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Changes in the microbial flora of Tenerife goats' milk cheese during ripening
PII:
ELSEVIER
Int. Dairy Journal 7 (1997) 635-641 ‘{: 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 09%6946/98/$19.00+0.00
SO958-6946(97)00065-4
Changes in the Microbial Flora of Tenerife Goats’ Milk Cheese During Ripening V. Z&ate*, F. Belda, C. PCrez and E. Cardell Departumento de Microhiologia y Biologia Celular, Universidad de La Laguna, La Laguna 38206, Tenertfe, Spain (Received
6 March
1997; accepted
6 October
1997)
ABSTRACT Changes in the microbial flora of Tenerife goats’ cheese, manufactured from raw milk without the addition of starter cultures, were studied during ripening. Lactic acid bacteria (lactococci, leuconostocs and lactobacilli) were the dominant microorganisms throughout ripening. Lactococcus Iactis ssp. lactis was the most common Lactococcus found (78.9% of isolates classified as lactococci). It reached maximum numbers in 2-day-old cheeses and decreased afterwards during ripening. Lactobacillus plantarum (56.9% of isolates classified as lactobacilli) and Lactobacillus paracasei ssp. paracasei (37.2% of isolates) were the main species of lactobacilli isolated. L. plantarum was present in high proportions in 2-day-old cheeses but decreased during ripening, whereas L. paracasei ssp. paracasei, present initially at low numbers, increased during ripening and was the predominant Lactobacillus in 60day-old cheeses. Leuconostoc mesenteroides ssp. dextranicum (81.8% of isolates classified as leuconostocs) was the main species identified as leuconostoc and its numbers decreased throughout ripening, particularly during the last month. Enterococcus faecalis and Enterococcus faecium, micrococci, moulds and yeasts were found at significant numbers and could contribute to the ripening process. Microorganisms used as an indicator of the bacteriological quality of the cheeses (Enterobacteriaceae, coliforms and faecal coliforms) and Staphylococcus aureus decreased sharply in the interior of the cheeses throughout ripening. Q 1998 Elsevier Science Ltd. All rights reserved
Keywords:
microbial
flora; lactic acid bacteria
INTRODUCTION
6 L of milk yield a cheese 15-18 cm in diameter, 81Ocm high and weighing 0.9-l .2 kg. Most of the cheeses are consumed fresh (after 2days), but some are ripened for 30 to 60days at 15-25°C and 75590% relative humidity. Because maturation acts as a natural selection process, during which possible pathogenic flora are normally inhibited by lactic acid bacteria (Nufiez et al., 1985) the consumption of fresh Tenerife cheese incurs a high risk of acquiring food-borne diseases. The aim of this study was to examine the changes in the microflora during the ripening of Tenerife goats’ milk cheese in order to identify the microorganisms which are present in high viable numbers during the ripening process. This is a preliminary step towards the preparation of a microbial starter suitable for the manufacture of Tenerife cheese on an industrial scale.
A number of fresh and ripened cheeses made from goats’ milk have been developed in Spain. The microbiological and physicochemical characteristics of some varieties such as Valdeteja (Gutierrez et ul., 1988), Majorero (Fontecha et al., 1990), Ibores (MasMayoral et al., 1991) Gredos (Medina et al., 1992), Valdeon (Lopez-Diaz et al., 1995) and Armada cheeses (Tornadijo et ul., 1995) have been studied. Tenerife cheese is a traditional farmhouse variety, with an annual production of about 1500 tonnes, manufactured by approximately 325 producers, from raw milk of local goats on the island of Tenerife. The milk is not heat-treated and it is not inoculated with a lactic culture. An animal rennet solution is prepared by homogenizing a small amount (approximately 1 g) of young goats’ stomach, previously salted and sun-dried, by hand or using a stem-type blender, in about 2030mL of tap water containing 5-10% NaCl. Coagulation takes place at 28-32°C for 3&60min. The coagulum is cut into pieces the size of a rice grain, left to settle in the bottom of a vat and the whey removed 15-20 min later. The curd is then transferred to clean aluminium rings and pressed by hand. The cheeses are salted by rubbing coarse salt on one side and the following day they are removed from the rings, inverted and salted on the other side. Approximately *Corresponding
MATERIALS
AND METHODS
Cheese manufacture
and sampling
Tenerife cheeses (0.9-1.2 kg) were manufactured at four different dairies by experienced cheesemakers following the traditional method, as described above. One batch of cheeses was manufactured at each dairy. For each batch, a sample of 2-, 30- and 60-day-old cheese (each sample consisted of one whole cheese), as well as a sample of the rennet and milk used in their
author. 635
636
V. Zrate
manufacture, were taken, transported to the laboratory under refrigeration (4°C) and kept at that temperature no longer than 24 h before analysis. Sampling of the surface of the cheeses was performed by scraping three different areas of the rind (Nufiez, 1978). Samples of the interiors of the cheeses were taken at a depth of 3cm below where the surface samples were taken (Poullet et al., 1991). In both cases, the three subsamples were pooled before analysis. Microbiological analysis
Cheese samples (10 g) were homogenized in 90 mL sterile 2% (w/v) of sodium citrate solution and decimal dilutions of rennet, milk and cheese homogenates prepared in l/4-strength Ringer solution (Unipath, Basingstoke, UK) were plated on the specific media required for the different microbial groups to be examined. Total viable counts were determined on plate count agar (PCA; Unipath) incubated at 30°C for 72 h. Lactococci were estimated on Ml7 agar (M17A; Unipath) incubated for 48 h at 30°C (Terzaghi and Sandine, 1975), and leuconostocs on Mayeux, Sandine and Elliker agar (MSEA; Mayeux et al., 1962) incubated for 72 h at 25°C. Rogosa agar (RA; Unipath) incubated at 30°C for 72 h in an anaerobic jar (Hz + CO2 Generbox; BioMerieux, Marcy l’Etoile, France) was used to estimate the numbers of lactobacilli, following the method of Nufiez (1978). Enterococci were enumerated on KF Streptococcus agar (KFSA; Unipath) incubated at 44°C for 48 h (Poullet et al., 1991), and Mcrococcaceae on mannitol salt agar (MSA; BioMCrieux) incubated for 72 h at 25°C (Chapman, 1945). Staphylococcus aureus was estimated on Baird-Parker agar (BPA; Unipath) plates incubated at 37°C for 48h (Baird-Parker, 1962) and confirmed by a positive coagulase test (BioMCrieux). Enterobacteriaceae were determined on violet red bile glucose agar (VRBGA; Unipath) plates incubated at 37°C for 24h, (Mossel et al., 1962). Coliforms were estimated according to Nufiez (1978) on violet red bile agar (VRBA; Unipath) incubated at 37°C for 24h, and faecal coliforms on the same medium but incubated at 44°C for 24 h, as described by Poullet et al. (1991). Yeasts and moulds were enumerated on potato dextrose agar (PDA; Unipath) acidified to pH 3.5 with tartaric acid and incubated at 26°C for 96 h (Nuiiez, 1978). Each dilution (1 mL) was inoculated, in duplicate, using the pour plate technique for PCA or the pour plate and overlay technique for M 17A, RA, KFSA, VRBGA and VRBA. Each dilution (0.1 mL) was inoculated, in duplicate, using the surface spread technique for MSEA, MSA, BPA and PDA. Results were expressed as logarithm (log) of colony forming units (cfu) per millilitre for milk and rennet, and log cfu g-’ for cheeses. Isolation and identification of bacterial species
