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Behaviour of Staphylococcus aureus strains FRI 137 and FRI 361 during the manufacture and ripening of manchego cheese
Int. Dairy Journal 3 (1993) 85-96
Nd Behaviour of Staphylococcus aureus Strains Fill 137 and FRI 361 During the Manufacture and Ripening of Manchego Cheese
A. O t e r o , M. C. G a r c i a , M. L. G a r c i a , J. A. S a n t o s & B. M o r e n o Department of Food Hygiene and Food Technology, Veterinary Faculty, University of Le6n, 24071-Le6n, Spain (Received 4 December 1991; revised version accepted 25 March 1992)
ABSTRACT The behaviour of Staphylococcus aureus strains FRI 137 (enterotoxin C 1 producer) and FRI 361 (enterotoxin C2 producer) during production and ripening of Manchego cheese manufactured with two levels of starter (0. O1 or 1%) was evaluated. Both strains grew well (four to eight generations), but only in the cheeses manufacturated with 0.01% of starter. The population of S. aureus increased during manufacture and decreased as ripening progressed. In spite of having reached S. aureus populations above 107 cfu/g, no C1 or C2 enterotoxin was detected. In the cheeses in which significant growth of S. aureus had occurred, thermonuclease was detected after pressing and it persisted during ripening. The presence of thermon uclease in Manchego cheese implies significant S. aureus multiplication, its use being recommended in a screening test.
INTRODUCTION Even t h o u g h the standard practices of refrigeration a n d pasteurization of milk (before cheesemaking) have greatly reduced cheese-induced illness, this product is still associated with outbreaks of staphylococcal food p o i s o n i n g (Bergdoll, 1989), T h e growth o f Staphylococcus aureus a n d in some cases the p r o d u c t i o n 85 Int. Dairy Journal 0958-6946/92/$05.00© 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland
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A. Otero, M. C. Garcia. M. L. Garcia, J. A. Santos, B. Moreno
of thermonuclease and enterotoxins during the manufacture and ripening of various types of cheese have been studied by several researchers (IDF, 1980; Khalid & Harrigan, 1984; Ntifiez et al., 1986). However, the results obtained have varied depending on, among other factors, the type of cheese and the nature and activity of the starter culture used. For this reason, any generalizations dealing with the behaviour of S. aureus in types of cheese different from those studied should not be made. The growth of staphylococcal populations in Manchego cheese has been studied by Rom~in-Pifiana (1975), Martinez-Moreno & Ntifiez (1976), Ord6fiez etal. (1978), Ramos etal. (1981) and Gaya etal. (1988), but the production ofenterotoxin in this type ofsheeps' milk cheese has been investigated only recently (G6mez-Lucia et al., 1986; Ntifiez et al., 1988). G6mez-Lucia et al. (1986) investigated the behaviour of an enterotoxin A producing strain of S. aureus ($6), but the majority of the S. a u r e u s enterotoxigenic strains of ovine origin produce only enterotoxin C (Guti6rrez et al., 1982). Moreover, results obtained by G6mez-Lucia et al. (1986) are not in accordance with those reported by Ntifiez et al. (1988), who studied the behaviour of an enterotoxin C producing strain of S. aureus and another multiple enterotoxin (A, B, C and D) producer. On the other hand, the manufacture of Manchego cheese from raw milk is frequent in Spain (Ntifiez et al., 1981). In this country the contamination by staphylococci of sheeps' milk, which is used in the manufacture of cheese, can reach high levels (Bautista et al., 1986). Moreover, the hygienic conditions of manufacture and/or ripening of the cheeses are often overlooked (Garcia et al., 1987). Determination of thermonuclease activity in foods as an indicator of the possible presence of enterotoxins is recommended, especially in fermented foods (National Research Council, 1985). Even though it is generally accepted that there is a good correlation between enterotoxin and thermonuclease (Tatini et al., 1976), on some occasions cheese samples free from thermonuclease, but containing enterotoxins, have been reported (Todd et al., 1981). The purpose of this study was to investigate the behaviour of two enterotoxin C producing S. a u r e u s strains during the production and ripening of Manchego cheese made with two levels of starter. MATERIALS AND METHODS Cultures S t a p h y l o c o c c u s aureus strains FRI 137 and FRI 361, producing entero-
toxins C~ and C2, respectively, were used separately to inoculate cheese
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
87
milk. Both strains were obtained from Professor M. S. Bergdoll, Food Research Institute, University of Wisconsin, Madison, USA. After 24 h growth at 37°C (with shaking) in '3 + 1' broth (3% NZ Amine NAK~ Humko Sheffield Chemical, Lynhurst, N J) and 1% yeast extract (Difco, Detroit, MI), strain FRI 137 produced 19pg/ml of enterotoxin C1, and strain FRI 361, 28.6pg/ml of enterotoxin C2. Corresponding figures for thermonuclease were 20.9jug/ml (strain FRI 137) and 0-8pg/ml (FRI 361), as reported in previous investigations (Otero et aL, 1990). Cultures of S. aureus were grown in brain heart infusion broth (BHI, Difco) at 37°C for 18 h and then washed twice in 0.1% peptone water (w/v). An appropriate volume of the preparation was added to pasteurized milk to provide about 10 4 t o 10 5 cfu/ml. Starter culture HM1 (Miles Martin, S.A.E., Madrid, Spain), containing Lactococcus lactis subsp, lactis, L. lactis subsp, cremoris and L. lactis subsp. lactis biovar, diacetylactis, was used in the manufacture of Manchego. The starter culture was incubated in sterile 12% skim milk (Unipath Ltd, Basingstoke, Hampshire) at 23°C for 16 h, and its activity was evaluated (taking into account the supplier's recommendations) on the basis of the acidity developed. For each S. aureus strain, cheeses were manufactured at two levels of starter: 0-01% (simulating an initial failure) and 1% (normal level).
