TACGCATTTCACC[GT]CTACA-3�) (Table 1), 1 Uof Eppendorf Master Taq polymerase, and 1 �l of template DNA. The ampli-fication program consisted of an initial denaturing step of 94°C for 4 min,followed by amplification using 30 cycles of 30 s at 94°C, 30 s at 50°C, and 60 sat 72°C and then a final extension at 72°C for 2 min. PCR products were purifiedwith a Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany) and weresequenced using an ABI Prism dye terminator cycle sequencing ready-reactionautomated sequencer (ABI 3100; Applied Biosystems). Homologies weresearched for in the BLASTN database (National Center for BiotechnologyInformation) using BLAST (2).

Species-specific PCR and multiple PCR. Species-specific PCR and multiplePCR were used to confirm the identities of the strains belonging to the sameRAPD genotype which were not identified by 16S rRNA gene sequencing. EachPCR was carried out using a 50-�l (total volume) mixture containing 5 �l of 10�Taq reaction buffer, 10 �l of 5� Taq Enhancer, 1.5 mM magnesium diacetate,each dNTP at a concentration of 400 �M, each of the two primers at a concen-tration of 0.4 �M, 1 U of Eppendorf Master Taq polymerase, and 1 �l oftemplate DNA. The species-specific primers used are shown in Table 1. Ampli-fication was performed by following the recommendations described previously(Table 1). The products were analyzed by electrophoresis in 1% agarose gels in1� TAE buffer.

TTGE. TTGE samples were prepared by nested PCR (37). First, a 700-bp frag-ment of the 16S rRNA gene was amplified as described above with primers WO1and WO12 (Table 1) but with 1.5 U of Eppendorf Master Taq polymerase and 2 �lof DNA template (100 ng). The PCR product was used to amplify the V3 region ofthe 16S rRNA gene, for which we used two different pairs of primers (6, 37): primersgc338f (5�-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3�) and 518r (5�-ATTACCGCGGCTGCTGG-3�) and primers HDA1-gc (5�-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAGT-3�) and HDA2 (5�-GTATTACCGCGGCTGCTGGCA-3�). The PCR was carried out using a 50-�l (totalvolume) mixture containing 5 �l of 10� Master Taq reaction buffer, 10 �l of 5� TaqEnhancer, 1.5 mM magnesium diacetate, each dNTP at a concentration of 400 �M,each primer at a concentration of 0.4 �M, 1.5 U of Eppendorf Master Taq poly-merase, and 1 �l of the amplified 700-bp fragment of the 16S rRNA gene. Theamplification program included an initial denaturing step of 94°C for 1 min and then30 cycles of DNA amplification (30 s at 94°C, 30 s at 52°C, and 30 s at 72°C) and afinal extension at 72°C for 5 min.

The PCR product of the V3 region was analyzed by TTGE using the Dcodeuniversal mutation detection system (Bio-Rad, Hercules, CA) as described byOgier et al. (37). The gels were photographed under UV light, and the differentbands were cut out with a sterile scalpel and reamplified with the same pair ofprimers. These PCR products were subject once more to TTGE analysis, and theproducts found to be pure were sequenced as described above.

LH-PCR. The 16S/23S rRNA intergenic spacer region specific to lactobacilliwas amplified using primers R16-1F and LbLMA1-R (17); the reverse primerLbLMA1-R was labeled fluorescently with 6-carboxyfluorescein. The PCR wascarried out using a 50-�l (total volume) mixture containing 5 �l of 10� MasterTaq reaction buffer, 10 �l of 5� Taq Enhancer, 1.5 mM magnesium diacetate,each dNTP at a concentration of 400 �M, each of the two primers at a concen-

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tration of 0.4 �M, 1 U of Eppendorf Master Taq polymerase, and 1 �l oftemplate DNA. The amplification program included an initial denaturing step of94°C for 3 min, followed by amplification for 30 cycles of 30 s at 94°C, 30 s at55°C, and 30 s at 72°C and then a final extension at 72°C for 5 min. The amplifiedDNA spacer regions were denatured by heating in formamide, and the lengths ofthe fragments were determined by means of automated fluorescent capillaryelectrophoresis with an ABI Prism 310 genetic analyzer (Applied Biosystems).Electropherogram peak values (i.e., fragment lengths) were calculated afterinterpolation with an internal size standard. The fingerprints were comparedelectronically with a database of fingerprints obtained for different Lactobacillusspecies (Table 2).

RESULTS

Microbiological analysis of the cheese samples. Microbialcounts obtained by plating serial dilutions of samples areshown in Table 3. The total LAB counts were always in therange from 107 to 108 CFU/g; the highest levels occurred at themid-ripening stage, and the lowest levels occurred at the end ofthe process. No significant differences between counts in the