For each batch, live to eight colonies were taken randomly for each sampling point in the interior of the cheeses (2, 30 and 60 days) from M17A, RA and MSEA plates. The isolates were purified in ManRogosa-Sharpe (de Man et al., 1960) (MRS; Unipath) agar and stored in MRS broth containing 20% (v/v) glycerol, at -80°C. A total of 180 strains (five from
et al. each sampling point, type of plate and batch) were further characterized. Although recent methodologies tend towards a higher number of isolates from each plate, the number of colonies selected was determined following protocols duly described in the applied scientific literature (Nufiez, 1978; Freitas et al., 1996). The criteria of Sharpe et al. (1966) were followed for differentiating lactococcci, enterococci, leuconostocs and lactobacilli. Identification at the species level of and Lactococcus Enterococcus was performed according to the criteria of Schleifer and Kilpper-Balz (1984), Schleifer et al. (1985) and Schleifer (1986). The API 50 CH system with API 50 CHL Medium (BioMCrieux) was used to identify the leuconostocs and lactobacilli at the species level. Physicochemical
analysis
Cheese pH, percentage moisture, NaCl content (percentage of total weight) and titratable acidity were assayed by standard methods (Bradley et al., 1993). Statistical treatment of results
Analyses of variance (ANOVAs) were performed on results obtained at different stages of ripening, with dairy and cheese age as main effects, using program SOLO 2.0, GLM-ANOVA (BMDP Statistical Software, Los Angeles, CA, USA).
RESULTS
AND DISCUSSION
Changes in the microbial group counts during ripening
The microbial counts in the raw goats’ milk used for the manufacture of Tenerife cheese, as well as in the interior of cheeses during ripening, are shown in Table 1. Mcrococaceae was the predominant microbial group in the rennet used in the manufacture of the cheeses, with levels 2-4 log units higher than that of the other and coliform groups, whereas Enterobacteriaceae numbers were low (around 1.5 log units as the average) (not shown). Lactic acid bacteria (mainly lactococci and leuconostocs) were the most abundant microbial groups in milk, being l-2 log units higher than the other groups. The counts for coliforms and faecal coliforms were similar to the values reported for goats’ milk used in the manufacture of Majorero, Gredos and Valdeon cheeses (Fontecha et al., 1990; Medina et al., 1992; Lopez-Diaz et al., 1995) and are a consequence of the lack of hygiene during milking and manipulation of the milk before cheesemaking. Due to the multiplication of bacteria and the retention of microorganisms in the curd after drainage of the whey (Tatini et al., 1971) most microbial groups increased by 2-3 log cycles from milk to 2-day-old cheeses. A similar increase in the microflora has been observed in goats’ and ewes’ cheeses (Fernandez de1 Pozo et al., 1988; Poullet et al., 1991; Medina et al., 1992) and was accompanied by a sharp decline in pH, due to the production of lactic acid (Table 3) by the high numbers of lactic acid bacteria (mainly lactococci and leuconostocs) (Table 1).
Changes in the microbialjora Table 1.
Medium
PCA M17A RA MSEA KFSA MSA BPA VRBGA VRBA VRBA PDA PDA
qf Tenerife goats’ milk cheese
637
Microbial Flora of the Interior of Tenerife Cheese During Ripening
Microbial group expected Total viable counts Lactococci Lactobacilli Leuconostocs Enterococci Micrococcaceae S. aureus Enterobacteriaceae Coliforms Faecal coliforms Yeasts Moulds
Milk”
Time of ripening (days)
5.54f2.44 5.39f2.62 3.59&l .32 5.10f2.70 3.75f3.12 4.52zt2.98 2.58&l .04 4.52f3.64 4.39f3.60 3.6211.16 3.78f 1.90 2.041tl.50
2b
3ob
60b
9.261t1.70 9.2411.54 5.38fl.80 7.6911.40 6.96f2.62 5.7913.12 3.141t4.26 7.12zt1.20 6.98f0.98 4.59f4.86 3.8913.86 2.20f2.38
9.2551.34 9.33Ito.44 7.16z!zO.86 7.68f1.44 6.97f1.78 5.6011.20 1.62f3.94 4.89f4.38 3.55zt5.96 1.91f5.32 4.66&l .86 2.87f2.50
8.74f0.46 8.62f0.58 7.5811.64 7.59f0.80 6.8511.44 5.91fl.46 Absence in 0.01 g 3.90zt4.42 3.31f5.62 cl 4.2811.46 3.9111.92
Data are average values&2 SD (95% CI) of four batches. Units: “log cfu mL_‘; blog cfu g ‘. (Devoyod and Miiller, 1969; Devoyod and Desmazeaud, 1970). Micrococcaceae were found at significant numbers in the cheeses at the three sampling times, particularly on the surface (Table 2) and, due to their lipolytic activity, may contribute to flavour development in these cheeses during ripening. S. aureus numbers decreased significantly (PC 0.05) in the interior of the cheeses with time and were not detected in 60-day-old cheeses. No significant change was seen in the number of yeasts throughout maturation whereas moulds, initially present at low numbers, increased during the last month of ripening. These groups may contribute to the ripening process by utilizing lactic acid, and by their proteolytic and lipolytic activity (Macedo et al., 1995). The numbers of microorganisms indicative of the bacteriological quality (Enterobacteriaceae, coliforms, faecal coliforms) were high, suggesting considerable contamination of the cheeses that may occur during milking and/or manufacturing. Furthermore, the dairy had a significant (P < 0.05) influence on the numbers of Enterobacteriaceae in the interior and surface of the cheeses. Ripening time had a significant (PC 0.05) influence on the numbers of Enterobacteriaceae, coliforms and faecal coliforms, with counts in the interior of fully ripened cheeses 34 log units lower than that of
