pcr–dgge fingerprints of microbial succession during a manufacture of traditional water buffalo...

PCR–DGGE fingerprints of microbial succession duringa manufacture of traditional water buffalo mozzarella cheese

D. Ercolini, G. Mauriello, G. Blaiotta, G. Moschetti and S. CoppolaDipartimento di Scienza degli Alimenti, Sezione di Microbiologia Agraria, Alimentare e Ambientale e di Igiene, Universita degli Studi di

Napoli �Federico II�, Portici, Italy

2003/0417: received 20 May 2003, revised 4 September 2003 and accepted 3 October 2003

ABSTRACT

D. ERCOLINI , G . MAURIELLO, G. BLA IOTTA, G. MOSCHETTI AND S. COPPOLA. 2003.

Aims: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial

diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the

natural starter to the achievement of the final product.

Methods and Results: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening

and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly

extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media

generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase

chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) experiments. The analysis of the DGGE

profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter

addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were

calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of

specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30�C showed high

viable counts and the highest diversity in species indicating their importance in the cheese making, which had not

been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile

suggesting to be effective in manufacture.

Conclusions: The PCR–DGGE analysis showed that in premium quality manufacture the NWC used as starter

had a strong influence on the microflora responsible for process development.

Significance and Impact of the Study: The molecular approach appeared to be valid as a tool to control process

development, starter effectiveness and product identity as well as to rank cheese quality.

Keywords: microbial diversity, natural whey culture, PCR–DGGE analysis, product identity, quality control,

starter effectiveness, tracing system, water buffalo mozzarella cheese.

INTRODUCTION

Mozzarella cheese is perhaps the most popular non-ripened

cheese in the world. Traditional mozzarella is mainly

produced in Italy although it is widely exported and also

industrially produced in other countries. Water buffalo

mozzarella cheese is a high moisture (55–62%) and high fat

in dry matter (>45%) cheese; it is characterized by a soft

body and a juicy appearance and by a pleasant, fresh, sour

and slightly nutty flavour. Water buffalo mozzarella cheese

from Campania (�Mozzarella di Bufala Campana�) received

the European certification Product of Designated Origin

Correspondence to: Dr Danilo Ercolini, Dipartimento di Scienza degli Alimenti,

Sezione di Microbiologia, Universita di Napoli �Federico II�, 80055 Portici, Italy

(e-mail: [email protected]).

ª 2003 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 96, 263–270 doi:10.1046/j.1365-2672.2003.02146.x

(DOP, EEC Regulation no. 1107 12th June 1996) and it is

regarded as a typical product of southern Italy.

The manufacture has been described in detail in previous

works (Coppola et al. 1988, 1990). Briefly, the cheese is

made from whole raw water buffalo milk by adding a natural

whey culture (NWC, from the previous day manufacture) as

starter. The raw milk is heated at 37�C, then rennet and

NWC are added. After a curd-ripening phase (4Æ0–4Æ5 h at

35–37�C), which occurs under whey, the optimal pH (4Æ9–

5Æ1) is reached and the drained curd is stretched in hot water

(90–95�C). The elastic product formed is then hand-

moulded in order to get the final typical round shape with

a hand-cut on one side, which gives it the name mozzarella

(from the Italian �mozzare� for hand-cutting).

The specific characteristics of the final product mainly

arise from the raw materials employed, the agro-ecosystem

of the area of production and the traditional technology of

manufacture. The traditional mozzarella is made from raw

water buffalo milk and the microflora occurring in such

complex environment is certainly one of the parameters

affecting the dairy manufacture. In the traditional proce-

dure the NWC is a natural microbial culture occurring in

the whey drained after curd ripening. Part of this whey is

stored and employed as starter in the manufacture of the

next day. In previous studies this starter has been

characterized using both traditional and molecular proce-

dures (Coppola et al. 1988, 1990; Ercolini et al. 2001b) and

defined as complex consortium of micro-organisms of great

importance for the quality of the traditional product. The

traditional technologies of cheese making are the most

difficult to control, especially when the cheese is produced

from raw milk or by adding natural starters. Therefore, it

would be interesting to develop new methods of quality

control capable of supporting a standardization of the

process for good quality products, while preserving their

typical traits. Tracing processes and traditional products

identity would be furthermore helpful for the protection of

territory claims and for consumer protection against

frauds.