Manufacture and sampling of cheese All cheeses were made on the same day and from the same batch of milk. Raw ewes' milk, with a pH of 6.70 and a count of 1.8 × 10v mesophiles/ ml, was used. After pasteurization, counts of mesophiles were 2-0 × 104 cfu/ml of milk. Sheeps' milk was pasteurized in a pilot plant-size plate pasteurizer at 72°C for 15 s, cooled at 30°C and inoculated with the lactic starter culture. The S. aureus strain (FRI 137 or FRI 361) and 0-1% CaC12 solution were added at separate stages. After 30 min, commercial rennet (Ha-La, Chr. Hansen, Copenhagen, Denmark) was also added and coagulation took place in 40 min. The coagulum was cut into 4 to 6 mm cubes and heated to 37°C over 15 min. After stirring (cubes of curd and whey) for 10 min at 37°C, three blocks of curd (20 cm diameter × 8 cm high) were obtained from each vat; these were pressed (3 kg/cm 2) for 18 to 20 h and salted by submersion in a 20% NaC1 (w/v) solution at 10°C for 48 h. The cheeses were ripened for 3 months at 10 to 12°C and 85% RH. The following samples were taken: (1) from milk after addition of S. aureus and lactic starter; (2) from the curd before pressing; (3) from the cheese after pressing; and (4) from the cheese after salting, and throughout ripening on day 7, 15, 30, 60 and 90. Sampling was carried out
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according to Law et al. (1973). Subsamples for thermonuclease (20 ml or 20g) and enterotoxin (100 ml or 100g) determinations were kept at -30°C until analysed. Cheeses without added S. aureus were also made as controls.
Analysis For each of the samples, a microbiological analysis, a pH measurement and a determination of thermonuclease and of enterotoxin were carried out. Enumeration o f bacteria a n d p H measurement
Samples (10 ml of milk or 10 g of curd or cheese) were prepared as recommended by the American Public Health Association (Richardson, 1985). Mesophilic aerobes were enumerated by the standard plate count (Richardson, 1985). Counts of S. aureus were effected on Baird-Parker medium (Unipath), according to ICMSF (1978). The pH values were determined using a pH meter (model PHM82, Radiometer, Copenhagen, Denmark) after homogenizing 12-5 g of cheese with 17 ml of distilled water (Perry & Sharpe, 1960). The above analyses were performed immediately after sampling. Thermonuclease determination
The enzyme was extracted according to the method of Cords & Tatini (1973), slightly modified in that 0.05 M glycine buffer at pH 10.0 was used instead of 0.05 M Tris buffer at pH 9.0 (Kamman & Tatini, 1977). Nuclease activity of the boiled extract (15 min) was determined as proposed by Ibrahim (1981). The amount of enzyme in cheeses was estimated as described by Otero et al. (1987a). The sensitivity of the assay was 1 ng of thermonuclease per 20 g of cheese. Enterotoxin assay
Enterotoxins were extracted according to the method of Freed et al. (1982) and concentrated by dialysis against polyethylene glycol 20M (Serva, Heidelberg, Germany). The sandwich ELISA technique ofFey et aL (1984) was used to detect and quantify enterotoxins C~ and C2. Reagents for the ELISA test were obtained from W. Bommeli, Bern, Switzerland. For control purposes, enterotoxins C~ and C2 (from the Food Research Institute, Madison, WI) were added to cheeses (enterotoxin free), extracted and quantified. The mean recovery of enterotoxin added was 67%. The minimum enterotoxin concentration detected was 1.5 ng/100 g of cheese.
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
89
RESULTS A N D DISCUSSION Results of the determinations made on Manchego cheese during manufacture and ripening are given in Tables 1 (S. aureus strain FRI 137 and 0.01% starter), 2 (FRI 137 and 1% starter), 3 (FRI 361 and 0.01% starter) and 4 (FRI 361 and 1% starter). Changes in aerobic mesophilic plate counts The changes observed were similar to those reported previously by Martinez-Moreno & Nrfiez (1976) and Ord6fiez et al. (1978). As might be expected, at the early stage of production, counts increased (up to salting) and then decreased during salting. Additional growth occurred in cheese during the first week of ripening, after which there was a decrease in population. Behaviour of S. a u r e u s An increase in the number ofS. a u r e u s occurred during the time between inoculation and the end of salting (S. a u r e u s strain FRI 361) or only until TABLE 1 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FRI 137 and 0.01% of Starter
Sample (days) Oh" b Ic 3d 7e 15e 30 e 60 e 90 e
Mesophiles S. aureus (log cfu/ml org) (logcfu/ml org) 4-84 5.53 8-54 8.15 8.64 8.08 7.70 7.34 7.38
4-46 5-30 6.79 6.79 4.54 3.64 2.65 <2 <2
Enterotoxin Thermonuclease C~ (n/g of cheese) ND ND ND ND ND ND ND ND ND
~0h = milk after inoculation with S. aureus and starter. bCurd before pressing. cSample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
ND ND 0.8 0.5 0.6 0.6 0-5 0.4 0.6
pH NI 6.74 5,25 5,15 5.16 5.21 5.22 5-19 5-19
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TABLE 2 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain F RI 137 and 1% of Starter
Sample (days) Oh~ b 1" 3d 7" 15e 30 e 60 e 90 e