Lactic acid bacteria were the dominant microorganisms throughout ripening. Lactococci decreased during the second month of ripening, whereas lactobacilli increased significantly (P c: 0.05) during maturation, probably due to the ability of this genus to grow at low pH values (Nufiez, 1978) and to resist high salt concentrations (Sharpe et al., 1966). Lactic acid bacteria influence the ripening process in various ways by the production of lactic acid, by a decrease in the oxidation-reduction potential, and by proteolysis and lipolysis (Steele, 1995). Numbers of enterococci, on which the particular dairy had a significant effect (P < O.Ol), were similar to those observed in other varieties of cheeses manufactured from raw ewes’ or goats’ milk (Fernandez de1 Pozo et al., 1988; Poullet et al., 1991; Tornadijo et al., 1995). The presence of a high number of enterococci could be due to poor hygienic practices during the manufacturing process, the presence of domestic animals in the dairy and the resistance of enterococci to adverse conditions (high temperatures, high salt concentration and high acid levels) (Mundt, 1986). Enterococci may influence the ripening process due to their caseinolytic and lipolytic activity and their ability to stimulate the production of acid by some lactococci and gas production by leuconostocs
Table 2. Medium
PCA M17A RA MSEA KFSA MSA BPA VRBGA VRBA VRBA PDA PDA
Microbial Flora of the Surface of Tenerife Cheese During Ripening
Microbial group expected Total viable counts Lactococci Lactobacilli Leuconostocs Enterococci Micrococcaceae S. aweus Enterobacteriaceae Coliforms Faecal coliforms Yeasts Moulds
Data are average yaluesf2 SD (95% CI) of four batches. Units: ^log cfu g
Time of ripening (days) 2”
30”
60”
8.65f2.24 8.73f1.94 5.1612.10 7.32f1.08 6.27f3.04 5.75kl.88 2.82f4.90 6.66zt1.40 6.27*1.08 5.59f0.90 5.2511.00 2.9811.94
9.4510.88 9.54f0.68 8.02zt1.38 8.43zt1.84 6.53f1.04 8.15zt0.62 c2 7.70+2.78 7.6012.82 5.67f0.34 6.901t1.52 5.04k4.22
8.78f0.54 8.6X&0.74 7.281tl.72 7.57-f-0.86 6.33h1.02 7.7010.66 Absence in 0.01 g 5.78f4.96 5.26f3.90 I .87&3.40 6.92f2.12 7.4450.86
638
V. Z&ate et al. Table 3.
Parameter
PH Moisture (%) NaCl content (% total weight) Titratable acidity (% lactic acid)
Physicochemical
Parameters of Tenerife Cheese During Ripening
Milk
6.42f0.34 0.22&O. 16 0.08f0.03
Time of ripening (days) 2
30
60
4.93f1.38” 4.95f0.56b 53.6*5.62a 51.3&9.84b 3.72*1.64a 6.54f 1.22b 0.17&0.16a 0. 19f0.06b
4.64f1.78a 4.70*0.66b 53.2&l 1.9a 44.0f6.92b 4.36&O/W 5.14&0.98b 0.24ztO. 14a 0.15f0.04b
4.65&l .98= 5.20&0.54b 51.0f8.94” 32.7&2.58b 6.10~t1.34~ 7.25f7.34b 0.23f0.12a 0. 12+0.06b
aInterior of the cheeses. bSurface of the cheeses. Data are average values+2 SD (95% CI) of four batches.
2day-old cheeses (Table 1). Various factors contribute to the decline of these groups in the interior of cheese during ripening; these include a significant (PC 0.01) increase in the concentration of NaCl and the inhibition of these bacteria by lactic acid bacteria (Nufiez et al., 1985), basically by causing a decrease in the pH and an increase in lactic acid concentration (Table 3). The numbers of these groups on the surface were about 2 log cycles higher than in the interior (Table 2) probably due to higher pH values (Table 3) and aeration. Species isolated and identified during ripening
Table 4 shows the species isolated on M17A, MSEA and RA from the interior of the cheeses, throughout ripening. The three isolation media used permitted the growth of the different groups of lactic acid bacteria but with different degrees of selectivity. M17A showed moderate selectivity for the isolation of lactococci (42% of the isolates obtained on this medium were classified as lactococci), considered as Gram-positive, catalase-negative, homofermentative cocci that grow at 10°C but not at 45°C. The selectivity of MSEA for the isolation of leuconostocs (Gram-positive, catalase-negative, heterofermentative cocci) was very low (only 17% of the isolates obtained on MSEA were classified as leuconostocs). These two media permitted the growth of a high number of enterococci (33 and 62% of the isolates, from M17A and MSEA, respectively), considered as Gram-positive, catalase-negative, homofermentative cocci that grow at 10°C and 45°C and in 6.5% NaCl. In contrast, RA showed a high selectivity for lactobacilli (Gram-positive, catalase-negative rods) because 70% of the isolates on this medium were classified as lactobacilli. These degrees of selectivity are in line with the results reported by other authors (Poullet et al., 1993; Tornadijo et al., 1995). Lactococcus lactis ssp. lactis was the most abundant Lactococcus found in Tenerife cheese (30 of 38 isolates classified as lactococci). This species reached maximum numbers in 2-day-old cheeses and then decreased during ripening (Table 4) probably as a consequence of the inhibitory effect of low pH and high NaCl content (Table 3), in a similar way as has been observed by other authors for different varieties of
Spanish cheese (Rodriguez-Medina et al., 1995; Tornadijo et al., 1995). The remaining eight lactococci were identified as L. lactis ssp. cremoris on the basis of their inability to hydrolyse arginine (Table 5). Homofermentative lactobacilli such as Lactobacillus pluntarum (29 of 51 isolates classified as lactobacilli) and L. paracasei ssp. paracasei (19 of 51 isolates) were the principal species of lactobacilli isolated. L. plantarum was present at high proportions in 2-day-old cheeses and decreased slightly during ripening, whereas L. paracasei ssp. paracusei, .present initially at low numbers, increased and was the predominant Lactobacillus in 60-day-old cheeses (Table 4). These two species were also the principal lactobacilli isolated from other varieties of cheese manufactured with raw milk (Nufiez, 1978; Macedo et al., 1995; Centeno et al., 1996; Freitas et al., 1996). On the other hand, heterofermentative lactobacilli, i.e., Lactobacillus brevis and L. brevis ssp. lindneri, were also isolated from Tenerife cheese although at very low numbers (one isolate each). Dextran-producing Leuconostoc leuconostocs, mesenteroides ssp. dextranicum (18 isolates) and L. mesenteroides ssp. mesenteroides (four isolates), were the leuconostocs identified in Tenerife cheese and both decreased throughout ripening, especially during the last month. In general, enterococci were very abundant in all the cheeses, with Enterococcus faecalis the predominant species, followed by Enterococcus faecium and, to a lesser extent, by Enterococcus avium (Table 4). Some of the enterococci isolates exhibited atypical patterns in some biochemical or growth characteristics: 18 out of 60 enterococci were unable to grow at pH 9.6; of 31 isolates identified as E. faecalis, seven did not produce
black colonies when grown in 0.04% potassium tellurite, three did not produce acid from melezitose and one fermented raflinose; among the 24 isolates identified as E. faecium, three were unable to produce acid from melibiose (Table 5). E. faecalis has also been described as the dominant Enterococcus species in other raw goats’ milk cheeses such as Majorero (Fontecha et al., 1990), Ibores (Mas and GonzilezCrespo, 1992) and Armada (Tornadijo et al., 1995). In contrast, E. faecium was the most abundant Enterococcus in Picante ewes’ and goats’ milk cheese (Freitas et al., 1996).
