Biochemical and microbiological characterization of artisan kid rennet extracts used for Cabrales cheese manufacture

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LWT 39 (2006) 605–612 www.elsevier.com/locate/lwt

Biochemical and microbiological characterization of artisan kid rennet extracts used for Cabrales cheese manufacture Ana B. Flo´reza, Ana M. Herna´ndez-Barrancoa, Isabel Marcosb, Baltasar Mayoa, a

Instituto de Productos La´cteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Asturias, Spain Consejo Regulador de la Denominacio´n de Origen Protegida ‘Queso de Cabrales’, 33555-Carren˜a de Cabrales, Asturias, Spain

b

Received 16 November 2004; accepted 1 March 2005

Abstract Four samples of artisanal kid rennet extracts from four different Cabrales cheese producers were biochemically and microbiologically characterized. Most samples had a very acidic pH (around pH 4.0), which may condition their biochemical and microbial variables. The milk-clotting activity of the extracts was assessed using a Formagraph apparatus. The properties of these artisanal rennets were found to be comparable to a 1/10 dilution of 1:10,000 commercial calf rennet; their enzymatic potentials, measured with a semi-quantitative commercial system (API ZYM), showed only very slight differences. A large population of lactobacilli was found in all artisanal kid rennet samples, whereas coliforms, enterococci, staphylococci and leuconostocs were only occasionally encountered. Sixty-four representative colonies were classified by PCR amplification and the sequencing of a stretch of their 16S rDNA genes. Strains of Lactobacillus plantarum completely dominated one of the extracts. In all others samples, strains of this homofermentative species and of the heterofermentative Lactobacillus brevis were present in similar amounts. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Kid rennet; Artisan rennet paste; Traditional cheeses; Blue-veined cheese; Cabrales cheese

1. Introduction Cheese is produced worldwide using commercial calf rennet or rennet substitutes (of both microbial and recombinant origin) as a coagulant. However, in some places in the Mediterranean basin (Italy, Greece, Spain), the traditional way of curdling the milk through the use of farmhouse rennet extracts or rennet pastes persists (Rampilli & Barzaghi, 1995). The use of such coagulants has repeatedly been shown to preserve the original and distinctive flavours of traditional cheeses in which a strong flavour is required (Irigoyen, Izco, Iba´n˜ez, & Torre, 2002; Bustamante et al., 2003). Strong aroma and flavour has been correlated with a major lypolytic activity of artisanal rennets, which causes the release of higher concentrations of short-chain fatty acids, especially butyric acid (Mattioli, Corresponding author. Tel.: +34 985 89 21 31; fax: +34 985 89 22 33. E-mail address: [email protected] (B. Mayo).

Corberi, Bergamaschi, & Suss, 1970; Barzaghi, Davoli, Rampilli, & Contarini, 1997; Bustamante et al., 2003; Piredda & Addis, 2003; Virto et al., 2003). The preservation of traditional bouquets is especially important in those cheeses with Protected Designation of Origin (PDO) status. In Spain, part of the production of several PDO cheeses (e.g. Roncal [Irigoyen, Izco, Iba´n˜ez, & Torre, 2001], Idiaza´bal [Bustamante et al., 2000], Majorero [de la Fuente, Fontecha, & Jua´rez, 1993] and Cabrales [Nu´n˜ez, 1978]) is still made with artisanal lamb or kid rennet pastes or extracts. Rennet pastes are, without doubt, a source of enzymes that may eventually play a role during ripening. Consequently, there have been continued efforts to technologically and biochemically characterize artisanal rennets (for a review, see Rampilli & Barzaghi, 1995). They may also inoculate the milk with microorganisms, which could have both sanitary and technological implications—unwanted bacteria could cause spoilage or might even be pathogenic. However, if rennet extracts