Mesophiles (log cfu/ml or g)
Enterotoxin Cz
Thermonuclease
pH
(log cfu/ml or g)
6.40 7-30 8.38 8.08 8.41 8.04 7.30 7-00 6-89
5.30 6-45 6.53 6.04 4.36 4.26 3.60 2.30 <2
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
NI 6-58 5.15 5.16 5.14 5.19 5.12 5.13 5.17
S. aureus
a0h = milk after inoculation with S. aureus and starter. hCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
TABLE 3 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FR1361 and 0.01% of Starter
Sample
Oha --~ 1' 3a 7e 15~ 30 e 60 e 90 e
Mesophiles (log cfu/ml or g)
Enterotoxin C:
Thermonuclease (ng/g of cheese)
pH
(log cfu/ml or g)
4-83 5.82 8.77 8.30 8.60 7-96 7.63 7.00 7.00
4-23 5.59 7.43 7.59 7.23 7.18 6.28 5.18 4.45
ND ND ND ND ND ND ND ND ND
ND ND 1.6 0-2 0.2 0.2 0.2 0.2 0.2
NI 6-64 5.25 5.13 5.22 5.26 5.20 5-25 5.24
S. aureus
a0h = milk after inoculation with S. aureus and starter. hCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
91
TABLE 4 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FR1361 and 1% of Starter
Sample (days) Oh~ b 1" 3d 7e 15e 30 e 60 e 90 e
Mesophiles S. aureus (log cfu/ml or g) (log cfu/ml or g) 6-40 7.25 8.26 8.08 8.64 8.11 7.39 6-00 5.75
5.23 6-20 6.43 6.51 5.45 5.34 4-66 3-76 3.41
Enterotoxin C2
Thermonuclease
pH
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
NI 6.65 5-17 5-08 5.12 5.10 5.02 5.04 5-07
~0h = milk after inoculation with S. aureus and starter. bCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
the end of pressing (S. a u r e u s strain FRI 137). Afterwards, the number decreased for both strains during ripening and for strain FRI 137 even to below 102/g. The increase in staphylococcal counts in curd over those in inoculated milk is largely attributable to the physical concentration of S. a u r e u s cells in the coagulum. According to Tatini et al. (1971), this phenomenon usually represents a 10-fold increase. The general patterns of the growth and survival for the test strains were different, depending on the level of starter. With 1%of starter, the number of staphylococci hardly increased, whereas with 0.01% starter there were increases of up to eight generations for strain FRI 361 and four or five for strain FRI 137 (excluding the 10-fold increase in numbers caused by concentration on converting milk into cheese). Taking into account that strains FRI 137 and FRI 361 grow in ewes' milk up to levels of 9-18 log cfu/ml and 10-07 log cfu/ml (Otero et al., 1987b), the above data indicate, once again, that use of an effective starter culture is critical in order to control S. a u r e u s growth in cheese (Stadhouders et al., 1978). A gradual decrease in the numbers of S. a u r e u s during ripening is a common fact for ripened cheeses (Ntifiez & Martinez-Moreno, 1976). According to other results obtained by us (Otero et al., 1988) and other workers (Stadhouders et al., 1978), this may be the result of competition
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A. Otero, M. C. Garcia, M, L. Garcia, J. A. Santos, B. Moreno
with lactic acid bacteria. During ripening of Manchego cheese, Gaya et al. (1988) also reported a strong effect o f S t r e p t o c o c c u s lactis on counts of S. a u r e u s (a 5.8-fold reduction). Undoubtedly, other factors, such as lactic acid and salt content, low pH, etc., have also negative effects on survival of S. a u r e u s (Reiter et al., 1964). The decrease in the numbers ofS. a u r e u s during storage of Manchego cheese to below 102/g, as well as the different resistance of staphylococcal strains to environmental conditions associated with ripening, confirms the low reliability ofS. a u r e u s determination for assessment of the extent of staphylococcal growth in Manchego cheese. Enterotoxin
Enterotoxin C1 or C2 could not be detected in any of the cheeses, even when counts of S. a u r e u s in some samples obtained from low-starter cheeses were greater than 107/g. A similar finding has been reported by NOfiez et al. (1988) for Manchego cheese inoculated with t w o S. a u r e u s strains producing enterotoxins A, B, C and D, and C, respectively. Data obtained previously (Otero et al., 1988) suggest that the influence of lactic acid bacteria on enterotoxin C1 or C2, by inhibition of toxin synthesis and destruction of produced toxins, explains the lack of detectable amounts of enterotoxins in Manchego cheese despite the attainment of high S. a u r e u s populations. Another explanation for these results may be that the maximal S. a u r e u s populations attained did not reach the minimal levels associated with the presence of detectable amounts of enterotoxins in tyndallized ewe's milk inoculated with pure cultures of strains FRI 137 or FRI 361 (Otero et al., 1987b). Lower S. a u r e u s populations produced enterotoxin A in a similar study on Manchego cheese (G6mez-Lucia et al., 1986). It must be noted, however, that synthesis of enterotoxin A seems to be less affected by adverse environmental conditions than that of other enterotoxins (Notermans et al., 1984). Differences in the type of strain ofS. a u r e u s as well as in the type of starter culture used by G6mez-Lucia et al. (1986), Nffiez et al. (1988) and ourselves may also contribute to the above divergent results. Thermonuclease