A molecular evaluation of the microbiota of several

mozzarella cheeses has been reported showing the potential

of a polymerase chain reaction–denaturing gradient gel

electrophoresis (PCR–DGGE) approach in discriminating

different qualities of cheese (Coppola et al. 2001).

In this study the succession of microbial populations

during the whole process of a premium quality traditional

water buffalo mozzarella cheese manufacture has been

monitored by cultivation coupled with molecular methods.

The aim was to monitor the process and the starter

effectiveness by collecting a series of fingerprints of the

microbial diversity occurring at different steps to control the

contribution of lactic acid bacteria (LAB) to the achievement

of the final product.

MATERIALS AND METHODS

Dairy samples

The samples were collected from a dairy producing top

quality traditional water buffalo mozzarella cheese PDO

(protected designation origin), located in Campania region,

southern Italy. Samples of raw milk before and after the

starter addition, starter (NWC), curd at the end of the

ripening, whey after draining, stretched curd and final

mozzarella cheese were aseptically collected, cooled at 4�C,

and analysed within 6 h.

Microbial enumeration and collectionof cells in bulk

Serial dilutions of each sample in quarter strength Ringer’s

solution (Oxoid) were used to inoculate plates of: MRS

agar; Rogosa agar and M17 agar (Oxoid) as culture media

widely employed to cultivate LAB. Two series of agar

plates were inoculated and incubated at 30 and 44�C for

48 h. Rogosa agar plates were incubated anaerobically

using an Anaerogen kit (Oxoid). Portions (0Æ1 ml) of

appropriate dilutions were spread plated in triplicate.

Colonies were counted and the results were calculated as

the means of three determinations. After the counts, the

plates were used for bulk formation as previously des-

cribed (Ercolini et al. 2001b). For each dilution, all the

colonies present on the surface of the plate were suspen-

ded in a suitable volume of quarter strength Ringer’s

solution to reach 1 unit of optical density (600 nm),

harvested with a sterile pipette and stored by freezing at

)20�C (Ercolini et al. 2001b). When necessary, 1 ml of the

bulk was used for DNA extraction as described below.

The bulk analysis was performed only on the samples of

initial raw water buffalo milk (M), NWC, curd at the end

of ripening (C) and final product (FP).

DNA extraction

Total DNA extraction from the dairy samples was conducted

as previously described (Ercolini et al. 2001b). The dairy

samples were fivefold diluted in TE (Tris–EDTA) buffer and

the protocol was applied to 1 ml of suspension. Moreover, the

protocol was also applied to aliquots of 1 ml bulk suspension

of colonies from the plates (Ercolini et al. 2001b). The

protocol described by the manufacturer of the Wizard DNA

purification kit (Promega, Madison, WI, USA) was applied as

follows: 1 ml of sample was centrifuged at 17 000 g for 5 min

at 4�C and the resulting pellet was resuspended in 100 ll of

TE buffer (100 mmol l)1 Tris, 10 mmol l)1 EDTA); then

160 ll of 0Æ5 mol l)1 EDTA/nuclei lysis solution (Wizard

DNA purification kit; Promega) in 1/4Æ16 ratio, 5 ll of

RNAse (10 mg ml)1; Sigma) and 20 ll of pronase E

264 D. ERCOLINI ET AL.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 263–270, doi:10.1046/j.1365-2672.2003.02146.x

(20 mg ml)1; Sigma) were added, and the mixture was

incubated for 60 min at 37�C. After incubation, 1 volume of

ammonium acetate 5 mol l)1 was added to the sample that was

then centrifuged at 17 000 g for 5 min at 4�C. The superna-

tant was precipitated with 0Æ7 volume of isopropanol and

centrifuged at 29 000 g for 5 min. Finally, the pellet was

dried and resuspended in 50 ll of DNA rehydration

solution by incubation at 55�C for 45 min.

PCR–DGGE analysis

Primers spanning the 200-bp V3 region of the 16S ribosomal

DNA of Escherichia coli were used in PCR amplification as

previously described (Ercolini et al. 2001a). A GC-clamp was

added to the forward primer, according to Muyzer et al.

(1993). Amplification was performed in a programmable

heating incubator (MJ Research Inc., Waltham, MA, USA).