0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.03.019

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and pastes could be colonized by the cheese-related starter or adjunct microorganisms, desirable biochemical changes could occur during maturation (Steele, 1995). Reports on the microbial characterization of artisanal rennets are, however, rather scarce: Mattioli et al. (1970) studied the microbiota of Provolone cheese made with commercial rennet and lipase and compared it to that obtained through the use of traditional rennet pastes; Cosseddu and Pisanu (1980) studied the incidence of staphylococci in artisanal lamb and goat rennet pastes from Sardinia; Dellaglio, Arrizza, and Ledda (1981) classified the lactobacilli populations of lamb rennet pastes used for the manufacture of Pecorino Romano cheese; Martı´ nez, Tesone, and Quevedo (1986) reported the microbial quality of bovine rennet commercialized in Argentina; and finally, Irigoyen et al. (2002) examined the microbial quality of lamb rennet pastes used in Roncal manufacture. Cabrales cheese, which has enjoyed a PDO since 1981 is by far the most famous of the Spanish blue-veined cheeses. It is made in the mountainous area of the ‘Picos de Europa’ in Asturias (Northern Spain) from cows’ milk, with seasonal additions of ewes’ and goats’ milk. The traditional manufacture of the cheese involves curdling mixtures of evening and morning milks at 28–30 1C with a farm-made kid rennet extract. Neither starter cultures nor Penicillium roqueforti spores are added (Nu´n˜ez, 1978). After a short period of drying, maturation takes place in natural caves (at constant temperature and humidity) within the area of manufacture. Several aspects of the microbial and biochemical characteristics of this cheese have been studied (Nu´n˜ez & Medina, 1979; Nu´n˜ez, 1978; Nu´n˜ez & Medina, 1980; Nu´n˜ez, Medina, Gaya, & Dias-Amado, 1981; Alonso, Jua´rez, Ramos, & Martı´ n-A´lvarez, 1987; Flo´rez, Lo´pezDı´ az, & Mayo, 2005). In this paper we report the microbiological and biochemical characterization of the traditional kid rennet extracts currently used by some Cabrales producers. This study was undertaken in order to assess the microbial safety of these extracts, and to determine whether they influence the microbial evolution that occurs during manufacture and ripening and the biochemical changes that take place during maturation (Nu´n˜ez, 1978; Flo´rez et al., 2005).

extract were used in cheese manufacture to coagulate around 300 l of fresh milk. 2.2. Biochemical analyses The pH of the rennet pastes was measured directly using a pH meter (Crison Instruments, Barcelona, Spain), following the guidelines of the FIL-IDF standard 104A. Milk-clotting activity: Clotting time (r), curd firming time (k20 ) and curd firmness measured 30 min after the addition of rennet (a30 ) (Fig. 1) were recorded at 35 1C in duplicate in the same milk sample using a Formagraph (Foss Electric, Hillerød, Denmark), following the manufacturer’s recommendations. Determination of enzyme activities: Enzyme activities were assessed using a semi-quantitative method (API ZYM, bioMe´rieux, Montalieu-Vercieu, France). In short, 65 ml of each rennet suspensions were inoculated into the wells of an API-ZYM strip and incubated at 30 1C for 4 h. Reactives and procedures recommended by the supplier were used to score enzymatic activities. 2.3. Microbial analyses Tenfold dilutions of each sample were prepared with sterile Ringer’s solution (VWR International,

r

k20

2. Material and methods 2.1. Preparation of kid rennet extracts After slaughter, kid abomasi were air dried in a ventilated room. When needed, 4–5 dried kid stomachs were cut into strips and left to steep in 5 l of acidified cheese whey for at least 24 h. The rennet extracts were then refrigerated until use. Some 500–700 ml of this

a30 Fig. 1. A typical diagram obtained at 35 1C by the Formagraph. The clotting time (r) indicates the period of time until the actual formation of the gel starts. The curd firming time (k20 ) indicates in minutes the time from start of the gel development and until a width of 20 mm is reached. The final curd firmness (a30 ) is indicated by the width after 30 min from adding of rennet.