No detectable thermonuclease was observed in any of the cheeses produced with 1% starter inoculum. This results from the inhibition of staphylococcal growth (Tables 2 and 4). Of the cheeses made with 0.01% starter activity, thermonuclease was detected in all samples obtained
Behaviour orS. aureus during manufacture and ripening of Manchego cheese
93
after pressing. During ripening, the thermonuclease content did not change significantly in cheeses made from milk inoculated with strain FRI 137 (Table 1), whereas extensive thermonuclease inactivation occurred in cheeses inoculated with strain FRI 361 (Table 3). The fact that the highest amounts of enzyme obtained in Manchego cheese were 1.2 ng/g of cheese (strain FRI 361) and 0.8 ng/g of cheese (strain FRI 137), while similar figures in '3 + 1' broth were 0.8 and 20.9 #g/ml, respectively (Otero et al., 1990), suggests that the substrate and/or starter had an inhibitory influence on thermonuclease production, which was stronger for strain FRI 137 than for strain FRI 361. Inactivation of thermonuclease in Manchego cheese has also been observed by Ntifiez et al. (1988). Since thermonuclease was detected when extensive growth ofS. aureus occurred and it remained detectable after 90 days of storage, the presence of thermonuclease could be used to assess staphylococcal growth and possible starter failure in Manchego cheese. However, the amount of enzyme found does not provide reliable information due to the variability of thermonuclease production by S. aureus, which depends on the test strain and growth conditions. Although the presence of thermonuclease was not associated with that of enterotoxin C~ or C2 in our study, it would seem advisable not to rule out the validity of this determination in screening tests for Manchego cheese. A positive result indicates that S. aureus may have reached a hazardous level, which, although it has not resulted in the presence ofenterotoxin C~ or C2, may have done so for other enterotoxins (G6mez-Lucia et al., 1986). ACKNOWLEDGEMENTS This work was supported in part by a grant from the Spanish CAICYT, Project No. 4466-79. Junta de Castilla y Le6n (Consejeria de Educaci6n y Cultura) and Diputaci6n Provincial de Le6n have also contributed. We are indebted to Professor M. S. Bergdoll and to Miles-Martin S.A.E. for providing enterotoxins, antisera and S. aureus strains, and the starter culture HM1, respectively. We are also indebted to Drs M. Nfifiez and M. de Paz, INIA, Madrid, Spain, for helping us in cheese making. REFERENCES Bautista, L., Bermejo, M. P. & Nfifiez, M. (1986). Seasonal variation and characterization of Micrococcaceae present in ewes' raw milk. J. Dairy Res., 53, 1-5.
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Bergdoll, M. S. (1989). Staphylococcus aureus. In Foodborne Bacterial Pathogens, ed. M. P. Doyle. Marcel Dekker, New York, pp. 463-523. Cords, B. R. & Tatini, S. R. (1973). Applicability of heat-stable deoxyribonuclease assay for assessment of staphylococcal growth and the likely presence of enterotoxin in cheese. J. Dairy Sci., 56, 1512-19. Fey, H., Pfister, H. & ROegg, O. (1984). Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C and D. J. Clin. Microbiol., 19, 34-8. Freed, R. C., Evenson, M. L., Reiser, R. F. & Bergdoll, M. S. (1982). Enzymelinked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol., 44, 1349-55. Garcia, M. C., Otero, A., Garcia, M. L. & Moreno, B. (1987). Microbiological quality and composition of two types of Spanish sheep's milk cheeses (Manchego and Burgos varieties). J. Dairy Res., 54, 551-7. Gaya, P., Medina, M., Bautista, L. & Ntifiez, M. (1988). Influence of lactic starter inoculation, curd heating and ripening temperature on Staphylococcus aureus behaviour in Manchego cheese. Int. J. Food Microbiol., 6, 24957. G6mez-Lucia, E., Blanco, J. L., Goyache, J., Fuente, R. de la, Vazquez, J. A., Ferri, E. F. R. & Su~irez, G. (1986). Growth and enterotoxin A production by Staphylococcus aureus $6 in Manchego type cheese. J. Appl. Bacteriol., 61, 499-503. Gutirrrez, L. M., Menes, I., Garcia, M. L., Moreno, B. & Bergdoll, M. S. (1982). Characterization and enterotoxigenicity of staphylococci isolated from ovine mastitic milk in Spain. J. Food Protect., 45, 1282-9. Ibrahim, G. F. (1981). A simple sensitive method for determining staphylococcal thermonuclease in cheese. J. Appl. Bacteriol., 51, 307-12. ICMSF (1978). Microorganisms in Foods. 1. Their Significance and Methods of Enumeration, ed. International Commission on Microbiological Specifications for Foods. University of Toronto Press, Toronto, 434 pp. IDF (1980). Behaviour of pathogens in cheese. I.D.F. Document No. 122, International Dairy Federation, Brussels, 24 pp. Kamman, J. K. & Tatini, S. R. (1977). Optimal conditions for assay of staphylococcal nuclease. J. Food Sci.. 42, 421-4. Khalid, A. S. & Harrigan, W. F. (1984). Investigation into the effect of salt level on growth of, and toxin production by, Staphylococcus aureus in Sudanese cheese. Lebensm.-wiss. Technol., 17, 99-103. Law, B. A., Sharpe, M. E., Mabbitt, L. A. & Cole, C. B. (1973). Microflora of Cheddar cheese and some of the metabolic products. In SamplingMicrobiological Monitoring of Environments, eds R. G. Board & D. W. Lovelock. Academic Press, London, pp. 1-9. Martinez-Moreno, J. L. & Ntifiez, M. (1976). Flora microbiana del queso Manchego. II. Evolucirn de la flora microbiana de quesos Manchegos industriales. An. INIA, Ser. Gen., (4), 33-40. National Research Council (1985). An Evaluation of the Role of Microbiological Criteriafor Foods and Food Ingredients. National Academy Press, Washington, DC, 436 pp. Notermans, S., Tips, P. & Heuvelman, C. J. (1984). Einfluss der milieu