Each mixture (final volume, 25 ll) contained 20 ng of tem-

plate DNA, each primer at a concentration of 0Æ2 lmol l)1,

each deoxynucleoside triphosphate at a concentration of

0Æ25 mmol l)1, 2Æ5 mmol l)1 MgCl2, 2Æ5 ll of 10x PCR

buffer and 2Æ5 U of Taq polymerase (Invitrogen). Template

DNA was denatured for 5 min at 94�C. A �touchdown� PCR

was performed (Muyzer et al. 1993) to increase the specificity

of amplification and to avoid the formation of spurious

by-products. PCR products were analysed by DGGE using a

Bio-Rad Dcode apparatus (Bio-Rad, Hercules, CA, USA).

Parallel electrophoresis experiments were performed at 60�Cby using gels containing a 25–50% urea-formamide denatur-

ing gradient [100% corresponded to 7 mol l)1 urea and 40%

(w/v) formamide] increasing in the direction of electrophor-

esis. After the electrophoresis the gels were stained in

ethidium bromide solution for 5 min, washed in distilled

water for 15 min and observed. Bands were automatically

detected by using the software Phoretic 1 advanced version

3Æ01 (Phoretix International Limited, Newcastle upon Tyne,

UK). The PCR–DGGE protocol was applied to the DNA

directly extracted from the dairy samples as well as from

DNA extracted from colonies collected in bulk.

Indexes

Simple mathematical indexes were calculated for each

fingerprint arising from PCR–DGGE analysis of the bulk

suspension from countable plates of appropriate media. The

bands considered in the analysis were only the ones

automatically detected by the software Phoretic 1.

Indexes of biodiversity:

IB1 ¼ n=nM

IB2 ¼ n=nB

IB3 ¼ IB1=nB

The biodiversity indexes (IB1, IB2 and IB3) were meant to

express the degree of microbial complexity for each bulk

analysed. The discriminative value was the number of bands in

the profile compared with the top of biodiversity detected (IB1)

or corrected for the microbial diversity relative to the specific

medium to which the profile belonged to (IB2 and IB3).

Index of similarity with the curd profile (Nei and Li

1979):IC1 ¼ 2nCs=n þ nC

where n is the number of DGGE bands in the profile; nM is

the number of bands counted in the bulk profile with the

maximum number of bands; nB is the total number of bands

counted on the specific medium the profiles refer to; nCs is

the number of bands of the profile also detected in the

profile of the curd from the same medium; nC is the number

of bands in the profile of the curd from the same medium. In

calculating the nB value, the bands migrating the same

distance in the gel were counted only once.

RESULTS

The results of viable counts are summarized in Table 1.

The raw water buffalo milk was found to be rich of

LAB. The curd and the NWC used as starter were found

to have a strong concentration of both thermophilic and

mesophilic micro-organisms; the whey collected after curd

Table 1 Viable counts of bacterial groups for different samples collected during water buffalo mozzarella cheese manufacture

Sample

Log CFU g)1 or ml)1 (S.DS.D.)

M17 at 30�C M17 at 44�C MRS at 30�C MRS at 44�C Rogosa at 30�C

Raw water buffalo milk 6Æ30 (0Æ22) 4Æ45 (0Æ20) 6Æ23 (0Æ20) 4Æ48 (0Æ16) 4Æ88 (0Æ21)

Milk after starter addition 6Æ39 (0Æ12) 4Æ64 (0Æ22) 6Æ28 (0Æ22) 4Æ63 (0Æ12) 4Æ69 (0Æ13)

Natural whey culture 7Æ78 (0Æ15) 6Æ34 (0Æ17) 7Æ49 (0Æ19) 6Æ25 (0Æ11) 6Æ50 (0Æ23)

Curd 8Æ08 (0Æ28) 6Æ32 (0Æ15) 8Æ17 (0Æ25) 6Æ20 (0Æ14) 7Æ04 (0Æ28)

Whey from curd draining 7Æ15 (0Æ24) 5Æ18 (0Æ18) 6Æ71 (0Æ17) 5Æ87 (0Æ24) 4Æ07 (0Æ27)

Stretched curd 5Æ36 (0Æ13) 2Æ39 (0Æ25) 5Æ76 (0Æ23) 3Æ39 (0Æ24) 3Æ82 (0Æ25)

Final product 4Æ04 (0Æ13) 2Æ58 (0Æ21) 3Æ78 (0Æ20) 2Æ43 (0Æ24) 2Æ84 (0Æ19)

The data are the mean values based on three replicates. The values in parentheses are standard deviations.