TABLE 1. Primer pairs used in this study

Primer Sequence Specificity Reference

WO1_for 5�-AGAGTTTGATC(AC)TGGCTC-3� 16S rRNA gene 37WO12_rev 5�-TACGCATTTCACC(GT)CTACA-3�

Lacto1 5�-GTAACTACCGAGAAAGGG-3� Lactococcus 13Lacto2 5�-ATCTCTAGGAATAGCAC-3�

Entero1 5�-CCCGGCTCAACCGGG-3� Enterococcus 13Entero2 5�-CTCTAGAGTGGTCAA-3�

R16-1F 5�-CTTGTACACACCGCCCGTCA-3� Lactobacillus 17LbLMA1-R 5�-CTCAAAACTAAACAAAGTTTC-3�

ddl-E1 5�-ATCAAGTACAGTTAGTCTT-3� E. faecalis 19ddl-E2 5�-ACGATTCAAAGCTAACTG-3�

ddl-F1 5�-TAGAGACATTGAATATGCC-3� E. faecium 19ddl-F2 5�-TCGAATGTGCTACAATC-3�

DuHiF 5�-TTATGTCCCTGTTTTGAAAAATCAA-3� E. hirae 29HiR 5�-TTTTGTTAGACCTCTTCCGGA-3�

DuHiF 5�-TTATGTCCCTGTTTTGAAAAATCAA-3� Enterococcus durans 29DuR 5�-TGAATCATATTGGTATGCAGTCCG-3�

St1 5�-CACTATGCTCAGAATACA-3� S. thermophilus 31St2 5�-CGAACAGCATTGATGTTA-3�

His1 5�-CTTCGTTATGATTTTACA-3� L. lactis 12His2 5�-CAATATCAACAATTCCAT-3�

casei 5�-TGCACTGAGATTCGACTTAA-3� L. casei 46Y2 5�-CCCACTGCTGCCTCCCGTAGGAGT-3�

para 5�-CACCGAGATTCAACATGG-3� L. paracasei 46Y2 5�-CCCACTGCTGCCTCCCGTAGGAGT-3�

rham 5�-TGCATCTTGATTTAATTTTG-3� L. rhamnosus 46Y2 5�-CCCACTGCTGCCTCCCGTAGGAGT-3�

planF 5�-CCGTTTATGCGGAACACCTA-3� L. plantarum 45pREV 5�-TCGGGATTACCAAACATCAC-3�

brevF 5�-CTTGCACTGATTTTAACA-3� L. brevis 26brevR 5�-GGGCGGTGTGTACAAGGC-3�

TABLE 2. LH-PCR fragment lengths obtained fordifferent lactobacilli

Species or strain Sourcea Fragment length(s)(�1 bp)

L. paraplantarum 1885 CNRZ 191, 208L. parabuchneri Isolated from cheese 203L. fermentum 14933 ATCC 203L. delbrueckii 11842 ATCC 207L. plantarum 748 CECT 207L. pentosus 4023 CECT 208L. curvatus 2739 NCFB 213L. coryniformis Isolated from cheese 213 (196)b

L. acidophilus 4356 ATCC 213 (196)L. sakei 2714 NCFB 214L. farciminis 571T CECT 216L. paracasei 4022T CECT 218L. rhamnosus Isolated from cheese 218L. brevis Isolated from cheese 218 (206, 208)

a CNRZ, National Centre for Zootechnical Research; ATCC, American TypeCulture Collection; CECT, Coleccion Espanola de Cultivos Tipo; NCFB, Na-tional Collection of Food Bacteria.

b The lengths of secondary peaks are indicated in parentheses.

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different culture media used were observed, although the low-est levels were obtained with BHA. Enterococci were found atlevels of 104 CFU/g during the first week but were not detectedduring the remaining ripening period. Staphylococci were de-tected only at the end of the ripening period, at levels of 104

CFU/g, and Enterobacteriaceae were not detected at allthroughout ripening. During cheese ripening the pH valueswere low (pH 5.44 after 1 week of ripening to pH 5.41 after 34weeks).

Microbial identification and cluster analysis. Numbers ofcolonies equal to the square roots of the total numbers of colonieson MRS medium, M17-glucose medium, BHA, and KFA platescontaining 30 to 300 colonies were randomly picked and re-streaked onto the same media to obtain pure cultures. Thus, atotal of 225 isolates (119 isolates from MRS agar, 54 isolates fromM17 agar, 41 isolates from BHA, and 11 isolates from KFA) werecollected at different times during the ripening period, and afterthey were tested for catalase and Gram staining, they were usedfor DNA extraction and RADP-PCR amplification. The level ofidentity for strain discrimination was set at 86% similarity fordifferent genotypes. The dendrograms deduced using the un-weighted-pair group method with arithmetic averages are shownin Fig. 1. After RAPD profiles were compared, a total of 51different genotypes were chosen for species identification andcluster analysis. At the end of the first week of ripening wedetected 26 different genotypes, which were designated G1-1 toG1-26. After 17 weeks of ripening 16 different genotypes werefound in the cheese, 10 of which were similar to genotypes iden-tified during the first week. The new genotypes that appeared inthis sample were designated G2-1 to G2-6. Finally, at week 34, 26genotypes were detected, 19 of which (G3-1 to G3-19) were foundonly at this late stage. A strain of each cluster was identified by16S rRNA gene sequencing, and the species identity of the othermembers of the cluster was confirmed by means of PCR ampli-fication using the species-specific probes shown in Table 1.