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begingungen auf das wachstum von Staphylococcus aureus und die enterotoxin-bildung. Fleischwirtschafi, 64, 1490, 1493-6. Ntifiez, M. & Martinez-Moreno, J. L. (1976). Flora microbiana del queso Manchego. I. Evoluci6n de la flora microbiana de quesos Manchegos artesanales. An. 1NIA, Ser. Gen., (4), 11-32. Ntifiez, M., Martinez-Moreno, J. L. & Medina, A. L. (1981). Ensayo de diversas cepas de Streptococcus lactis de diversa capacidad acidificante como fermentos para queso tipo Manchego.An. INIA, Ser. Ganadera, (12), 65-72. Ntifiez, M., Chavarri, F. J., Garcia, B. E. & Gayt~in, L. E. (1986). The effect of lactic starter inoculation and storage on the behaviour of Staphylococcus aureus and Enterobacter cloacae in Burgos cheese. Food Microbiol., 3, 23542. Ntifiez, M., Bautista, L., Medina, M. & Gaya, P. (1988). Staphylococcus aureus, thermonuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese. J. Appl. Bacteriol., 65, 29-34. Ord6fiez, J. A., Bameto, R. & Ramos, M. (1978). Studies on Manchego cheese ripened in olive oil. Milchwissenschafi, 33, 609-13. Otero, A., Garcia, M. L., Garcia, M. C. & Moreno, B. (1987a). Comparison of four methods for assay of staphylococcal thermonuclease.Arch. Lebensmittelhyg., 38, 132-5. Otero, A., Garcia, M. C., Garcia, M. L. & Moreno, B. (1987b). Production of staphylococcal enterotoxins Ci and C2 and thermonuclease in ewe's milk. Food Microbiol., 4, 339--45. Otero, A., Garcia, M. C., Garcia, M. L. & Moreno, B. (1988). Effect of growth of a commercial starter culture on growth of Staphylococcus aureus and thermonuclease and enterotoxins (C1 and C2) production in broth cultures. Int. J. Food Microbiol., 6, 107-14. Otero, A., Garcia, M. L., Garcia, M. L., Moreno, B. and Bergdoll, M. S. (1990). Production of staphylococcal enterotoxins CL and C2 and thermonuclease throughout the growth cycle. Appl. Environ. Microbiol., 56, 555-9. Perry, K. D. & Sharpe, M. E. (1960). Lactobacilli in raw milk and in Cheddar cheese. J. Dairy Res., 27, 267-75. Ramos, M., Barneto, R. & Ord6fiez, J. A. (1981). Evaluation of a specific starter for Manchego cheese production. Milchwissenscha)q, 36, 528-32. Reiter, B., Fewins, B. G., Fryer, T. F. & Sharpe, M. E. (1964). Factors affecting the multiplication and survival of coagulase positive staphylococci in Cheddar cheese. J. Dairy Res., 31, 261-72. Richardson, G. H. (ed.) (1985). Standard Methods for the Examination of Dairy Products, 15th edn, American Public Health Association, Washington, DC, 425 pp. Rom~in-Pir~ana, M. (1975). Etude de la flore microbienne du fromage espagnol "Manchego". I. Son evolution au cours de la fabrication et de l'affinage. Lait, 55, 401-13. Stadhouders, J., Cordes, M. M. & van Schouwenburg-van Foeken, A. W. J. (1978). The effect of manufacturing conditions on the development of staphylococci in cheese. Their inhibition by starter bacteria. Neth. Milk Dairy J.. 32, 193-203. Tatini, S. R., Jezeski, J. J. Morris, H. A., Olson, J. C., Jr. & Casman, E. P. (1971).
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Production of staphylococcal enterotoxin A in Cheddar and Colby cheeses. J. Dairy Sci., 54, 815-25.
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Nd Behaviour of Staphylococcus aureus Strains Fill 137 and FRI 361 During the Manufacture and Ripening of Manchego Cheese
A. O t e r o , M. C. G a r c i a , M. L. G a r c i a , J. A. S a n t o s & B. M o r e n o Department of Food Hygiene and Food Technology, Veterinary Faculty, University of Le6n, 24071-Le6n, Spain (Received 4 December 1991; revised version accepted 25 March 1992)
ABSTRACT The behaviour of Staphylococcus aureus strains FRI 137 (enterotoxin C 1 producer) and FRI 361 (enterotoxin C2 producer) during production and ripening of Manchego cheese manufactured with two levels of starter (0. O1 or 1%) was evaluated. Both strains grew well (four to eight generations), but only in the cheeses manufacturated with 0.01% of starter. The population of S. aureus increased during manufacture and decreased as ripening progressed. In spite of having reached S. aureus populations above 107 cfu/g, no C1 or C2 enterotoxin was detected. In the cheeses in which significant growth of S. aureus had occurred, thermonuclease was detected after pressing and it persisted during ripening. The presence of thermon uclease in Manchego cheese implies significant S. aureus multiplication, its use being recommended in a screening test.