Results are the total from four batches No.. Number of isolates.
L. lactis ssp. lactis L. lactis ssp. cremoris E. ,faecalis E. faecium E. avium Unidentified streptococci L.mesenteroides ssp.mesenteroides L. mesenteroides ssp. dextranicum L. plantarum L. paracasei ssp. paracasei L. brevis L. brevis ssp. lindneri Unidentified lactobacilli Non-lactic acid bacteria Total
6 _
of cheeses.
1 20
_ _
_ _ _ _ 20
5 1 3 1
1 7 1 _
2
1 _
11 _
20
_ _
1
5 1 9
1
1 2 _
1 60
_
13 2 1 13 1 3 10 2 12 2 _
1.7 100
_ _
21.7 3.3 1.7 21.7 I.7 5.0 16.6 3.3 20.0 3.3
20
_
2 1 _
1 2 1 1 6
6
20
_ _
2 1 2 1
2 2 8 2
20
1 1 _
6 7 _
2 2 1 _ _
60
1
1
3 7 2 9 8 _
1
10 4 10 4
100
1.7 1.7 _
16.7 6.6 16.7 6.6 1.7 5.0 11.7 3.3 15.0 13.3 _
20
~ ~~
6 2 4 3 3 2 ~ -
20
_ _ _ _
1
1 _
15 3
_
20
7 9 1
60
_
8 9 1
3 2 1 _
_ _ _
I
2 20
I
1
1
_
1
100
__
13.3 15.0 1.7
11.7 3.3 33.3 11.7 5.0 3.3 1.7 _
30 8 31 24 5 8 18 4 29 19 1 1 1 1 180
3‘ -. fi L& 2 R
a” G+
; a q 2 f% :: z
k. 2 S
640 Table 5.
V. Z&ate Biochemical
and Growth
Characteristics
et al.
of Lactococcus and Enterococcus Ripening
Strains Isolated
from Tenerife Cheese During
L. lactis ssp. lactis
L. lactis ssp. cremoris
E. faecalis
E. faecium
E. avium
30
8
31
24
5
No. of isolates CO2 from glucose Growth at 10°C Growth at 45°C Growth in pH 9.6 Growth in 4% NaCl Growth in 6.5% NaCl Growth in 0.1% methylene blue Growth in 0.04% K tellurite Survives 60°C 30 min Arginine hydrolysis Voges-Proskauer reaction P-Haemolysis Acid from: L-Arabinose Glucose Lactose Maltose Mannitol Melezitose Melibiose Raflinose Ribose Sorbitol Litmus milk testb
0” 30 0 0 30
0 8 0 0 8
0 31 31 22 31
0 24 24 17 24
0 5 5 3 5
0
0
31
24
4
29
8
31
24
0
0
0
24
16
0
30
8
31
24
5
30 13
0 4
31 31
24 24
0 5
0
0
4
0
0
2 30 28 30 14 6 2 0 30 1 RAC 18 AC 10 RA2
1 8 8 8 4 3 4 0 8 0 RAC 7 AC 1
aThe number included in the table correspond to the number bR, Reduction; A, acidification; C, coagulation.
ACKNOWLEDGEMENTS This work was financially supported by a grant from Exmo. Cabildo Insular de Tenerife. The authors are also grateful to ACORAN and Arico Sot. Coop. for their collaboration.
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0 31 31 31 31 28 0
1 31 19 RAC 28 RA 3 of positive
20 24 24 23 17 3 21 17 24 2 RAC 24
3 5 5 5 3 4 0 0 5 5 AC 3 RC 2
isolates in each test.
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Changes
in the microbial flora
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Mayeux, J. V., Sandine, W. E. and Elliker, P. R. (1962) A selective medium for detecting Leuconostoc organisms in mixed-strain starter cultures. Journal of Dairy Science 45, 655-656.
Medina, M., Gaya, P. and Nuiiez, M. (1992) Gredos goats’ milk cheese: microbiological and chemical changes throughout ripening. Journal of Dairy Research 59, 563566.
Mossel, D. A. A., Mengerink, W. H. J. and Scholts, H. H. (1962) Use of a modified MacConkey agar medium for the selective growth and enumeration of Enterobacteriaceae. Journal of Bacteriology 84, 38 1. Mundt, J. 0. (1986) Enterococci. In Bergey’s Manual of Systematic Bucteriology, ed. J. P. Butler, 6th edn, Vol. 2, pp. 106331065. Williams & Wilkins, Baltimore, MD. Nuiiez, M. (1978) Microflora of Cabrales cheese: changes during maturation. Journal of Dairy Research 45, 50 l-508. Nuiiez, M., Gaya, P. and Medina, M. (1985) Influence of manufacturing and ripening conditions on the survival of Enterobacteriaceae in Manchego cheese. Journal of Dairy Science 68, 794800. Poullet, B., Huertas, M., Sanchez, A., Caceres, P. and Larriba, G. (1991) Microbial study of Casar de Caceres cheese throughout ripening. Journal of Dairy Research 58, 231l 238.
Poullet, B., Huertas, M., Sanchez, A., Caceres, P. and Larriba, G. (1993) Main lactic acid bacteria isolated during ripening of Casar de Caceres cheese. Journal of Dairy Research 60, 1233127.
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641
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Rodriguez-Medina, M. L., Tornadijo, M. E., Carballo, J. and Martin-Sarmiento, R. (1995) Microbiological study of Leon cow-milk cheese, a Spanish craft variety. Journal oj Food Protection
Schleifer, Manual
57, 998-1006.
K. H. (1986)
Gram-positive
of Systematic
Bacteriology,
cocci. In Bergey’s ed. J. P. Butler, 6th & Wilkins, Baltimore,
edn, Vol. 2. pp. 999-l 103. Williams MD. Schleifer, K. H. and Kilpper-Balz, R. (1984) Transfer of Streptococcus faecalis and Streptococcus ,jaecium to the Genus Enterococcus nom. rev. as Enterococcus jaecalis comb. nov. and Enterococcus,faecium comb. nov.. International Journal
of Systematic
Bacteriology
34, 3 l-34.