Microbial evolution during cheese ripening. The designa-tions of different genotypes, the species assigned to each of thegenotypes, and the percentages of each species based on thetotal numbers of isolates at the three sampling times are shownin Fig. 1A, B, and C. Log counts were obtained by theoreticalcalculation on the basis of the proportions of the differentfrequencies of isolation of each genotype. Cluster analysis ofRAPD-PCR patterns revealed a high degree of diversity, dom-inated by Lactobacillus strains, which were the predominant

bacteria throughout the entire ripening period. Other bacterialspecies, such as Staphylococcus and Kocuria species, appearedonly at the mature stage. Six genotypes, L. brevis G1-4, L.paracasei G1-12 and G1-14, L. plantarum G1-15 and G1-20,and Lactobacillus farciminis G1-19, were found to be presentduring weeks 1 and 17 but did not appear at the end of ripen-ing, while three other genotypes, L. paracasei G2-2 and G2-3and Lactobacillus parabuchneri G2-1, were detected duringweeks 17 and 34 but not during the first week (Fig. 1A, B, andC). The highest titers found during the first week of ripeningwere the titers of genotypes G1-2 (L. brevis) and G1-15 (L.plantarum), while the lowest titers were the titers of entero-cocci (approximately 104 CFU/g), represented by Enterococcusfaecalis, Enterococcus malodoratus, Enterococcus hirae, and,above all, Enterococcus devriesei, During ripening, however,the Enterococcus species gradually disappeared (Fig. 1A and1C). At mid-ripening the highest levels were the levels ofgenotypes G1-15 (L. plantarum) and G1-10 (L. paracasei) (Fig.1B). During the last month of ripening the levels of the bac-teria that had dominated during the first week decreased, leav-ing genotypes G1-7 (L. brevis), G3-6, G1-10, G2-3, G1-13,G3-10, G3-3, G3-4, and G3-5 (all identified as L. paracasei),G2-1 (L. parabuchneri), and G3-15 (Staphylococcus equorum)as the best-represented genotypes (Fig. 1C).

According to the findings described above, L. paracasei wascertainly the most dominant bacterium (55.7%), followed by L.brevis (18.5%), L. parabuchneri (6.3%), and S. equorum (10.5%),while L. rhamnosus (1.3%), Lactobacillus coryniformis (1.3%),Staphylococcus epidermidis (1.3%), Kocuria spp. (1.3%), andCorynebacterium variabile (0.52%) were present at minor levels(Fig. 1C). The highest diversity within a single species was thediversity in L. paracasei, for which 16 different genotypes wereisolated (Fig. 1A, B, and C), followed by L. brevis (12 genotypes)and L. plantarum (9 genotypes). Nevertheless, the distribution ofLactobacillus diversity differed according to the stage of ripening.Thus, while during the earliest ripening stage L. plantarum and L.brevis (eight genotypes each) were the most diverse species, L.paracasei showed the highest intraspecific biodiversity from themid-ripening stage until full maturity. Furthermore, only fourgenotypes, genotypes belonging to L. brevis (G1-7 and G1-9) andL. paracasei (G1-10 and G1-13), showed high titers throughoutthe ripening process (Fig. 1A, B, and C).

Throughout the whole ripening period an increase in biodi-versity was observed, as demonstrated by the simultaneous

TABLE 3. Microbial counts determined during Cueva de la Magaha cheese ripening

Culture medium

Counts after the following ripening times:

1 wk 17 wk 34 wk

Log CFU/g SDa Log CFU/g SD Log CFU/g SD

MRS 7.67 0.05 8.00 0.04 7.19 0.02APT 7.74 0.02 7.92 0.07 7.23 0.03M-17 7.76 0.06 7.98 0.12 7.33 0.04BHA 7.05 0.18 7.88 0.04 6.81 0.08KFA 4.60 0.09 NDb NAc ND NAVogel-Johnson ND NA ND NA 4.80 0.18MacConkey ND NA ND NA ND NA

a The standard deviations were calculated after log transformation.b ND, not detected.c NA, not applicable.



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presence of L. parabuchneri, L. rhamnosus, and L. coryniformis,and in the final product even S. equorum, S. epidermidis, Kocu-ria sp., and C. variabile were detected.

TTGE analysis of bacterial microbiota in Cueva de la Ma-gaha cheese and quantification of representative LAB popula-tions. TTGE fingerprinting of the total bacterial community ofCueva de la Magaha cheese using two different pairs of primerswas carried out to amplify the V3 region of the 16S rRNA gene(Fig. 2). The electrophoresis profiles obtained with primersgc338f and 518r showed the presence of four principal bandsafter the first week, which were identified as L. plantarum(band 1), L. brevis (band 4), L. lactis (band 7), and S. ther-mophilus (band 8) (Fig. 2). Interestingly, during ripening, newspecies, including S. equorum (band 3), L. curvatus (band 6),and L. paracasei (band 9), were detected. The intensities of thebands corresponding to L. plantarum and L. brevis were highestat the end of ripening. On the other hand, the most intensebands during the early stage, corresponding to L. lactis and S.thermophilus, were much less intense during the later stages ofripening. The intensities of the bands for the remaining speciesincreased throughout the ripening period.

The same bacterial species were detected using the HDA1and HDA2 primers, except for L. coryneformis (band 5), which

was present only at the end of ripening. Several quantitativedifferences were observed, however. Thus, for example, themost intense bands after 34 weeks of ripening corresponded toL. plantarum and S. thermophilus. What is noteworthy are thedifferences in intensity between S. thermophilus and L. lactisdepending on the primers used (Fig. 2).