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Darmstadt, Germany) and plated in duplicate on specific media. Total bacterial counts: Total numbers were determined on Plate Count Agar (PCA; VWR International) containing 10 g l
1 skimmed milk powder, which was incubated at 30 1C for 72 h. Lactococci: Lactococci were enumerated on M17 agar (Terzaghi & Sandine, 1975) (Scharlau Chemie, Barcelona, Spain) after incubating at 30 1C for 48 h. Lactobacilli: Lactobacilli were scored on Man, Rogosa, and Sharpe agar (MRS; VWR International), pH 5.4, after 72 h of incubation at 30 1C in an enriched CO2 atmosphere (Gaspak; BBL, Benton Dickinson and Co., Franklin Lakes, NJ, USA). Leuconostocs: Leuconostocs were grown on Mayeux, Sandine, and Elliker agar (MSE; Biokar Diagnostics, Beauvais, France) containing 30 mg ml
1 vancomycin (Sigma, Sigma-Aldrich Co., St. Louis, MI, USA) and enumerated after 3–5 days of incubation at 25 1C. Yeasts and moulds: Dilutions of the rennet samples were plated onto the surface of Yeast-extract Glucose Chloramphenicol agar (YGC) (VWR International) and incubated for 3–5 days at 25 1C. Enterobacteria and coliforms: Violet Red Bile Lactose agar (VRBL) (VWR International) was used to enumerate enterobacteria and coliforms, using the pour-plate and overlay technique. Incubations were for 24–48 h at 30 1C. Enterococci: Enterococci were scored after 24 h of incubation at 44 1C in Slanetz and Bartley medium (S–B; VWR International). Staphylococci: Dilutions were plated on Baird–Parker agar (B–P; VWR International) and incubated for 24 h at 37 1C. Black colonies with or without egg yolk clearing were recorded. 2.4. Identification of dominant bacteria Sixty-four representative colonies of all morphologies were chosen at random from the agar plates of counting the dominant bacterial populations, purified by subculturing, and maintained at
80 1C in cryoprotective media. Cell extracts of the purified isolates were used as DNA templates in PCR reactions. Cell extracts were obtained and PCR performed essentially as described by Ward, Brown, and Davey (1998). Two PCR primers, Y1 (50 -TGG CTC AGG ACG AAC GCT GGC GGC-30 ) (position 20–43 on 16S rDNA, Escherichia coli numbering) and Y2 (50 -CCT ACT GCT GCC TCC CGT AGG AGT-30 ) (positions 361–338) (Young, Downer, & Eardly, 1991), were used to amplify a 348-bp stretch of DNA from representative strains. Y1 and Y2 are based on prokaryotic conserved regions embracing the V1 and V2 variable regions of the 16S rDNA gene (Neefs, van de Peer, de Rijk, Chapelle, & de Wachter, 1993). Amplicons were purified to remove unincorpo-

607

rated primers and nucleotides using Microcon PCR filters (Millipore, Bedford, MA), and sequenced by cycle extension in an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The sequences obtained were compared to others in public databases using the BLAST program (Altschul et al., 1997). The comparison and alignment of sequences to those deposited on GeneBank (http://www.ncbi.nlm.nih.gov/ BLAST/) and on the Ribosomal Database Project (http://rdp.cme.msu.edu/index.jsp) allows a precise genetic identification of the strains (Stackebrandt & Goebel, 1994; Palys, Nakamura, & Cohan, 1997).

3. Results and discussion 3.1. Biochemical characterization The pH of the extracts was 4.83, 4.10, 4.18, and 3.78 for rennet samples 1 through 4, respectively. The percentage of NaCl they contained was 0.35, 0.24, 1.59, and 0.32, respectively. The pH of the extracts was low, as expected for a whey-derived, naturally acidified product. When the weather is hot, some producers dilute the whey used to make the rennet extract with water, which may be the reason for the high pH in rennet extract number 1. Salt is not normally added to the extracts; however, some producers add some salt to the abomasi before drying, which could explain the NaCl content of rennet number 3. Other producers use the whey that drains from cheeses after their salting, which may alternatively explain the NaCl levels recorded for this extract. The enzymatic activities of the rennet extracts were analysed with the commercial API ZYM system at three different pHs (6.0, 5.0, and their current pH), mimicking some of the successive pHs of the milk and curd during manufacture and ripening. pH was adjusted using an NaOH (0.1 mol l
1) solution; the extracts were allowed to remain at their new pH for 1 h before the assay to allow renaturation of enzymes. For comparison, commercial 1:10,000 calf rennet (Nievi, Vizcaya, Spain) and its 1/10 dilution were subjected to the same analysis. The results are summarized in Table 1. Small differences were recorded between samples at the different pHs. For instance, rennet extract 1 showed high alkaline and acid phosphatase activity compared to all other artisanal rennets, while rennets 2 and 3 showed greater N-acetylb-glucosaminidase activity. The increased pH activated some enzymes (leucine arylamidase, b-galactosidase, and b-glucosidase) whereas it inactivated or reduced the activity of others (esterase C4, esterase-lipase C8, cysteine arylamidase, acid phosphatase, naphtol-AS-BIphosphohydrolase, and b-glucuronidase). As can be seen, the enzyme activities of commercial calf rennet (pH 5.47) were always greater (even for the 1/10