INTRODUCTION Even t h o u g h the standard practices of refrigeration a n d pasteurization of milk (before cheesemaking) have greatly reduced cheese-induced illness, this product is still associated with outbreaks of staphylococcal food p o i s o n i n g (Bergdoll, 1989), T h e growth o f Staphylococcus aureus a n d in some cases the p r o d u c t i o n 85 Int. Dairy Journal 0958-6946/92/$05.00© 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland
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of thermonuclease and enterotoxins during the manufacture and ripening of various types of cheese have been studied by several researchers (IDF, 1980; Khalid & Harrigan, 1984; Ntifiez et al., 1986). However, the results obtained have varied depending on, among other factors, the type of cheese and the nature and activity of the starter culture used. For this reason, any generalizations dealing with the behaviour of S. aureus in types of cheese different from those studied should not be made. The growth of staphylococcal populations in Manchego cheese has been studied by Rom~in-Pifiana (1975), Martinez-Moreno & Ntifiez (1976), Ord6fiez etal. (1978), Ramos etal. (1981) and Gaya etal. (1988), but the production ofenterotoxin in this type ofsheeps' milk cheese has been investigated only recently (G6mez-Lucia et al., 1986; Ntifiez et al., 1988). G6mez-Lucia et al. (1986) investigated the behaviour of an enterotoxin A producing strain of S. aureus ($6), but the majority of the S. a u r e u s enterotoxigenic strains of ovine origin produce only enterotoxin C (Guti6rrez et al., 1982). Moreover, results obtained by G6mez-Lucia et al. (1986) are not in accordance with those reported by Ntifiez et al. (1988), who studied the behaviour of an enterotoxin C producing strain of S. aureus and another multiple enterotoxin (A, B, C and D) producer. On the other hand, the manufacture of Manchego cheese from raw milk is frequent in Spain (Ntifiez et al., 1981). In this country the contamination by staphylococci of sheeps' milk, which is used in the manufacture of cheese, can reach high levels (Bautista et al., 1986). Moreover, the hygienic conditions of manufacture and/or ripening of the cheeses are often overlooked (Garcia et al., 1987). Determination of thermonuclease activity in foods as an indicator of the possible presence of enterotoxins is recommended, especially in fermented foods (National Research Council, 1985). Even though it is generally accepted that there is a good correlation between enterotoxin and thermonuclease (Tatini et al., 1976), on some occasions cheese samples free from thermonuclease, but containing enterotoxins, have been reported (Todd et al., 1981). The purpose of this study was to investigate the behaviour of two enterotoxin C producing S. a u r e u s strains during the production and ripening of Manchego cheese made with two levels of starter. MATERIALS AND METHODS Cultures S t a p h y l o c o c c u s aureus strains FRI 137 and FRI 361, producing entero-
toxins C~ and C2, respectively, were used separately to inoculate cheese
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
87
milk. Both strains were obtained from Professor M. S. Bergdoll, Food Research Institute, University of Wisconsin, Madison, USA. After 24 h growth at 37°C (with shaking) in '3 + 1' broth (3% NZ Amine NAK~ Humko Sheffield Chemical, Lynhurst, N J) and 1% yeast extract (Difco, Detroit, MI), strain FRI 137 produced 19pg/ml of enterotoxin C1, and strain FRI 361, 28.6pg/ml of enterotoxin C2. Corresponding figures for thermonuclease were 20.9jug/ml (strain FRI 137) and 0-8pg/ml (FRI 361), as reported in previous investigations (Otero et aL, 1990). Cultures of S. aureus were grown in brain heart infusion broth (BHI, Difco) at 37°C for 18 h and then washed twice in 0.1% peptone water (w/v). An appropriate volume of the preparation was added to pasteurized milk to provide about 10 4 t o 10 5 cfu/ml. Starter culture HM1 (Miles Martin, S.A.E., Madrid, Spain), containing Lactococcus lactis subsp, lactis, L. lactis subsp, cremoris and L. lactis subsp. lactis biovar, diacetylactis, was used in the manufacture of Manchego. The starter culture was incubated in sterile 12% skim milk (Unipath Ltd, Basingstoke, Hampshire) at 23°C for 16 h, and its activity was evaluated (taking into account the supplier's recommendations) on the basis of the acidity developed. For each S. aureus strain, cheeses were manufactured at two levels of starter: 0-01% (simulating an initial failure) and 1% (normal level).
Manufacture and sampling of cheese All cheeses were made on the same day and from the same batch of milk. Raw ewes' milk, with a pH of 6.70 and a count of 1.8 × 10v mesophiles/ ml, was used. After pasteurization, counts of mesophiles were 2-0 × 104 cfu/ml of milk. Sheeps' milk was pasteurized in a pilot plant-size plate pasteurizer at 72°C for 15 s, cooled at 30°C and inoculated with the lactic starter culture. The S. aureus strain (FRI 137 or FRI 361) and 0-1% CaC12 solution were added at separate stages. After 30 min, commercial rennet (Ha-La, Chr. Hansen, Copenhagen, Denmark) was also added and coagulation took place in 40 min. The coagulum was cut into 4 to 6 mm cubes and heated to 37°C over 15 min. After stirring (cubes of curd and whey) for 10 min at 37°C, three blocks of curd (20 cm diameter × 8 cm high) were obtained from each vat; these were pressed (3 kg/cm 2) for 18 to 20 h and salted by submersion in a 20% NaC1 (w/v) solution at 10°C for 48 h. The cheeses were ripened for 3 months at 10 to 12°C and 85% RH. The following samples were taken: (1) from milk after addition of S. aureus and lactic starter; (2) from the curd before pressing; (3) from the cheese after pressing; and (4) from the cheese after salting, and throughout ripening on day 7, 15, 30, 60 and 90. Sampling was carried out
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according to Law et al. (1973). Subsamples for thermonuclease (20 ml or 20g) and enterotoxin (100 ml or 100g) determinations were kept at -30°C until analysed. Cheeses without added S. aureus were also made as controls.
Analysis For each of the samples, a microbiological analysis, a pH measurement and a determination of thermonuclease and of enterotoxin were carried out. Enumeration o f bacteria a n d p H measurement
Samples (10 ml of milk or 10 g of curd or cheese) were prepared as recommended by the American Public Health Association (Richardson, 1985). Mesophilic aerobes were enumerated by the standard plate count (Richardson, 1985). Counts of S. aureus were effected on Baird-Parker medium (Unipath), according to ICMSF (1978). The pH values were determined using a pH meter (model PHM82, Radiometer, Copenhagen, Denmark) after homogenizing 12-5 g of cheese with 17 ml of distilled water (Perry & Sharpe, 1960). The above analyses were performed immediately after sampling. Thermonuclease determination
The enzyme was extracted according to the method of Cords & Tatini (1973), slightly modified in that 0.05 M glycine buffer at pH 10.0 was used instead of 0.05 M Tris buffer at pH 9.0 (Kamman & Tatini, 1977). Nuclease activity of the boiled extract (15 min) was determined as proposed by Ibrahim (1981). The amount of enzyme in cheeses was estimated as described by Otero et al. (1987a). The sensitivity of the assay was 1 ng of thermonuclease per 20 g of cheese. Enterotoxin assay
Enterotoxins were extracted according to the method of Freed et al. (1982) and concentrated by dialysis against polyethylene glycol 20M (Serva, Heidelberg, Germany). The sandwich ELISA technique ofFey et aL (1984) was used to detect and quantify enterotoxins C~ and C2. Reagents for the ELISA test were obtained from W. Bommeli, Bern, Switzerland. For control purposes, enterotoxins C~ and C2 (from the Food Research Institute, Madison, WI) were added to cheeses (enterotoxin free), extracted and quantified. The mean recovery of enterotoxin added was 67%. The minimum enterotoxin concentration detected was 1.5 ng/100 g of cheese.