To confirm and quantify the most representative bacterialpopulations identified by TTGE analysis of mature cheeses(after 34 weeks of ripening), two series of TTGE fingerprintswere obtained for total DNA extracted from enrichment cul-tures obtained by dilution of cheese in MRS broth and Rothebroth. None of the enrichment cultures yielded a fingerprintidentical to that of the total community DNA (Fig. 3) since theTTGE patterns of the diluted enrichment cultures containedfewer bands than those of the nonenriched original cheese. Asexpected, the number of bands decreased throughout the di-lution steps (Fig. 3). The dominant species, L. paracasei and L.brevis (bands 9 and 4), accounted for up to 107 cells g�1, S.equorum (band 3) accounted for up to 106 cells g�1 in MRSbroth and 105 cells g�1 in Rothe broth, and L. plantarum (band1) accounted for up to 106 cells g�1. Nevertheless, none of theL. paracasei and L. brevis bands could be detected at lowerdilutions (10�1 to 10�4). L. curvatus (band 6) accounted for up

FIG. 1. Dendrogram obtained after RAPD-PCR analysis of the 225 strains isolated during cheese ripening. (A) One week of ripening.(B) Seventeen weeks of ripening. (C) Thirty-four weeks of ripening. The genotypes and their levels (in log CFU/g) (in parentheses) are indicatedon the right. Log CFU/g values were obtained by theoretical calculation on the basis of proportions of the different frequencies of isolation of eachgenotype on or in different culture media.



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to 105 cells g�1 in MRS broth and 104 cells g�1 in Rothe broth.Other minor bacteria, such L. lactis (band 7) and E. faecalis/Enterococcus faecium, appeared only at dilutions of 10�2 to10�4 and 10�1 to 10�3, respectively. E. faecalis and E. faeciumcould not be detected in the TTGE fingerprints of maturecheese. Although bands corresponding to S. thermophilus, L.parabuchneri, S. epidermidis, Kocuria sp., and C. variabile couldnot be detected in TTGE preparations from any of the enrich-ment cultures, quantification of some of the bacteria, such asL. paracasei, L. brevis, L. plantarum, L. curvatus, L. lactis, E.faecalis, and E. faecium, was confirmed by species-specific PCRusing enrichment cultures (Fig. 3B).

Analysis of the Lactobacillus population by LH-PCR. Sepa-ration of the PCR products by capillary electrophoresis en-abled us to distinguish between fragments whose lengths dif-fered by only 1 bp. This method allowed us to fingerprint theLactobacillus diversity of the cheese during ripening (Fig. 4). Adatabase with reference strains (Table 2) was established, andthis was followed by performing LH-PCR with the total com-munity DNA during the different ripening periods. On thebasis of the peak sizes, four groups could be distinguished(peak 203, peaks 207 and 208, peaks 213 and 214, and peak218) (Table 2). Three Lactobacillus groups were commonlydetected during all the ripening stages: L. plantarum (peaks207 and 208), L. curvatus/L. coryniformis (peak 213), and L.paracasei/L. rhamnosus/L. brevis (peak 218). Similar quantities

of the three Lactobacillus groups were detected during earlyripening, and the signal for the L. plantarum group was alwaysthe most intense signal. The remaining groups produced sim-ilar signals during the mid-ripening period, but in the finalproduct the intensity of the signal of the L. paracasei/L. rham-nosus/L. brevis group increased compared to that of the L.curvatus/L. coryniformis group. Other minor peaks (peaks 191,194, and 201) were also detected in the mature cheese. Peak191 could be assigned to Lactobacillus paraplantarum, but theother two minor peaks could not be identified using our data-base.

DISCUSSION

Currently, the microbiological diversity in cheese is of inter-est, and it has been the subject of many studies, which havegenerally used classical culture methods relying on investiga-tion of phenotypic characteristics. Very little is known aboutthe biochemistry or microbial content of a great many tradi-tional Spanish cheeses (22). Nevertheless, such informationabout the cheese-making process could benefit the growth oflocal industries and the marketing of their traditional products,together with improving the hygienic safety of the final prod-uct, thus avoiding spoilage of raw materials.

We studied the microbial communities of Cueva de la Ma-gaha cheese, monitoring them throughout their 8 months of

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ripening using a combination of classical and molecular tech-niques with the intention of minimizing the biases inherent inboth culture-dependent and culture-independent methods.The concentration of microorganisms in Cueva de la Magahacheese was high, ranging from 4.6 to 7.98 log CFU/g, valueswhich correspond to those usually found in cheeses duringripening (4, 20, 39, 42). During the first week the LAB levelswere 107 CFU/g, and they increased by 1 logarithmic unitduring the mid-ripening period to finally stabilize once more at107 CFU/g. Low levels of enterococci were isolated during theinitial ripening period but not thereafter. The presence of thisbacterial group in cheese seems to vary depending upon thetype of cheese, and this presence has been associated withthe use of raw milk (11, 33, 38) and also the season when thecheese is made. As far as non-LAB populations are concerned,no Enterobacteriaceae were detected and 104 CFU/g of staph-ylococci were found in the final product.

A collection of bacterial isolates obtained from cheese dur-ing the ripening period was used as the starting material forthis study. Biochemical characterization, although useful forgeneral identification purposes, is less able to distinguish be-tween individual strains; thus, we used molecular techniques,which are powerful tools for fingerprinting the specific DNA

patterns of a single strain. PCR-based DNA fingerprintingmethods using arbitrary primers, such as arbitrary primer PCRand RAPD-PCR, have been used for studying genomic DNApolymorphisms of LAB and thus for distinguishing betweendifferent strains in a population. The use of RAPD to investi-gate the diversity of the isolates from the cheese allowed us todistinguish 51 different genotypes among the strains isolated(mainly lactobacilli), which were present at various percent-ages throughout the ripening period. These genotypes weresubsequently identified by 16S rRNA sequencing or species-specific PCR amplification. Overall, the strains present at thebeginning of ripening were different from those present at theend, although a few (two or three strains of each Lactobacillusspecies) persisted throughout the process. Changes in environ-mental parameters, such as humidity, salt concentration, pH(pH 5.44 at the beginning of the process to pH 5.41 at the endof ripening), and the presence of proteins, fatty acids, freeamino acids, and other nutrients, could well explain the suc-cession of strains observed, a succession that has been reportedfor other cheeses, such as Cheddar cheese (20). When weanalyzed our results, we found that most of the genotypesappeared either at the beginning (26 genotypes) or at the endof ripening (19 genotypes not detected previously plus 7 geno-