4.83 5.00 6.00

4.10 5.00 6.00

4.18 5.00 6.00

3.78 5.00 6.00

5.47

Rennet 1

Rennet 2

Rennet 3

Rennet 4

Calf rennet

10

20

10 5 5

5 2.5 2.5

10 5 2.5

20 20 20

5

10

— 2.5 2.5

5 2.5 2.5

5 2.5 2.5

5 2.5 2.5

Esterase (C4)

5

10

— 2.5 2.5

5 2.5 2.5

5 2.5 2.5

5 2.5 2.5

Esterase Lipase (C8)

5

20

— — 2.5

— — —

— 2.5 2.5

— — 2.5

Leucine arylamidase

—

2.5

— — —

— — —

— — —

— — —

Valine arylamidasa

10

20

10 5 5

5 2.5 2.5

10 5 2.5

20 20 20

Cysteine arilamidase

30

40

2.5 2.5 5

5 2.5 2.5

5 5 2.5

20 20 20

Acid phosphatase

30

40

2.5 2.5 2.5

5 2.5 2.5

5 5 2.5

5 2.5 2.5

Naphtol-AS-BIphosphohydrolase

Activity was recorded as the approximate nanomoles of hydrolized substrate. Activity on substrates for lipase C14, trypsin, a- a-chymotrypsin enzymes was not observed. — activity not detected.

Calf rennet 1/10

pH

Alcaline phosphatase

2.5

10

— — —

— — —

— — —

— — —

agalactosidase

10

30

— 2.5 5

2.5 10 5

2.5 10 20

5 20 20

bgalactosidase

10

40

— 2.5 —

5 2.5 2.5

10 2.5 2.5

5 2.5 2.5

bglucuronidase

2.5

30

2.5 2.5 5

2.5 — —

2.5 — —

2.5 2.5 2.5

aglucosidase

5

10

— 2.5 5

2.5 — —

— — 2.5

— 2.5 2.5

bglucosidase

20

30

— 5 10

20 5 5

20 10 5

5 2.5 2.5

N-acethyl-bglucosaminidase.

—

—

— — —

5 2.5 2.5

— — —

— — —

amannosidase

5

10

— — —

— — 2.5

— — —

— — —

afucosidase

608

Sample

Enzyme

Table 1 Enzymatic activities measured with the API ZYM system of the different artisanal rennets

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dilution) than those of the artisanal rennets. Normalization by the protein content of the extracts gives a similar result (data not shown). No clear qualitative or quantitative differences in enzymatic activities were seen among the artisanal rennet samples. Furthermore, neither the percentage of retention of these activities in the cheese matrix nor their role in ripening are currently known. The coagulation characteristics of the rennet extracts were examined using a Formagraph. Table 2 shows the mean values for the examined variables [clotting time (r), curd firming time (k20 ) and curd firmness measured 30 min after rennet addition (a30 )] from duplicate assays with the same milk. The coagulating strength of the artisanal kid rennet samples was around that of the 1/10 dilution of the commercial calf rennet (strength around 1:1000), except for rennet number 3 which showed reduced activity compared to the others. All of them had

Table 2 Biochemical characterization of the different artisanal rennets samples with the Formagraph Sample

Dilution

Formagraph parametersa (in cm) r

k20

a30

Rennet 1

1 1/2 pH 6.0

0.80 1.85 1.00

0.60 1.10 0.75

4.80 4.30 4.50

Rennet 2

1 1/2 pH 6.0

1.00 2.25 2.00

0.65 1.25 1.10

4.15 4.15 4.40

Rennet 3

1 1/2 pH 6.0

1.55 4.30 4.00

0.95 2.30 2.10

4.20 1.90 2.20

Rennet 4

1 1/2 pH 6.0

0.70 2.00 2.15

0.60 1.05 1.10

4.10 4.30 4.45

Calf rennet

1 (pH 5.47) 1/10 1/20

0.80 0.75 1.80

0.60 0.65 1.00

5.00 4.85 4.60

a

Average of two independent analyses with the same milk sample.