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
89
RESULTS A N D DISCUSSION Results of the determinations made on Manchego cheese during manufacture and ripening are given in Tables 1 (S. aureus strain FRI 137 and 0.01% starter), 2 (FRI 137 and 1% starter), 3 (FRI 361 and 0.01% starter) and 4 (FRI 361 and 1% starter). Changes in aerobic mesophilic plate counts The changes observed were similar to those reported previously by Martinez-Moreno & Nrfiez (1976) and Ord6fiez et al. (1978). As might be expected, at the early stage of production, counts increased (up to salting) and then decreased during salting. Additional growth occurred in cheese during the first week of ripening, after which there was a decrease in population. Behaviour of S. a u r e u s An increase in the number ofS. a u r e u s occurred during the time between inoculation and the end of salting (S. a u r e u s strain FRI 361) or only until TABLE 1 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FRI 137 and 0.01% of Starter
Sample (days) Oh" b Ic 3d 7e 15e 30 e 60 e 90 e
Mesophiles S. aureus (log cfu/ml org) (logcfu/ml org) 4-84 5.53 8-54 8.15 8.64 8.08 7.70 7.34 7.38
4-46 5-30 6.79 6.79 4.54 3.64 2.65 <2 <2
Enterotoxin Thermonuclease C~ (n/g of cheese) ND ND ND ND ND ND ND ND ND
~0h = milk after inoculation with S. aureus and starter. bCurd before pressing. cSample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
ND ND 0.8 0.5 0.6 0.6 0-5 0.4 0.6
pH NI 6.74 5,25 5,15 5.16 5.21 5.22 5-19 5-19
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TABLE 2 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain F RI 137 and 1% of Starter
Sample (days) Oh~ b 1" 3d 7" 15e 30 e 60 e 90 e
Mesophiles (log cfu/ml or g)
Enterotoxin Cz
Thermonuclease
pH
(log cfu/ml or g)
6.40 7-30 8.38 8.08 8.41 8.04 7.30 7-00 6-89
5.30 6-45 6.53 6.04 4.36 4.26 3.60 2.30 <2
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
NI 6-58 5.15 5.16 5.14 5.19 5.12 5.13 5.17
S. aureus
a0h = milk after inoculation with S. aureus and starter. hCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
TABLE 3 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FR1361 and 0.01% of Starter
Sample
Oha --~ 1' 3a 7e 15~ 30 e 60 e 90 e
Mesophiles (log cfu/ml or g)
Enterotoxin C:
Thermonuclease (ng/g of cheese)
pH
(log cfu/ml or g)
4-83 5.82 8.77 8.30 8.60 7-96 7.63 7.00 7.00
4-23 5.59 7.43 7.59 7.23 7.18 6.28 5.18 4.45
ND ND ND ND ND ND ND ND ND
ND ND 1.6 0-2 0.2 0.2 0.2 0.2 0.2
NI 6-64 5.25 5.13 5.22 5.26 5.20 5-25 5.24
S. aureus
a0h = milk after inoculation with S. aureus and starter. hCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
Behaviour of S. aureus during manufacture and ripening of Manchego cheese
91
TABLE 4 Aerobic Mesophilic Counts, Staphylococci, Thermonuclease Content and Enterotoxin in Manchego Cheese Made with Ewe's Milk Inoculated with S. aureus Strain FR1361 and 1% of Starter
Sample (days) Oh~ b 1" 3d 7e 15e 30 e 60 e 90 e
Mesophiles S. aureus (log cfu/ml or g) (log cfu/ml or g) 6-40 7.25 8.26 8.08 8.64 8.11 7.39 6-00 5.75
5.23 6-20 6.43 6.51 5.45 5.34 4-66 3-76 3.41
Enterotoxin C2
Thermonuclease
pH
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
NI 6.65 5-17 5-08 5.12 5.10 5.02 5.04 5-07
~0h = milk after inoculation with S. aureus and starter. bCurd before pressing. "Sample after pressing. dSample after salting. eSamples on 7th to 90th day of ripening. N D = not detectable. NI = not investigated.