Schleifer, K. H., Kraus, J., Dvorak, C., Kilpper-Balz, R., Collins, M. D. and Fisher, W. (1985) Transfer of Streptoc,occus lactis and related streptococci to the genus Lactococcus gen. nov.. Systematic and Applied Microbiology 6, 1833195.
Sharpe, M. E., Identification
Fryer, T. F. and Smith, D. G. (1966) of lactic acid bacteria. In Identification Methods ,jor Microbiologists, eds B. M. Gibbs and F. A. Skinner, pp. 65-79. Academic Press, London. Steele, J. L. (1995) Contribution of lactic acid bacteria to cheese ripening. In Chemistry of StructureFunction Relationships in Cheese, eds. E. L. Malin and M. H. Tunick, pp. 2099220. Plenum Press, New York. Tatini, S. R., Jezeski. J. J., Morris, H. A., Olson, J. C. and Casman, E. P. (1971) Production of staphylococcal enterotoxin A in Cheddar and Colby cheeses. Journal of Dairy Science
54, 8 155825.
Terzaghi, B. E. and Sandine, W. E. (1975) Improved for lactic streptococci and their bacteriophages. Microbiology
medium Applied
29, 807-8 13.
Tornadijo, M. E., Fresno, J. M., Bernardo, A., MartinSarmiento, R. and Carballo, J. (1995) Microbiological changes throughout the manufacturing and ripening of a Spanish goat’s raw milk cheese (Armada variety). Lait 75, 551-570.
ELSEVIER
Int. Dairy Journal 7 (1997) 635-641 ‘{: 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 09%6946/98/$19.00+0.00
SO958-6946(97)00065-4
Changes in the Microbial Flora of Tenerife Goats’ Milk Cheese During Ripening V. Z&ate*, F. Belda, C. PCrez and E. Cardell Departumento de Microhiologia y Biologia Celular, Universidad de La Laguna, La Laguna 38206, Tenertfe, Spain (Received
6 March
1997; accepted
6 October
1997)
ABSTRACT Changes in the microbial flora of Tenerife goats’ cheese, manufactured from raw milk without the addition of starter cultures, were studied during ripening. Lactic acid bacteria (lactococci, leuconostocs and lactobacilli) were the dominant microorganisms throughout ripening. Lactococcus Iactis ssp. lactis was the most common Lactococcus found (78.9% of isolates classified as lactococci). It reached maximum numbers in 2-day-old cheeses and decreased afterwards during ripening. Lactobacillus plantarum (56.9% of isolates classified as lactobacilli) and Lactobacillus paracasei ssp. paracasei (37.2% of isolates) were the main species of lactobacilli isolated. L. plantarum was present in high proportions in 2-day-old cheeses but decreased during ripening, whereas L. paracasei ssp. paracasei, present initially at low numbers, increased during ripening and was the predominant Lactobacillus in 60day-old cheeses. Leuconostoc mesenteroides ssp. dextranicum (81.8% of isolates classified as leuconostocs) was the main species identified as leuconostoc and its numbers decreased throughout ripening, particularly during the last month. Enterococcus faecalis and Enterococcus faecium, micrococci, moulds and yeasts were found at significant numbers and could contribute to the ripening process. Microorganisms used as an indicator of the bacteriological quality of the cheeses (Enterobacteriaceae, coliforms and faecal coliforms) and Staphylococcus aureus decreased sharply in the interior of the cheeses throughout ripening. Q 1998 Elsevier Science Ltd. All rights reserved
Keywords:
microbial
flora; lactic acid bacteria
INTRODUCTION
6 L of milk yield a cheese 15-18 cm in diameter, 81Ocm high and weighing 0.9-l .2 kg. Most of the cheeses are consumed fresh (after 2days), but some are ripened for 30 to 60days at 15-25°C and 75590% relative humidity. Because maturation acts as a natural selection process, during which possible pathogenic flora are normally inhibited by lactic acid bacteria (Nufiez et al., 1985) the consumption of fresh Tenerife cheese incurs a high risk of acquiring food-borne diseases. The aim of this study was to examine the changes in the microflora during the ripening of Tenerife goats’ milk cheese in order to identify the microorganisms which are present in high viable numbers during the ripening process. This is a preliminary step towards the preparation of a microbial starter suitable for the manufacture of Tenerife cheese on an industrial scale.
A number of fresh and ripened cheeses made from goats’ milk have been developed in Spain. The microbiological and physicochemical characteristics of some varieties such as Valdeteja (Gutierrez et ul., 1988), Majorero (Fontecha et al., 1990), Ibores (MasMayoral et al., 1991) Gredos (Medina et al., 1992), Valdeon (Lopez-Diaz et al., 1995) and Armada cheeses (Tornadijo et ul., 1995) have been studied. Tenerife cheese is a traditional farmhouse variety, with an annual production of about 1500 tonnes, manufactured by approximately 325 producers, from raw milk of local goats on the island of Tenerife. The milk is not heat-treated and it is not inoculated with a lactic culture. An animal rennet solution is prepared by homogenizing a small amount (approximately 1 g) of young goats’ stomach, previously salted and sun-dried, by hand or using a stem-type blender, in about 2030mL of tap water containing 5-10% NaCl. Coagulation takes place at 28-32°C for 3&60min. The coagulum is cut into pieces the size of a rice grain, left to settle in the bottom of a vat and the whey removed 15-20 min later. The curd is then transferred to clean aluminium rings and pressed by hand. The cheeses are salted by rubbing coarse salt on one side and the following day they are removed from the rings, inverted and salted on the other side. Approximately *Corresponding
MATERIALS
AND METHODS
Cheese manufacture
and sampling
Tenerife cheeses (0.9-1.2 kg) were manufactured at four different dairies by experienced cheesemakers following the traditional method, as described above. One batch of cheeses was manufactured at each dairy. For each batch, a sample of 2-, 30- and 60-day-old cheese (each sample consisted of one whole cheese), as well as a sample of the rennet and milk used in their
author. 635
636