609

a similar curd firming time to that of the 1/10 commercial dilution (around 0.6). However, firmness at 30 min was clearly less when any of the artisanal samples were used. Neutralization of the extracts worsened all coagulation variables (r was increased and k20 and a30 reduced) suggesting that the chymosin activity responsible for curdling is not related to any of the enzymes assayed by the API ZYM system. 3.2. Microbial characterization Since milk bacteria and bacteria from the kid stomach are allowed to grow in the whey, large populations of bacteria were expected. Table 3 shows the log counts of the microbial groups examined in the four rennet extracts. Total aerobic counts ranged from 1.70  107–1.21  108 cfu ml
1. Equivalent numbers of bacteria were enumerated on MRS and M17, which are normally used for lactobacilli and lactococci counting, respectively (Table 1). A variable population of yeasts was found in all four samples, ranging from 1.14  103 to 1.16  105 cfu ml
1. The exception was rennet sample 2, in which a single type of bacterium (which turned to be strains of Acetobacter cerevisiae, see below) grew on the YCG plates. Species of leuconostocs were only recovered from rennet extract number 1 (1.65  103 cfu ml
1), as were coliforms (3.51  106 cfu ml
1). Enterococci were detected in rennet extract number 3 (1.35  103 cfu ml
1), and staphylococci in rennets 1 and 3 (1.61  105 and 1.62  102 cfu ml
1, respectively). None of the staphylococcal colonies showed a coagulase-positive phenotype. The microbial counts for these rennet extracts are quite different from those of artisanal or commercial rennets to which high salt concentrations (usually between 10% and 25%) are added as a preservative (Mattioli et al., 1970; Martı´ nez et al., 1986; Irigoyen et al., 2001; Virto et al., 2003). The higher pH of rennet extract 1 may aid the survival of quite a large population of coliforms; these were detected in no other sample. As can be seen from Table 3, the majority of the viable microorganisms belonged to the most typical lactic acid bacteria genera. However,

Table 3 Log counts of the microbial groups examined in the four artisanal kid rennets extracts analyzed Microbial log counts (cfu ml
1) Sample Rennet Rennet Rennet Rennet

1 2 3 4

Average7SD nd, not detected.

Total bacteria

Lactobacilli

Lactococci

Leuconostoc

Enterococci

Staphylococci

Coliforms

Yeasts and moulds

8.01 7.23 7.83 7.32

7.83 7.29 7.82 7.54

8.15 7.31 7.90 7.45

3.22 nd nd nd

nd nd 3.13 nd

5.21 nd 2.22 nd

6.55 nd nd nd

5.01 nd 5.04 3.16

7.6270.40

7.6270.25

7.7070.38

0.871.60

0.7871.56

1.8672.46

1.6673.27

4.0871.12

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in microscopic observations of the cells from the MRS, M17 and MSE media, only rods were seen, suggesting that lactobacilli were the only cultivatable lactic acid bacteria present in all extracts. In order to characterize the microbial components that dominate the rennet extracts, 64 representative colonies (20, 18, 17, and 9 strains from samples 1–4, respectively) were isolated from the PCA (25), M17 (17), MRS (16), VRBL (3), and YGC (3) agar plates. Enterococci, staphylococci and leuconostoc populations were encountered only occasionally and in low numbers. For this reason, although members of these genera could be in some instances technologically relevant, these populations were not classified further. Isolates were all subjected to molecular classification by amplification and sequencing of a stretch of their 16S rRNA gene. The level of identity obtained by the BLAST program between sequences from the rennet isolates and those from type strains on the databases was always higher than 97%, a percentage which is considered for strains belonging to a single species (Stackebrandt & Goebel, 1994). The results are summarized in Table 4. The following species were found: Lactobacillus plantarum (33 isolates), Lactobacillus brevis (16), Hafnia alvei (7), Acetobacter cerevisiae (3), Lactobacillus kefiri (2), and Acetobacter orientalis (1). Two isolates could only be assigned to the genus level, one to Lactobacillus and the other to Cellulomonas. The above-mentioned species were not, however, evenly distributed among the rennet samples. For instance, L. plantarum species completely