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types also found at the beginning or at the mid-ripening stage).After 17 weeks of ripening, only six new genotypes were de-tected compared to the genotypes found after the first week,three of which reappeared during week 34. In any case, lacto-bacilli predominated throughout the ripening period, and only0.05% of the strains isolated from the first samples were en-terococci. At the end of the ripening period, 11% of the iso-lates were staphylococci, which may be considered a normalconsequence of the lower humidity of the cheese at this stage,given the higher tolerance of Staphylococcus spp. to dryness.Furthermore, Kocuria and Corynebacterium each accounted for1% of the total strains isolated during this period. Brine isoften a source of contamination of cheese by salt-tolerantmicroorganisms, such as staphylococci, micrococci, entero-cocci, and corynebacteria.

During the early ripening period, the predominant species wereL. brevis (38.97%), L. plantarum (34%), and L. paracasei (25.8%),while during late ripening the predominant strains were L. para-casei (55.7%) and L. brevis (18.5%). We detected high intraspe-cific biodiversity in L. paracasei from mid-ripening to the end ofripening, although L. plantarum and L. brevis showed the highestintraspecific biodiversity at first. L. plantarum could be consideredone of the predominant species in the first week of ripening, butthe L. plantarum populations then declined in favor of L. para-casei. Sanchez et al. (43) reported similar results for isolates ob-tained from several farmhouse Manchego cheeses, and Zarate etal. (47) reported that in Tenerife goat’s milk cheeses there werehigh proportions of L. plantarum in 2-day-old cheese but that the

count decreased during ripening, whereas L. paracasei waspresent initially at low densities but the concentration increasedduring ripening so that this species became the dominant Lacto-bacillus species in 60-day-old cheeses. In other Spanish freshcheeses, however, L. plantarum was the main Lactobacillus spe-cies in the mature cheese (7). These differences could easily beattributed to the distinct microbial compositions of these cheeses,because the activity, growth, and survival of a species are deter-mined mainly by the other microorganisms present in its environ-ment (23). In this respect the microbiota of any particular cheeseis determined by the source of the milk, the manufacturing pro-cess, and the hygienic practices observed during milking, cheesemaking, and ripening (7).

Cheeses, especially farmhouse style cheeses, can be consid-ered natural ecosystems. It has been proved that some micro-organisms in natural ecosystems are extremely difficult to cul-ture (24). Thus, in addition to culture-dependent methods, wedirectly applied molecular techniques to total cheese DNA inorder to obtain a more complete understanding of the ecologyof Cueva de la Magaha cheese. Since the results are simple tointerpret and mistakes due to problems related to growth fail-ure in culture media are avoided, dominant strains duringfermentation can be easily detected and identified in this way.In this work we used TTGE fingerprinting to monitor thepopulation dynamics during cheese ripening, and to circum-vent the biases inherent in subjective interpretation, we con-firmed the species assignments of the bands by direct sequenc-ing. With the two specific primer pairs for the V3 region of the16S rRNA gene used in these TTGE studies, we obtaineddifferent band intensities depending on which pair was used,and the best results were obtained with HDA1-gc and HDA2.Nevertheless, all the profiles obtained by TTGE allowed us todistinguish between the main bacterial groups; L. lactis and S.thermophilus were the primary species detected, and L. plan-tarum and L. brevis were present at lower levels. It is worthmentioning that L. lactis and S. thermophilus were not detectedby direct isolation. Our failure to recover these bacteria, whichare generally ubiquitous in cheese, might be explained by theirinability to grow on solid media because they were eitherstressed or in a viable but noncultivable (VBNC) state. In fact,their presence was established by the strategic use of the cul-ture-independent method TTGE. During ripening the increas-ing number of TTGE bands revealed an increase in diversity.For the final product nine bands appeared, corresponding to L.plantarum, L. brevis, L. coryniformis, L. curvatus, L. paracasei,L. lactis, S. thermophilus, and S. equorum. It is quite surprisingthat the band corresponding to L. plantarum was very welldefined in the final product, while this species was not isolatedat this time. High levels of L. paracasei, on the other hand,were isolated from all the samples, while this species was de-tected by TTGE at the end of ripening only as a very weakband. So, although in general our findings suggest that TTGEanalysis can provide better results for species present at highconcentrations, amplification results may vary depending onthe efficacy of DNA extraction, which depends in turn on thebacterial species and food matrices (1, 40). Alternatively, tar-get competition by primers during PCR may occur, as sug-gested by Heuer and Smalla (27). Accordingly, Muyzer et al.(36) and Heuer and Smalla (27) reported that species repre-senting less that 1% of the total community would not be

FIG. 2. TTGE of PCR-amplified 16S rRNA genes in Cueva de laMagaha cheese samples after 1, 17, and 34 weeks of ripening. Bands 1and 2, L. plantarum; band 3, S. equorum; band 4, L. brevis; band 5, L.coryniformis; band 6, L. curvatus; band 7, L. lactis; band 8, S. thermophi-lus; band 9, L. paracasei.