V. Zrate
manufacture, were taken, transported to the laboratory under refrigeration (4°C) and kept at that temperature no longer than 24 h before analysis. Sampling of the surface of the cheeses was performed by scraping three different areas of the rind (Nufiez, 1978). Samples of the interiors of the cheeses were taken at a depth of 3cm below where the surface samples were taken (Poullet et al., 1991). In both cases, the three subsamples were pooled before analysis. Microbiological analysis
Cheese samples (10 g) were homogenized in 90 mL sterile 2% (w/v) of sodium citrate solution and decimal dilutions of rennet, milk and cheese homogenates prepared in l/4-strength Ringer solution (Unipath, Basingstoke, UK) were plated on the specific media required for the different microbial groups to be examined. Total viable counts were determined on plate count agar (PCA; Unipath) incubated at 30°C for 72 h. Lactococci were estimated on Ml7 agar (M17A; Unipath) incubated for 48 h at 30°C (Terzaghi and Sandine, 1975), and leuconostocs on Mayeux, Sandine and Elliker agar (MSEA; Mayeux et al., 1962) incubated for 72 h at 25°C. Rogosa agar (RA; Unipath) incubated at 30°C for 72 h in an anaerobic jar (Hz + CO2 Generbox; BioMerieux, Marcy l’Etoile, France) was used to estimate the numbers of lactobacilli, following the method of Nufiez (1978). Enterococci were enumerated on KF Streptococcus agar (KFSA; Unipath) incubated at 44°C for 48 h (Poullet et al., 1991), and Mcrococcaceae on mannitol salt agar (MSA; BioMCrieux) incubated for 72 h at 25°C (Chapman, 1945). Staphylococcus aureus was estimated on Baird-Parker agar (BPA; Unipath) plates incubated at 37°C for 48h (Baird-Parker, 1962) and confirmed by a positive coagulase test (BioMCrieux). Enterobacteriaceae were determined on violet red bile glucose agar (VRBGA; Unipath) plates incubated at 37°C for 24h, (Mossel et al., 1962). Coliforms were estimated according to Nufiez (1978) on violet red bile agar (VRBA; Unipath) incubated at 37°C for 24h, and faecal coliforms on the same medium but incubated at 44°C for 24 h, as described by Poullet et al. (1991). Yeasts and moulds were enumerated on potato dextrose agar (PDA; Unipath) acidified to pH 3.5 with tartaric acid and incubated at 26°C for 96 h (Nuiiez, 1978). Each dilution (1 mL) was inoculated, in duplicate, using the pour plate technique for PCA or the pour plate and overlay technique for M 17A, RA, KFSA, VRBGA and VRBA. Each dilution (0.1 mL) was inoculated, in duplicate, using the surface spread technique for MSEA, MSA, BPA and PDA. Results were expressed as logarithm (log) of colony forming units (cfu) per millilitre for milk and rennet, and log cfu g-’ for cheeses. Isolation and identification of bacterial species
For each batch, live to eight colonies were taken randomly for each sampling point in the interior of the cheeses (2, 30 and 60 days) from M17A, RA and MSEA plates. The isolates were purified in ManRogosa-Sharpe (de Man et al., 1960) (MRS; Unipath) agar and stored in MRS broth containing 20% (v/v) glycerol, at -80°C. A total of 180 strains (five from
et al. each sampling point, type of plate and batch) were further characterized. Although recent methodologies tend towards a higher number of isolates from each plate, the number of colonies selected was determined following protocols duly described in the applied scientific literature (Nufiez, 1978; Freitas et al., 1996). The criteria of Sharpe et al. (1966) were followed for differentiating lactococcci, enterococci, leuconostocs and lactobacilli. Identification at the species level of and Lactococcus Enterococcus was performed according to the criteria of Schleifer and Kilpper-Balz (1984), Schleifer et al. (1985) and Schleifer (1986). The API 50 CH system with API 50 CHL Medium (BioMCrieux) was used to identify the leuconostocs and lactobacilli at the species level. Physicochemical
analysis
Cheese pH, percentage moisture, NaCl content (percentage of total weight) and titratable acidity were assayed by standard methods (Bradley et al., 1993). Statistical treatment of results
Analyses of variance (ANOVAs) were performed on results obtained at different stages of ripening, with dairy and cheese age as main effects, using program SOLO 2.0, GLM-ANOVA (BMDP Statistical Software, Los Angeles, CA, USA).
RESULTS
AND DISCUSSION
Changes in the microbial group counts during ripening
The microbial counts in the raw goats’ milk used for the manufacture of Tenerife cheese, as well as in the interior of cheeses during ripening, are shown in Table 1. Mcrococaceae was the predominant microbial group in the rennet used in the manufacture of the cheeses, with levels 2-4 log units higher than that of the other and coliform groups, whereas Enterobacteriaceae numbers were low (around 1.5 log units as the average) (not shown). Lactic acid bacteria (mainly lactococci and leuconostocs) were the most abundant microbial groups in milk, being l-2 log units higher than the other groups. The counts for coliforms and faecal coliforms were similar to the values reported for goats’ milk used in the manufacture of Majorero, Gredos and Valdeon cheeses (Fontecha et al., 1990; Medina et al., 1992; Lopez-Diaz et al., 1995) and are a consequence of the lack of hygiene during milking and manipulation of the milk before cheesemaking. Due to the multiplication of bacteria and the retention of microorganisms in the curd after drainage of the whey (Tatini et al., 1971) most microbial groups increased by 2-3 log cycles from milk to 2-day-old cheeses. A similar increase in the microflora has been observed in goats’ and ewes’ cheeses (Fernandez de1 Pozo et al., 1988; Poullet et al., 1991; Medina et al., 1992) and was accompanied by a sharp decline in pH, due to the production of lactic acid (Table 3) by the high numbers of lactic acid bacteria (mainly lactococci and leuconostocs) (Table 1).
Changes in the microbialjora Table 1.