MICROBIAL SUCCESSION IN MOZZARELLA CHEESE 265


ripening had a concentration of about 1 log lower than the

curd, probably as result of cell retention in the curd

matrix. The microflora counted in the process significantly

decreased after the hot stretching step and the brine

soaking. On the basis of the counts on M17 agar, both

mesophilic and thermophilic lactic streptococci were

affected by the stretching, which displayed a significant

decrease in counts. Rogosa agar after incubation at 44�Cdid not give countable plates and thus was not considered

for the bulk analysis (data not shown).

Figure 1 shows the fingerprints obtained after the PCR–

DGGE analysis of the DNA directly extracted from the

dairy samples. The raw water buffalo milk profile consisted

of at least 15 detectable bands (Fig. 1; lane 1); all the other

samples displayed a fingerprint identical to the NWC profile

(lanes 3–6).

The analysis of the microbial diversity was also extended

to the cultivable community and the results are displayed

in Fig. 2a–e. Each panel shows profiles of the countable

dilutions from specific culture media. Only the countable

plates were considered for the analysis because only those

dilutions would have been taken into account in a

traditional isolation procedure. Simple indexes were calcu-

lated for each profile in order to achieve an objective

interpretation of the results in terms of degree of

biodiversity and technological importance of the sample.

Two sets of indexes, described in the �Materials and

methods� section, were taken into account. The biodiversity

indexes (IB1, IB2 and IB3) were meant to express the

degree of microbial complexity for each bulk analysed. Of

course, the discriminative value was the number of bands

in the profile which was considered as compared with the

top of biodiversity detected (IB1) or corrected for the

microbial diversity relative to the specific medium which

the profile belonged to (IB2 and IB3). A Pearson correlation

index (CI) was calculated for IB1 vs IB2, IB1 vs IB3 and

IB2 vs IB3; it was found that the CI was always very close

to 1 (data not shown). As the IB indexes were related, the

use of the three of them could not be useful because they

would express the same degree of diversity. Therefore, the

simplest IB1 was chosen for the interpretation of data. The

IB1 values for all the samples and media are depicted in

Fig. 3. The index is comprised between 0 and 1 and

according to the IB1 values the degree of diversity of each

sample can be arbitrarily divided in three groups: (i) high,

(ii) medium and (iii) low microbial diversity (IB1 > 0Æ7,

0Æ4 < IB1 < 0Æ7 and IB1 < 0Æ4, respectively). As shown in

Fig. 3, dairy samples plated on the same medium showed

about the same degree of complexity except for the samples

from MRS agar at 44�C which gave different IB1 values.

Moreover, the highest diversity was shown by the

mesophilic LAB, giving IB1 values higher than 0Æ7 for all

the samples on MRS and Rogosa at 30�C. The samples of

milk plated onto MRS and M17 and incubated at 44�Calso gave medium values of IB1 indicating that a consid-

erable diversity of thermophilic species was present in the

raw water buffalo milk. In addition, mesophilic streptococci

showed low microbial diversity.

IB1 values were plotted vs IC1 values and the resulting

graph is depicted in Fig. 4. This graph allows the

interpretation of two sets of data at the same time. The

plot is divided in order to highlight the area characterized

by high microbial complexity (IB1 > 0Æ7) and high

similarity with the curd after ripening (IC1 > 0Æ7). As

already observed in Fig. 3, the mesophilic LAB collected

from MRS and Rogosa incubated at 30�C displayed the

highest degree of microbial diversity grouping in the top

part of the plot. However, in the right part of the plot all

the samples with high IC1 values are gathered. Of course,

all the curd samples have IC1 values equal to 1. All the

NWC patterns showed high similarity with the curd

laying in the right part of the plot. Particularly, NWC

bulk from M17 at 44�C showed profiles identical to the

corresponding curd. Among all the other samples, only

the milk sample from M17 at 30�C displayed high

similarity with the curd.