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visible in denaturing gradient gel electrophoresis profiles of themicrobial community, and Ogier et al. (37) indicated thatTTGE analysis results reflect only the most abundant mi-crobes. To obviate this problem, we considered the possibilitythat a preenrichment step before DNA extraction might helpus to recover less dominant microorganisms, as suggested byGiraffa and Neviani (24). Thus, we carried out TTGE analysesof DNA extracted after we enriched the cheese dilutions inbroth in order to supplement our findings, in a semiquantita-tive way, for the main culturable microbial groups. A compar-ison of TTGE profiles obtained for the total DNA of thecheese community and for DNA extracted from enrichmentcultures revealed some important differences. For example, E.faecalis and E. faecium, which could not be isolated from ma-ture cheese, were detected in enriched MRS and Rothe broth

media. We cannot ignore the possibility that these bacteriamight have been present in a VBNC state, which would havemade it difficult to isolate them directly in a selective medium.In fact, a VBNC state has been induced experimentally in E.faecalis (14), and under suitable conditions the cells are able torecover from the debilitated state. This may well explain whywe did not isolate enterococci from selective plates (KFA),although growth was observed in a general medium (MRSbroth) and even in selective broth (Rothe broth). This was alsotrue for L. curvatus, which was detected at titers of 104 and 105

CFU/g in Rothe broth and MRS broth, respectively, only whenthe culture-independent technique was used. Similarly, L.plantarum was determined to be present at a level of 106

CFU/g by QPCR, whereas it could not be isolated from maturecheese.

FIG. 3. Estimation of relative abundance of bacterial species obtained from enrichment cultures in MRS broth and Rothe broth for mature cheese.(A) TTGE fingerprinting of PCR-amplified 16S rRNA gene fragments of DNA extracted from enrichment cultures. The numbers indicate the bandnumbers for total community DNA, as follows: band 1, L. plantarum; band 3, S. equorum; band 4, L. brevis; band 5, L. coryniformis; band 6, L. curvatus;band 7, L. lactis; band 9, L. paracasei. (B) Quantification of representative bacterial species by specific PCR. The lane numbers at the top of each gelindicate enrichment of different dilutions (10�1 to 10�7) in broth media. Lane M contained a 1-kbp DNA ladder (Biotools B&M Labs, Madrid, Spain),lane M1 contained a 100-bp DNA ladder (Biotools B&M Labs, Madrid, Spain), and lane Q contained genomic DNAs from cheese.



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FIG. 4. LH-PCR of lactobacilli present in Cueva de la Magaha cheese. (A) One week of ripening. (B) Seventeen weeks of ripening.(C) Thirty-four weeks of ripening.

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In the same way, L. lactis, although not detected by directisolation on agar, was determined to be present by QPCR-TTGE at titers of 104 CFU/g in mature cheese, thus confirmingthe results obtained by temperature gradient gel electrophore-sis analysis. Nevertheless, the approximate levels of lactococcifound are much lower than the levels expected as the levels ofthese bacteria generally are more than 108 CFU ml�1 duringmilk curding. Our interpretation of this finding is that L. lactismay be especially susceptible to the environmental conditionsformed by the microbiota in this cheese (for instance, aceticacid produced by heterofermentative lactobacilli, which wouldkill most of the lactococci or else drive them into a VBNCstate).

Finally, our monitoring of the Lactobacillus composition byLH-PCR revealed the increasing predominance of L. plantarumthroughout ripening, followed by abundant representation of theL. rhamnosus/L. paracasei/L. brevis group. Apart from this, L.curvatus/L. coryniformis, L. paraplantarum, and two other uniden-tified lactobacilli were found in the mature cheeses.

This study provides a complete view of the composition ofthe microbial community in Cueva de la Magaha cheese ob-tained using a polyphasic approach combining culture-depen-dent and culture-independent methods. Such a combined ap-proach was vital for obtaining a realistic view of the existingmicrobial ecosystem because of the proven variations betweenthe results obtained by the two types of methods. Our resultssuggest that the failure of culture-dependent methods may beexplained by overshadowing of minor populations by the pre-dominant populations and also by the transition to a VBNCstate, probably caused by environmental stress.

ACKNOWLEDGMENTS

This work was supported by research project CAL02-078 of theInstituto Nacional de Investigacion y Tecnologıa Agraria y Alimentaria(INIA, Ministerio de Ciencia y Tecnologıa) and by the Research Planof the Junta de Andalucıa (research group CVI 0160). A. M. Martın-Platero had a grant from the Consejerıa de Innovacion, Ciencia yEmpresa (Junta de Andalucıa).

We thank the Cueva de la Magaha cheese factory for supplying thesamples and J. Purswani for her assistance with the manuscript. Wealso thank J. Trout for reviewing the English text.

REFERENCES

1. Abriouel, H., N. Ben Omar, R. Lucas, M. Martınez-Canamero, S. Keleke,and A. Galvez. 2006. Culture-independent analysis of the microbial compo-sition of the African traditional fermented foods poto poto and degue byusing three different DNA extraction methods. Int. J. Food Microbiol. 111:228–233.

2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

3. Andrighetto, C., G. Marcazzan, and A. Lombardi. 2004. Use of RAPD-PCRand TTGE for the evaluation of biodiversity of whey cultures for GranaPadano cheese. Lett. Appl. Microbiol. 38:400–405.