Medium
PCA M17A RA MSEA KFSA MSA BPA VRBGA VRBA VRBA PDA PDA
qf Tenerife goats’ milk cheese
637
Microbial Flora of the Interior of Tenerife Cheese During Ripening
Microbial group expected Total viable counts Lactococci Lactobacilli Leuconostocs Enterococci Micrococcaceae S. aureus Enterobacteriaceae Coliforms Faecal coliforms Yeasts Moulds
Milk”
Time of ripening (days)
5.54f2.44 5.39f2.62 3.59&l .32 5.10f2.70 3.75f3.12 4.52zt2.98 2.58&l .04 4.52f3.64 4.39f3.60 3.6211.16 3.78f 1.90 2.041tl.50
2b
3ob
60b
9.261t1.70 9.2411.54 5.38fl.80 7.6911.40 6.96f2.62 5.7913.12 3.141t4.26 7.12zt1.20 6.98f0.98 4.59f4.86 3.8913.86 2.20f2.38
9.2551.34 9.33Ito.44 7.16z!zO.86 7.68f1.44 6.97f1.78 5.6011.20 1.62f3.94 4.89f4.38 3.55zt5.96 1.91f5.32 4.66&l .86 2.87f2.50
8.74f0.46 8.62f0.58 7.5811.64 7.59f0.80 6.8511.44 5.91fl.46 Absence in 0.01 g 3.90zt4.42 3.31f5.62 cl 4.2811.46 3.9111.92
Data are average values&2 SD (95% CI) of four batches. Units: “log cfu mL_‘; blog cfu g ‘. (Devoyod and Miiller, 1969; Devoyod and Desmazeaud, 1970). Micrococcaceae were found at significant numbers in the cheeses at the three sampling times, particularly on the surface (Table 2) and, due to their lipolytic activity, may contribute to flavour development in these cheeses during ripening. S. aureus numbers decreased significantly (PC 0.05) in the interior of the cheeses with time and were not detected in 60-day-old cheeses. No significant change was seen in the number of yeasts throughout maturation whereas moulds, initially present at low numbers, increased during the last month of ripening. These groups may contribute to the ripening process by utilizing lactic acid, and by their proteolytic and lipolytic activity (Macedo et al., 1995). The numbers of microorganisms indicative of the bacteriological quality (Enterobacteriaceae, coliforms, faecal coliforms) were high, suggesting considerable contamination of the cheeses that may occur during milking and/or manufacturing. Furthermore, the dairy had a significant (P < 0.05) influence on the numbers of Enterobacteriaceae in the interior and surface of the cheeses. Ripening time had a significant (PC 0.05) influence on the numbers of Enterobacteriaceae, coliforms and faecal coliforms, with counts in the interior of fully ripened cheeses 34 log units lower than that of
Lactic acid bacteria were the dominant microorganisms throughout ripening. Lactococci decreased during the second month of ripening, whereas lactobacilli increased significantly (P c: 0.05) during maturation, probably due to the ability of this genus to grow at low pH values (Nufiez, 1978) and to resist high salt concentrations (Sharpe et al., 1966). Lactic acid bacteria influence the ripening process in various ways by the production of lactic acid, by a decrease in the oxidation-reduction potential, and by proteolysis and lipolysis (Steele, 1995). Numbers of enterococci, on which the particular dairy had a significant effect (P < O.Ol), were similar to those observed in other varieties of cheeses manufactured from raw ewes’ or goats’ milk (Fernandez de1 Pozo et al., 1988; Poullet et al., 1991; Tornadijo et al., 1995). The presence of a high number of enterococci could be due to poor hygienic practices during the manufacturing process, the presence of domestic animals in the dairy and the resistance of enterococci to adverse conditions (high temperatures, high salt concentration and high acid levels) (Mundt, 1986). Enterococci may influence the ripening process due to their caseinolytic and lipolytic activity and their ability to stimulate the production of acid by some lactococci and gas production by leuconostocs
Table 2. Medium
PCA M17A RA MSEA KFSA MSA BPA VRBGA VRBA VRBA PDA PDA
Microbial Flora of the Surface of Tenerife Cheese During Ripening
Microbial group expected Total viable counts Lactococci Lactobacilli Leuconostocs Enterococci Micrococcaceae S. aweus Enterobacteriaceae Coliforms Faecal coliforms Yeasts Moulds
Data are average yaluesf2 SD (95% CI) of four batches. Units: ^log cfu g
Time of ripening (days) 2”
30”
60”
8.65f2.24 8.73f1.94 5.1612.10 7.32f1.08 6.27f3.04 5.75kl.88 2.82f4.90 6.66zt1.40 6.27*1.08 5.59f0.90 5.2511.00 2.9811.94
9.4510.88 9.54f0.68 8.02zt1.38 8.43zt1.84 6.53f1.04 8.15zt0.62 c2 7.70+2.78 7.6012.82 5.67f0.34 6.901t1.52 5.04k4.22
8.78f0.54 8.6X&0.74 7.281tl.72 7.57-f-0.86 6.33h1.02 7.7010.66 Absence in 0.01 g 5.78f4.96 5.26f3.90 I .87&3.40 6.92f2.12 7.4450.86
638
V. Z&ate et al. Table 3.
Parameter
PH Moisture (%) NaCl content (% total weight) Titratable acidity (% lactic acid)
Physicochemical
Parameters of Tenerife Cheese During Ripening
Milk
6.42f0.34 0.22&O. 16 0.08f0.03
Time of ripening (days) 2
30
60
4.93f1.38” 4.95f0.56b 53.6*5.62a 51.3&9.84b 3.72*1.64a 6.54f 1.22b 0.17&0.16a 0. 19f0.06b
4.64f1.78a 4.70*0.66b 53.2&l 1.9a 44.0f6.92b 4.36&O/W 5.14&0.98b 0.24ztO. 14a 0.15f0.04b
4.65&l .98= 5.20&0.54b 51.0f8.94” 32.7&2.58b 6.10~t1.34~ 7.25f7.34b 0.23f0.12a 0. 12+0.06b
aInterior of the cheeses. bSurface of the cheeses. Data are average values+2 SD (95% CI) of four batches.