1 2 3 4 5 6

Fig. 1 PCR-DGGE profiles of dairy samples during water buffalo

mozzarella cheese manufacture. Lanes: 1, raw milk; 2, milk after NWC

addition; 3, NWC; 4, curd after ripening; 5, stretched curd; 6, final

product



DISCUSSION

In this study the microbial succession during the manufac-

ture of traditional water buffalo mozzarella cheese was

investigated by PCR–DGGE analysis of the DNA directly

extracted from the dairy samples. Moreover, LAB popula-

tions were also monitored by analysing the PCR–DGGE

profiles of bulk colonies recovered from the countable plates

of three different media incubated at 37 and 44�C.

The PCR–DGGE analysis of mixed bacterial popula-

tions is widely used in environmental microbiology (Muy-

zer 1999). The amplification of variable regions of the 16S

rDNA followed by DGGE analysis leads to fingerprints of

the microbial community characterized by a correspon-

dence between bands and microbial species. This approach

has been recently applied to food and food-related

ecosystems where the microbial community was identified

from PCR–DGGE profiles after a direct DNA extraction

from food samples (Ampe et al. 1999; Cocolin et al. 2001;

Ercolini et al. 2001b, 2003; Randazzo et al. 2002). How-

ever, in some of these studies it was demonstrated that the

analysis of the cultivable community can give support to

the analysis of the sole microbial DNA directly extracted

from food (Ercolini et al. 2001b, 2003; Randazzo et al.

2002). In this study, PCR–DGGE fingerprints were

obtained from countable bulk colonies from M17, MRS

and Rogosa agar plates incubated at different temperatures.

This approach was previously explored (Ercolini et al.

2001b, 2003) and it was considered a rapid and useful

support for the PCR–DGGE analysis performed in situ.

Moreover, in previous studies we demonstrated that

statistical analysis of PCR–DGGE profiling results has

good potential in differentiating dairy products (Coppola

et al. 2001; Ercolini et al. 2002) and also in ascertaining the

geographical origin of natural starters (NWC) for mozza-

rella cheese PDO (Mauriello et al. 2003). This was applied

to monitor a mozzarella cheese making process in this

study.

Fig. 2 PCR-DGGE profiles of bulk from

media used for viable counts of LAB. (a) Bulk

on Rogosa agar after incubation at 30�C;

(b) bulk on MRS agar after incubation at

30�C; (c) bulk on MRS agar after incuba-

tion at 44�C; (d) bulk on M17 agar after

incubation at 44�C; (e) bulk on M17 agar after

incubation at 30�C. Lanes: 1, raw water

buffalo milk; 2, NWC; 3, curd after ripening;

4, final product



Analysing the Fig. 1 it is immediately clear that, as

expected, the NWC used as starter had a great influence on

the whole process. In fact, the complex pattern of the raw

water buffalo milk is soon simplified after the starter

addition. Throughout the process, all the samples displayed

a fingerprint identical to the one shown by the NWC.

Noteworthy, the Fig. 1 shows that all the microbial entities

composing the NWC are detected from the starter addition

until the end of the process.

The analysis of the cultivable community provided with

the following data. According to IB1 values, it was shown

that the mesophilic LAB represented the group with the

highest diversity in this process. It has been generally

recognized that the microflora involved in the manufactur-

ing of water buffalo mozzarella cheese mainly consisted of

thermophilic species (Limsowtin et al. 1995; Ottogalli 1998),

while the mesophilic lactobacilli were usually not considered

(Coppola et al. 1988, 1990; Parente et al. 1997). However, in

this case, mesophilic LAB able to grow on Rogosa and MRS

at 30�C appeared to be a significant group. They gave a high

number of CFU and also displayed the highest number of

bands in DGGE analysis of bulk (Fig. 2) resulting in high

values of IB1 indexes (Fig. 3). This is consistent with the

high levels of the mesophilic Lactobacillus fermentum and

L. crispatus in NWC found in a previous study (Ercolini

et al. 2001b).