4. Antonsson, M., G. Molin, and Y. Ardo. 2003. Lactobacillus strains isolatedfrom Danbo cheese as adjunct cultures in a cheese model system. Int. J. FoodMicrobiol. 85:159–169.

5. Baruzzi, F., M. Morea, A. Matarante, and P. S. Cocconcelli. 2000. Changesin the Lactobacillus community during Ricotta forte cheese natural fermen-tation. J. Appl. Microbiol. 89:807–814.

6. Ben Omar, N., and F. Ampe. 2000. Microbial community dynamics duringproduction of the Mexican fermented maize dough pozol. Appl. Environ.Microbiol. 66:3664–3673.

7. Beresford, T. P., N. A. Fitzsimons, N. L. Brennan, and T. M. Cogan. 2001.Recent advances in cheese microbiology. Int. Dairy J. 11:259–274.

8. Bulut, C., H. Gunes, B. Okuklu, S. Harsa, S. Kilic, H. S. Coban, and A. F.Yenidunya. 2005. Homofermentative lactic acid bacteria of a traditionalcheese, Comlek peyniri from Cappadocia region. J. Dairy Res. 72:19–24.

9. Callon, C., L. Millet, and M. C. Montel. 2004. Diversity of lactic acid bacteriaisolated from AOC Salers cheese. J. Dairy Res. 71:231–244.

10. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2000. Development ofa rapid method for the identification of Lactobacillus spp. isolated fromnaturally fermented Italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis. Lett. Appl. Microbiol. 30:126–129.

11. Coppola, R., M. Nanni, M. Succi, A. Sorrentino, M. Iorizzo, C. Chiavari, andL. Grazia. 2000. Microbiological characteristics of Parmigiano Reggianocheese during the cheese making and the first months of the ripening. Lait80:479–490.

12. Corroler, D., N. Desmasures, and M. Gueguen. 1999. Correlation betweenpolymerase chain reaction analysis of the histidine biosynthesis operon,randomly amplified polymorphic DNA analysis and phenotypic character-ization of dairy Lactococcus isolates. Appl. Microbiol. Biotechnol. 51:91–99.

13. Deasy, B. M., M. C. Rea, G. F. Fitzgerald, T. M. Cogan, and T. P. Beresford.2000. A rapid PCR based method to distinguish between Lactococcus andEnterococcus. Syst. Appl. Microbiol. 23:510–522.

14. Del Mar Lleo, M., S. Pierobon, M. C. Tafi, C. Signoretto, and P. Canepari.2000. mRNA detection by reverse transcription-PCR for monitoring viabilityover time in an Enterococcus faecalis viable but nonculturable populationmaintained in a laboratory microcosm. Appl. Environ. Microbiol. 66:4564–4567.

15. Demarigny, Y., E. Beuvier, A. Dasen, and G. Duboz. 1996. Influence of rawmilk microflora on the characteristics of Swiss-type cheeses. I. Evolution ofmicroflora during ripening and characterization of facultatively heterofer-mentative lactobacilli. Lait 76:371–387.

16. De Vuyst, L., and E. J. Vandamme. 1994. Antimicrobial potential of lacticacid bacteria, p. 91–142. In L. De Vuyst and E. J. Vandamme (ed.), Bacte-riocins of lactic acid bacteria: microbiology, genetics and applications.Blackie Academic and Professional, London, United Kingdom.

17. Dubernet, S., N. Desmasures, and M. Gueguen. 2002. A PCR-based methodfor identification of lactobacilli at the genus level. FEMS Microbiol. Lett.214:271–275.

18. Duthoit, F., J. J. Godon, and M. C. Montel. 2003. Bacterial communitydynamics during production of registered designation of origin Salers cheeseas evaluated by 16S rRNA gene single-strand conformation polymorphismanalysis. Appl. Environ. Microbiol. 69:3840–3848.

19. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopep-tide resistance genotypes and identification to the species level of clinicallyrelevant enterococci by PCR. J. Clin. Microbiol. 33:24–27.

20. Fitzsimons, N. A., T. M. Cogan, S. Condon, and T. Beresford. 2001. Spatialand temporal distribution of non-starter lactic acid bacteria in Cheddarcheese. J. Appl. Microbiol. 90:600–608.

21. Fox, P. F., P. L. H. McSweeney, and C. M. Lynch. 1998. Significance ofnonstarter lactic acid bacteria in Cheddar cheese. Aust. J. Dairy Technol.53:83–89.

22. Freitas, C., and F. X. Malcata. 2000. Microbiology and biochemistry ofcheeses with appelation d’origine protegee and manufactured in the Iberianpeninsula from ovine and caprine milks. J. Dairy Sci. 83:584–602.

23. Giraffa, G. 2004. Studying the dynamics of microbial populations during foodfermentation. FEMS Microbiol. Rev. 28:251–260.

24. Giraffa, G., and E. Neviani. 2001. DNA-based, culture-independent strate-gies for evaluating microbial communities in food-associated ecosystems. Int.J. Food Microbiol. 67:19–34.

25. Giraffa, G., and L. Rossetti. 2004. Monitoring of the bacterial composition ofdairy starter cultures by RAPD-PCR. FEMS Microbiol. Lett. 237:133–138.

26. Guarneri, T., L. Rossetti, and G. Giraffa. 2001. Rapid identification ofLactobacillus brevis using the polymerase chain reaction. Lett. Appl. Micro-biol. 33:377–381.