2day-old cheeses (Table 1). Various factors contribute to the decline of these groups in the interior of cheese during ripening; these include a significant (PC 0.01) increase in the concentration of NaCl and the inhibition of these bacteria by lactic acid bacteria (Nufiez et al., 1985), basically by causing a decrease in the pH and an increase in lactic acid concentration (Table 3). The numbers of these groups on the surface were about 2 log cycles higher than in the interior (Table 2) probably due to higher pH values (Table 3) and aeration. Species isolated and identified during ripening
Table 4 shows the species isolated on M17A, MSEA and RA from the interior of the cheeses, throughout ripening. The three isolation media used permitted the growth of the different groups of lactic acid bacteria but with different degrees of selectivity. M17A showed moderate selectivity for the isolation of lactococci (42% of the isolates obtained on this medium were classified as lactococci), considered as Gram-positive, catalase-negative, homofermentative cocci that grow at 10°C but not at 45°C. The selectivity of MSEA for the isolation of leuconostocs (Gram-positive, catalase-negative, heterofermentative cocci) was very low (only 17% of the isolates obtained on MSEA were classified as leuconostocs). These two media permitted the growth of a high number of enterococci (33 and 62% of the isolates, from M17A and MSEA, respectively), considered as Gram-positive, catalase-negative, homofermentative cocci that grow at 10°C and 45°C and in 6.5% NaCl. In contrast, RA showed a high selectivity for lactobacilli (Gram-positive, catalase-negative rods) because 70% of the isolates on this medium were classified as lactobacilli. These degrees of selectivity are in line with the results reported by other authors (Poullet et al., 1993; Tornadijo et al., 1995). Lactococcus lactis ssp. lactis was the most abundant Lactococcus found in Tenerife cheese (30 of 38 isolates classified as lactococci). This species reached maximum numbers in 2-day-old cheeses and then decreased during ripening (Table 4) probably as a consequence of the inhibitory effect of low pH and high NaCl content (Table 3), in a similar way as has been observed by other authors for different varieties of
Spanish cheese (Rodriguez-Medina et al., 1995; Tornadijo et al., 1995). The remaining eight lactococci were identified as L. lactis ssp. cremoris on the basis of their inability to hydrolyse arginine (Table 5). Homofermentative lactobacilli such as Lactobacillus pluntarum (29 of 51 isolates classified as lactobacilli) and L. paracasei ssp. paracasei (19 of 51 isolates) were the principal species of lactobacilli isolated. L. plantarum was present at high proportions in 2-day-old cheeses and decreased slightly during ripening, whereas L. paracasei ssp. paracusei, .present initially at low numbers, increased and was the predominant Lactobacillus in 60-day-old cheeses (Table 4). These two species were also the principal lactobacilli isolated from other varieties of cheese manufactured with raw milk (Nufiez, 1978; Macedo et al., 1995; Centeno et al., 1996; Freitas et al., 1996). On the other hand, heterofermentative lactobacilli, i.e., Lactobacillus brevis and L. brevis ssp. lindneri, were also isolated from Tenerife cheese although at very low numbers (one isolate each). Dextran-producing Leuconostoc leuconostocs, mesenteroides ssp. dextranicum (18 isolates) and L. mesenteroides ssp. mesenteroides (four isolates), were the leuconostocs identified in Tenerife cheese and both decreased throughout ripening, especially during the last month. In general, enterococci were very abundant in all the cheeses, with Enterococcus faecalis the predominant species, followed by Enterococcus faecium and, to a lesser extent, by Enterococcus avium (Table 4). Some of the enterococci isolates exhibited atypical patterns in some biochemical or growth characteristics: 18 out of 60 enterococci were unable to grow at pH 9.6; of 31 isolates identified as E. faecalis, seven did not produce
black colonies when grown in 0.04% potassium tellurite, three did not produce acid from melezitose and one fermented raflinose; among the 24 isolates identified as E. faecium, three were unable to produce acid from melibiose (Table 5). E. faecalis has also been described as the dominant Enterococcus species in other raw goats’ milk cheeses such as Majorero (Fontecha et al., 1990), Ibores (Mas and GonzilezCrespo, 1992) and Armada (Tornadijo et al., 1995). In contrast, E. faecium was the most abundant Enterococcus in Picante ewes’ and goats’ milk cheese (Freitas et al., 1996).
Results are the total from four batches No.. Number of isolates.
L. lactis ssp. lactis L. lactis ssp. cremoris E. ,faecalis E. faecium E. avium Unidentified streptococci L.mesenteroides ssp.mesenteroides L. mesenteroides ssp. dextranicum L. plantarum L. paracasei ssp. paracasei L. brevis L. brevis ssp. lindneri Unidentified lactobacilli Non-lactic acid bacteria Total
6 _
of cheeses.
1 20
_ _
_ _ _ _ 20
5 1 3 1
1 7 1 _
2
1 _
11 _
20
_ _
1
5 1 9
1
1 2 _
1 60
_
13 2 1 13 1 3 10 2 12 2 _
1.7 100
_ _
21.7 3.3 1.7 21.7 I.7 5.0 16.6 3.3 20.0 3.3
20
_
2 1 _
1 2 1 1 6
6
20
_ _
2 1 2 1
2 2 8 2
20
1 1 _
6 7 _
2 2 1 _ _
60
1
1
3 7 2 9 8 _
1
10 4 10 4
100
1.7 1.7 _
16.7 6.6 16.7 6.6 1.7 5.0 11.7 3.3 15.0 13.3 _
20
~ ~~
6 2 4 3 3 2 ~ -
20
_ _ _ _
1
1 _
15 3
_
20
7 9 1
60
_
8 9 1
3 2 1 _
_ _ _
I
2 20
I
1
1
_
1
100
__
13.3 15.0 1.7
11.7 3.3 33.3 11.7 5.0 3.3 1.7 _
30 8 31 24 5 8 18 4 29 19 1 1 1 1 180
3‘ -. fi L& 2 R
a” G+
; a q 2 f% :: z
k. 2 S
640 Table 5.
V. Z&ate Biochemical
and Growth
Characteristics
et al.
of Lactococcus and Enterococcus Ripening
Strains Isolated
from Tenerife Cheese During
L. lactis ssp. lactis
L. lactis ssp. cremoris
E. faecalis
E. faecium
E. avium
30
8
31
24
5
No. of isolates CO2 from glucose Growth at 10°C Growth at 45°C Growth in pH 9.6 Growth in 4% NaCl Growth in 6.5% NaCl Growth in 0.1% methylene blue Growth in 0.04% K tellurite Survives 60°C 30 min Arginine hydrolysis Voges-Proskauer reaction P-Haemolysis Acid from: L-Arabinose Glucose Lactose Maltose Mannitol Melezitose Melibiose Raflinose Ribose Sorbitol Litmus milk testb
0” 30 0 0 30
0 8 0 0 8
0 31 31 22 31
0 24 24 17 24
0 5 5 3 5
0
0
31
24
4
29
8
31
24
0
0
0
24
16
0
30
8
31
24
5
30 13
0 4
31 31
24 24
0 5
0
0
4
0
0
2 30 28 30 14 6 2 0 30 1 RAC 18 AC 10 RA2
1 8 8 8 4 3 4 0 8 0 RAC 7 AC 1
aThe number included in the table correspond to the number bR, Reduction; A, acidification; C, coagulation.
ACKNOWLEDGEMENTS This work was financially supported by a grant from Exmo. Cabildo Insular de Tenerife. The authors are also grateful to ACORAN and Arico Sot. Coop. for their collaboration.
REFERENCES Baird-Parker, A. C. (1962) An improved diagnostic and selective medium for isolating coagulase-positive staphylococci. Journal of Applied Bacteriology 25, 12-19. Bradley, R. L., Arnold, E., Barbano, D. M., Semerad, R. G., Smith, D. E. and Vines, B. K. (1993) Chemical and physical methods. In Standard Methods for the Examination of Dairy Products, ed. R. T. Marshall, pp. 433-53 1. American Public Health Association, Washington, DC. Centeno, J. A., Cepeda, A. and Rodriguez-Otero, J. L. (1996) Lactic acid bacteria isolated from Arzua cows’ milk cheeses. International Dairy Journal 6, 65-78. Chapman, G. H. (1945) The significance of sodium chloride in studies of staphylococci. Journal af Bacteriology 50, 201203. de Man, J. C., Rogosa, M. and Sharpe, M. E. (1960) A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 130-l 35.
0 31 31 31 31 28 0
1 31 19 RAC 28 RA 3 of positive
20 24 24 23 17 3 21 17 24 2 RAC 24
3 5 5 5 3 4 0 0 5 5 AC 3 RC 2
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