dominated rennet sample 3, whereas they were found in amounts similar to L. brevis in all other samples. The distribution of lactobacilli species obtained in this work is quite different to that reported by Dellaglio et al. (1981) who found that the heterofermentative Lactobacillus reuteri and Lactobacillus fermentum species dominated the rennet pastes used to manufacture Pecorino Romano cheese. These differences may be attributable to differences in the technology used to produce the rennet that might select distinct species (Calandrelli et al., 1997; Bustamante et al., 2003; Virto et al., 2003). Hafnia alvei was found only in rennet 1, in which a large population of coliforms was present. This species of enterobacteria was recovered from both PCA and VRBL plates, indicating that it reached similar numbers to those of lactobacilli. As for any other artisanal product, the differences between samples in terms of microbial composition and numbers were quite large. Such differences have been noted during the microbial characterization of different batches of Cabrales cheese (Nu´n˜ez, 1978; Nu´n˜ez & Medina, 1979; Flo´rez et al., 2005), suggesting that the rennet extracts used might be important sources of microorganisms. In fact, L. plantarum and L. brevis are the most commonly reported lactobacilli in this cheese (Nu´n˜ez & Medina, 1979; Flo´rez et al., 2005). Furthermore, Lo´pez and Mayo (1997) reported a similar distribution of homofermentative and heterofermentative lactobacilli in artisanal cheeses. The typing of a reasonable number of rennet and cheese strains—which

Table 4 Species distribution and sample and media of procedence of the classified strains from the artisanal rennets Species

Rennet sample Rennet 1

Rennet 2

Rennet 3

Rennet 4

Total

Lactobacillus plantarum

8 (3 M17, 3 MRS, 2 PCA)

6 (3 M17, 2 PCA, 1 MRS)

17 (7 PCA, 5 M17, 5 MRS)

2 (2 M17)

33

Lactobacillus brevis

5 (2 PCA, 2 MRS, 1 M17)

7 (4 PCA, 2 M17, 1 MRS)

4 (4 PCA)

16

1 (MRS)

1 (1 MRS)

2

1 (1 MRS)

1

Lactobacillus kefiri Lactobacillus spp. Hafnia alvei

7 (3 PCA, 3 VRBL, 1 M17)

Acetobacter cerevisiae

7 3 (3 YGC)

3

Acetobacter orientalis

1 (1 PCA)

Cellulomonas spp. Total

1 (1MRS) 20

18

1 1

17

9

64

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is currently underway—will undoubtedly solve the question of whether or not they are related. In the present experiment, none of the 64 strains examined, including the 17 from the MRS medium, were Lactococcus spp. The species of this genus are more acidsensitive than lactobacilli (Mundt, 1996), and they probably cannot survive for long periods at the low pH of the extracts. However, since lactococci are dominant during the manufacture and ripening of Cabrales cheese (Flo´rez & Mayo, unpublished; Nu´n˜ez, 1978; Flo´rez et al., 2005), it follows that these bacteria must reach the cheese milk from another source. L. plantarum was the most abundant bacteria in the rennet extracts examined. This species is frequently found as a dominant nonstarter lactic acid bacteria in cheeses made from raw milk (Beresford, Fitzsimons, Brennan, & Cogan, 2001; Crow, Curry, & Hayes, 2001). Strains of this species display similar biochemical characteristics and a very high level of homology in their 16S rRNA sequences (ca. 99%) (Collins et al., 1991), although it has been demonstrated to contain three groups on the bases of DNA–DNA hybridization: L. plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum (Dellaglio, Bottazzi, & Vescovo, 1975). To reliably distinguish among these three related groups, a multiplex PCR assay has been recently developed (Torriani, Felis, & Dellaglio, 2001). Preliminary data indicate that L. pentosus is not present in the rennet extracts. By contrast, L. plantarum and L. paraplantarum appear in equal numbers (data not shown). In conclusion, rennet extracts are probably the source of the majority of the nonstarter lactic acid bacteria in Cabrales cheese. These bacteria have been linked to the strong flavours of other traditional cheeses, although they are also known to produce undesirable flavours as well (Fox, McSweeney, & Lynch, 1998; Beresford et al., 2001). Thus, the microbial composition of the rennet extracts used might ultimately be related to the final quality of the cheese produced. While displaying similar enzymatic activities, rennet extracts showed a high microbial variability, which suggests that this may be one of the main reasons of fluctuation in the overall Cabrales quality. In spite of this, from a hygienic-sanitary point of view, artisanal kid rennet extracts appeared to be safe, as neither pathogens nor opportunistic microorganisms were identified.

Acknowledgement This work was supported by a research project from the Spanish Ministry of Science and Technology (reference PTR1995-0556-OP).

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