27. Heuer, H., and K. Smalla. 1997. Application of denaturing gradient gelelectrophoresis (DGGE) and temperature gradient gel electrophoresis(TGGE) for studying soil microbial communities, p. 353–373. In J. D. vanElsas, J. T. Trevors, and E. M. H. Wellington (ed.), Modern soil microbiol-ogy. Marcel Dekker, New York, NY.

28. Klein, G., A. Pack, C. Bonaparte, and G. Reuter. 1998. Taxonomy andphysiology of probiotic lactic acid bacteria. Int. J. Food Microbiol. 41:103–125.

29. Knijff, E., F. Dellaglio, A. Lombardi, C. Andrighetto, and S. Torriani. 2001.Rapid identification of Enterococcus durans and Enterococcus hirae by PCRwith primers targeted to the ddl genes. J. Microbiol. Methods 47:35–40.

30. Lazzi, C., L. Rossetti, M. Zago, E. Neviani, and G. Giraffa. 2004. Evaluationof bacterial communities belonging to natural whey starters for GranaPadano cheese by length heterogeneity-PCR. J. Appl. Microbiol. 96:481–490.

31. Lick, S., K. Drescher, and K. J. Heller. 2001. Survival of Lactobacillus delbrueckiisubsp. bulgaricus and Streptococcus thermophilus in the terminal ileum offistulated Gottingen minipigs. Appl. Environ. Microbiol. 67:4137–4143.

32. Lopez, S., and B. Mayo. 1997. Identification and characterization of homo-fermentative mesophilic Lactobacillus strains isolated from artisan starter-free cheeses. Lett. Appl. Microbiol. 25:233–238.

33. Marino, M., M. Maifreni, and G. Rondinini. 2003. Microbiological charac-



.asm.org/

Dow

nloaded from

http://aem.asm.org/

terization of artisanal Montasio cheese: analysis of its indigenous lactic acidbacteria. FEMS Microbiol. Lett. 229:133–140.

34. Martın-Platero, A. M., E. Valdivia, M. Maqueda, and M. Martınez-Bueno.2007. Fast, convenient and economical method for isolating genomic DNAfrom lactic-acid bacteria using a modification of the “protein salting-out”procedure. Anal. Biochem. 366:102–104.

35. Medina, R., M. Katz, S. Gonzalez, and G. Oliver. 2001. Characterization ofthe lactic acid bacteria in ewe’s milk and cheese from northwest Argentina.J. Food Prot. 64:559–563.

36. Muyzer, G. A., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling ofcomplex microbial populations by denaturing gradient gel electrophoresisanalysis of polymerase chain reaction-amplified genes coding for 16S rRNA.Appl. Environ. Microbiol. 59:695–700.

37. Ogier, J. C., O. Son, A. Gruss, P. Tailliez, and A. Delacroix-Buchet. 2002.Identification of the bacterial microflora in dairy products by temporal tem-perature gradient gel electrophoresis. Appl. Environ. Microbiol. 68:3691–3701.

38. Olarte, C., S. Sanz, E. Gonzalez-Fandos, and P. Torre. 2000. The effect of acommercial starter culture addition on the ripening of an artisanal goat’scheese (Cameros cheese). J. Appl. Microbiol. 88:421–429.

39. Østlie, H. M., L. Eliassen, A. Florvaag, and S. Skeie. 2004. Phenotypic andPCR-based characterization of the microflora in Norvegia cheese duringripening. Int. J. Food Microbiol. 94:287–299.

40. Perez-Pulido, R., N. Ben Omar, H. Abriouel, R. Lucas-Lopez, M. Martınez-Canamero, and A. Galvez. 2005. Microbiological study of lactic acid fermen-

tation of caper berries by molecular and culture-dependent methods. Appl.Environ. Microbiol. 71:7872–7879.

41. Poznanski, E., A. Cavazza, F. Cappa, and P. S. Cocconcelli. 2004. Indigenousraw milk microbiota influences the bacterial development in traditionalcheese from an alpine natural park. Int. J. Food Microbiol. 92:141–151.

42. Randazzo, C. L., S. Torriani, A. D. L. Akkermans, W. M. de Vos, and E. E.Vaughan. 2002. Diversity, dynamics, and activity of bacterial communitiesduring production of an artisanal Sicilian cheese as evaluated by 16S rRNAanalysis. Appl. Environ. Microbiol. 68:1882–1892.

43. Sanchez, I., S. Sesena, J. M. Poveda, L. Cabezas, and L. Palop. 2006. Geneticdiversity, dynamics, and activity of Lactobacillus community involved in tra-ditional processing of artisanal Manchego cheese. Int. J. Food Microbiol.107:265–273.

44. Sanchez, J. I., L. Rossetti, B. Martınez, A. Rodrıguez, and G. Giraffa. 2006.Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations.J. Microbiol. Methods 65:268–277.

45. Torriani, S., G. E. Felis, and F. Dellaglio. 2001. Differentiation of Lactoba-cillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequenceanalysis and multiplex PCR assay with recA gene-derived primers. Appl.Environ. Microbiol. 67:3450–3454.

46. Ward, L. J. H., and M. J. Timmins. 1999. Differentiation of Lactobacilluscasei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerasechain reaction. Lett. Appl. Microbiol. 29:90–92.

47. Zarate, V., F. Belda, C. Perez, and E. Cardell. 1997. Changes in the microbialflora of Tenerife goat’s milk cheese during ripening. Int. Dairy J. 7:635–641.



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