0 0·2 0·4 0·6 0·8 1

Rogosa at 30°C

MRS at 30°C

MRS at 44°C

M17 at 44°C

M17 at 30°C

IB1

Fig. 3 Bars diagram showing the distribution

of IB1 values of DGGE profiles of bulk from

all the dairy samples collected during the

cheese making. Lines indicate the arbitrary

subgroups according to IB1 values. (j), Milk;

(j), NWC; (() curd; (j), final product

0·1

0·2

0·3

0·4

0·5

0·6

0·7

0·8

0·9

1

0 0·1 0·2 0·3 0·4 0·5 0·6 0·7 0·8 0·9 1IC1

IB1

Curd

Curd

Curd

Curd

Curd

NWC

NWC

NWC

NWC

NWC

Milk

Milk

Milk

Milk

Milk

FP

FP

FP FP

FP

Fig. 4 Scatter plot of the similarity indexes IC1

and IB1 of DGGE profiles of bulk from different

media and growth condition. (d), Rogosa at

30�C; (j), MRS at 30�C; (m), MRS at 44�C;

((), M17 at 44�C; (s), M17 at 30�C



Mesophilic lactic streptococci monitored on M17 incuba-

ted at 30�C showed high viable counts but low microbial

diversity (IB1 values lower than 0Æ4; Fig. 3), suggesting that

only a narrow group of species is involved in the process. By

contrast, Morea et al. (1999) found a significant diversity of

species of both mesophilic and thermophilic streptococci in

mozzarella cheese made from cow milk.

The index IC1 (Nei and Li 1979) was calculated in order

to estimate the similarity of each dairy sample with the curd

after ripening. This index had a technological meaning. For

milk and NWC samples the IC1 values represented an

estimation of the technological importance of that type of

organisms for the process. In fact, the micro-organisms still

present in the curd are considered to be responsible for the

acidification of milk and curd ripening with the consequent

release of compounds important for texture and flavour of

the final product (Moio et al. 1993; Mauriello et al. 2001,

2003). However, for the final product samples, IC1 values

represented the influence of the hot-stretching phase and its

effect on the microflora of the curd. Showing the highest

IC1 values, the NWC profiles contained a significant

number of bands that occurred also in the curd profile.

Hence, the microbial species of the NWC strongly

contribute to the process and, once added to the milk, out

compete the other species arising from milk and environ-

ment of production leading to curd ripening and influencing

the quality of the final product. The competition is mainly

because of the technological conditions such as temperature,

short time of ripening (4 h) and use of raw milk as

substrate. Traditional mozzarella cheese is made from

nonpasteurized milk and this is one of the conditions

recommended by the official rules of production followed

by the PDO water buffalo mozzarella producers. However,

milk profiles showed high or medium level of microbial

diversity but often had only few bands in common with

curd suggesting that the species occurring in the milk did

not grow during the ripening remaining at concentrations

lower than the species occurring in the NWC. The

technological influence of the NWC as resulted from the

IC1 estimation is absolutely consistent with the results of

the PCR–DGGE analysis of the DNA directly extracted

from the dairy samples (Fig. 1). The NWC has been

already defined as a complex ecosystem (Coppola et al.1988, 1990) and it is believed to represent the strength of

the traditional mozzarella cheese manufacture. The micro-

flora of the NWC mainly arises from milk as well as the

environment of the farm and processing areas. However,

only few species are naturally selected as capable of utilizing

milk nutrients, providing acidification and metabolic activ-

ities leading to typical flavours and texture. The use of

the NWC also identifies the product identity and it has

been shown to be linked to the geographical origin

(Mauriello et al. 2003).

In this study, the fate of the starter and its effectiveness

could be checked in 24 h and it has been ascertained that the

microflora of the NWC dominates in all the samples

collected during the process. The water buffalo mozzarella

cheese manufacture monitored in this study led to a top

quality product. Therefore the microbial succession could be

registered as fingerprints of microbial groups involved in a

premium quality production. This procedure might find a

useful application for the general monitoring of nonpremi-

um quality products where the poor quality arises from the

lack of development of the NWC or one or more of

the microbial groups targeted in this study. Consequently,

the procedure might allow ranking product quality when

nonpremium products are found.

This method can be easily applied to other plants allowing

process development and starter effectiveness to be checked

by analysing dairy samples by PCR–DGGE. The molecular

approaches can be considered a step forward for the

innovation of tracing systems in food technology and may

play an important role in the quality control of traditional

products allowing the preservation of their typical identity

and the consumer protection when territory claims are

involved.

ACKNOWLEDGEMENTS

This work was financed by a grant of National Research

Council (CNR), Rome, Italy (grant Agenzia 2000 no.

G00B58E) and by a grant of MURST (Rome, Italy). The

authors would like to thank Immacolata Tagliamonte for

technical collaboration.

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pcr–dgge fingerprints of microbial succession during a manufacture of traditional water buffalo...

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