the end of pressing (S. a u r e u s strain FRI 137). Afterwards, the number decreased for both strains during ripening and for strain FRI 137 even to below 102/g. The increase in staphylococcal counts in curd over those in inoculated milk is largely attributable to the physical concentration of S. a u r e u s cells in the coagulum. According to Tatini et al. (1971), this phenomenon usually represents a 10-fold increase. The general patterns of the growth and survival for the test strains were different, depending on the level of starter. With 1%of starter, the number of staphylococci hardly increased, whereas with 0.01% starter there were increases of up to eight generations for strain FRI 361 and four or five for strain FRI 137 (excluding the 10-fold increase in numbers caused by concentration on converting milk into cheese). Taking into account that strains FRI 137 and FRI 361 grow in ewes' milk up to levels of 9-18 log cfu/ml and 10-07 log cfu/ml (Otero et al., 1987b), the above data indicate, once again, that use of an effective starter culture is critical in order to control S. a u r e u s growth in cheese (Stadhouders et al., 1978). A gradual decrease in the numbers of S. a u r e u s during ripening is a common fact for ripened cheeses (Ntifiez & Martinez-Moreno, 1976). According to other results obtained by us (Otero et al., 1988) and other workers (Stadhouders et al., 1978), this may be the result of competition
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A. Otero, M. C. Garcia, M, L. Garcia, J. A. Santos, B. Moreno
with lactic acid bacteria. During ripening of Manchego cheese, Gaya et al. (1988) also reported a strong effect o f S t r e p t o c o c c u s lactis on counts of S. a u r e u s (a 5.8-fold reduction). Undoubtedly, other factors, such as lactic acid and salt content, low pH, etc., have also negative effects on survival of S. a u r e u s (Reiter et al., 1964). The decrease in the numbers ofS. a u r e u s during storage of Manchego cheese to below 102/g, as well as the different resistance of staphylococcal strains to environmental conditions associated with ripening, confirms the low reliability ofS. a u r e u s determination for assessment of the extent of staphylococcal growth in Manchego cheese. Enterotoxin
Enterotoxin C1 or C2 could not be detected in any of the cheeses, even when counts of S. a u r e u s in some samples obtained from low-starter cheeses were greater than 107/g. A similar finding has been reported by NOfiez et al. (1988) for Manchego cheese inoculated with t w o S. a u r e u s strains producing enterotoxins A, B, C and D, and C, respectively. Data obtained previously (Otero et al., 1988) suggest that the influence of lactic acid bacteria on enterotoxin C1 or C2, by inhibition of toxin synthesis and destruction of produced toxins, explains the lack of detectable amounts of enterotoxins in Manchego cheese despite the attainment of high S. a u r e u s populations. Another explanation for these results may be that the maximal S. a u r e u s populations attained did not reach the minimal levels associated with the presence of detectable amounts of enterotoxins in tyndallized ewe's milk inoculated with pure cultures of strains FRI 137 or FRI 361 (Otero et al., 1987b). Lower S. a u r e u s populations produced enterotoxin A in a similar study on Manchego cheese (G6mez-Lucia et al., 1986). It must be noted, however, that synthesis of enterotoxin A seems to be less affected by adverse environmental conditions than that of other enterotoxins (Notermans et al., 1984). Differences in the type of strain ofS. a u r e u s as well as in the type of starter culture used by G6mez-Lucia et al. (1986), Nffiez et al. (1988) and ourselves may also contribute to the above divergent results. Thermonuclease
No detectable thermonuclease was observed in any of the cheeses produced with 1% starter inoculum. This results from the inhibition of staphylococcal growth (Tables 2 and 4). Of the cheeses made with 0.01% starter activity, thermonuclease was detected in all samples obtained
Behaviour orS. aureus during manufacture and ripening of Manchego cheese
93
after pressing. During ripening, the thermonuclease content did not change significantly in cheeses made from milk inoculated with strain FRI 137 (Table 1), whereas extensive thermonuclease inactivation occurred in cheeses inoculated with strain FRI 361 (Table 3). The fact that the highest amounts of enzyme obtained in Manchego cheese were 1.2 ng/g of cheese (strain FRI 361) and 0.8 ng/g of cheese (strain FRI 137), while similar figures in '3 + 1' broth were 0.8 and 20.9 #g/ml, respectively (Otero et al., 1990), suggests that the substrate and/or starter had an inhibitory influence on thermonuclease production, which was stronger for strain FRI 137 than for strain FRI 361. Inactivation of thermonuclease in Manchego cheese has also been observed by Ntifiez et al. (1988). Since thermonuclease was detected when extensive growth ofS. aureus occurred and it remained detectable after 90 days of storage, the presence of thermonuclease could be used to assess staphylococcal growth and possible starter failure in Manchego cheese. However, the amount of enzyme found does not provide reliable information due to the variability of thermonuclease production by S. aureus, which depends on the test strain and growth conditions. Although the presence of thermonuclease was not associated with that of enterotoxin C~ or C2 in our study, it would seem advisable not to rule out the validity of this determination in screening tests for Manchego cheese. A positive result indicates that S. aureus may have reached a hazardous level, which, although it has not resulted in the presence ofenterotoxin C~ or C2, may have done so for other enterotoxins (G6mez-Lucia et al., 1986). ACKNOWLEDGEMENTS This work was supported in part by a grant from the Spanish CAICYT, Project No. 4466-79. Junta de Castilla y Le6n (Consejeria de Educaci6n y Cultura) and Diputaci6n Provincial de Le6n have also contributed. We are indebted to Professor M. S. Bergdoll and to Miles-Martin S.A.E. for providing enterotoxins, antisera and S. aureus strains, and the starter culture HM1, respectively. We are also indebted to Drs M. Nfifiez and M. de Paz, INIA, Madrid, Spain, for helping us in cheese making. REFERENCES Bautista, L., Bermejo, M. P. & Nfifiez, M. (1986). Seasonal variation and characterization of Micrococcaceae present in ewes' raw milk. J. Dairy Res., 53, 1-5.
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Production of staphylococcal enterotoxin A in Cheddar and Colby cheeses. J. Dairy Sci., 54, 815-25.
Tatini, S. R., Cords, B. R. & Gramoli, J. (1976). Screening for staphylococcal enterotoxins in food. Food Technol., 40, 64-74. Todd, E., Szabo, R., Robern, H., Gleeson, T., Park, C. & Clark, D. S. (1981). Variation in counts, enterotoxin levels and TNase in Swiss-type cheese contaminated with Staphylococcus aureus. J. Food Protect., 44, 839-52, 856.