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Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben, a Moroccan traditional fresh cheese
International Dairy Journal 12 (2002) 933–938
Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben, a Moroccan traditional fresh cheese Abed Hamama*, Nisrine El Hankouri, Mohamed El Ayadi Institut Agronomique et V!et!erinaire Hassan II, B.P. 6202 Rabat-Instituts, Rabat, Morocco Received 19 November 2001; accepted 23 July 2002
Abstract The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated. With an inoculum level of 103 cfu mL1, S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 103 cfu mL1. With a higher inoculum of 105 cfu mL1, S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 105 cfu mL1. Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Fresh cheese; Staphylococcus aureus; Lactic starters; Nisin
1. Introduction Jben is a Moroccan fresh cheese (unripened), traditionally made from raw milk and consumable within 10 or 15 days after manufacture. It contains about 37.5 g 100 g1 total solids and 16.5 g 100 g1 fat (Hamama & Bayi, 1991). Jben has been found to present a doubtful hygienic quality. Several investigations have reported the detection of pathogens such as Listeria monocytogenes (El Marrakchi, Hamama, & El Othmani, 1993), Salmonella (Hamama, 1989), Yersinia enterocolitica (Hamama, El Marrakchi, & El Othmani, 1992) and S. aureus (Hamama, 1989) in this product. Although data regarding the incidence of staphylococcal intoxications from Jben are not available, type C enterotoxins have been detected from two of 30 Jben samples (Hamama, 1989). *Corresponding author. E-mail address: [email protected] (A. Hamama).
Moroccan raw milk used for making Jben usually has high total bacterial counts (106–107 cfu mL1) and fermentation takes place at room temperature (15–251C). This may not favour the growth of S. aureus and production of enterotoxins. Nevertheless, S. aureus behaviour depends upon other factors such as its initial population, the nature of the competing microflora and conditions of fermentation. In the present work, we investigated the fate of an enterotoxigenic S. aureus during making of the Moroccan fresh cheese, Jben, in the presence of either a nisin-producing Lactococcus lactis strain or a commercial lactic starter. Nisin-producing lactococci have been already used to prevent the growth of Gram-positive pathogens in many dairy fermented products, especially in cheeses (Ferrera & Lund, 1996; Hirsh, Grinsted, Chapman, & Mattick, 1951; Maisnier-Patin, Deschamps, Tatini, & Richard, 1992; Roberts, Zottola, & Mc Kay, 1992; Rodriguez, Gaya, Nunez, & Medina, 1998; Sulzer & Busse, 1991).
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A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
2. Materials and methods
Cheeses were then surface salted with 1 g 100 g1 NaCl before storing at 41C.
2.1. Microorganisms 2.3. Sampling and analyses An enterotoxin type C-producing strain of S. aureus (J10) previously isolated from Jben (Hamama, 1989), was used in this study. A 24 h culture of this organism, grown in brain heart infusion (BHI; Difco Laboratories, Detroit, MI, USA) broth at 371C, was obtained and used to make different dilutions. The optical density (OD 340 nm) was measured with a spectrophotometer (Spectronic 21, Milton Roy Company, USA) in order to prepare a standard curve of the type: Y ¼ aX þ b; where Y refers to the population of S. aureus and X to the corresponding OD. The equation obtained (Y ¼ 80:9X þ 0:26) was used to calculate the appropriate dilutions of S. aureus to obtain the desired inocula of 1 103 and 1 105 S. aureus cfu mL1 of milk. A nisin Z-producing strain of L. lactis subsp. lactis (UL730), kindly offered by Pr. Ronald Simard (University of Laval, Que! bec, Canada) was subcultured twice in sterile reconstituted milk for 24 h at 301C before use as a culture for the manufacture of Jben. 2.2. Cheese manufacture Jben was prepared in three trials from pasteurized (751C, 1 min) recombined milk (3.5 g fat 100 mL1 milk) made from low-heat non-fat milk powder and industrial butter, both produced by the dairy cooperative ‘‘ExtraLait’’ of Ke! nitra, Morocco. For each trial, the milk was divided into four portions of 4 L each. Milk portion 1 was inoculated with 103 S. aureus cfu mL1 and L. lactis UL730 (2 mL 100 mL1) starter culture, portion 2 with 103 S. aureus cfu mL1 and a commercial mesophilic lactic starter (Ezal, Lacto-Labo, Dange! Saint-Romain, France), portion 3 with 105 S. aureus cfu mL1 and L. lactis UL730 (2 mL 100 mL1) culture, and portion 4 with 105 S. aureus cfu mL1 and a commercial mesophilic lactic starter. Portions 2 and 4 were used as controls and were designated as nisin () milks and portions 1 and 3 as nisin (+) milks. Inoculated milks were left at room temperature (18–221C) for 20 h to allow lactic fermentation. At a milk pH of 6.1–6.2, commercial rennet (1/10,000 strength) and calcium chloride (Merck, Darmstadt, Germany) were then added at levels of 0.1 mL L1 and 0.2 g L1, respectively, and coagulation occurred in about 30 min at room temperature. The pH was monitored hourly until a value of 4.4–4.6 was reached. Whey drainage was initiated by pouring the coagulum into 1 kg-capacity cylindrical moulds using a scoop, and was continued for at least 48 h at room temperature until the appropriate texture of Jben was obtained.
For each of the three replicate cheese-making trials, two samples of milk, 24-h coagulum, 72-h coagulum and cheese were examined for the following analyses: pH, titratable acidity, lactococci count, S. aureus count, presence of staphylococcal thermonuclease (TNase) (screening test for potential presence of enterotoxin) and enterotoxin in TNase-positive samples only (since TNase-negative samples are not expected to contain enterotoxin (Tatini, 1981), and nisin activity. 2.3.1. pH and titratable acidity determinations These parameters were determined according to the methods recommended by Serres, Amariglio, and Petransxiene (1973) for milk and fresh cheese. 2.3.2. TNase testing TNase was extracted using a sample of 40 mL or 40 g of milk, coagulum, or cheese adjusted to pH 4.5 (Tatini, Cords, & Gramoli, 1976), where necessary with a solution of 3 n HCl. Samples were then centrifuged at 5635g for 30 min at 51C using a refrigerated centrifuge (Dupont Instruments, USA). The resultant supernatant was heated for 60 min at 1001C (Tatini, Hoover, & Lachica, 1984) to eliminate non-staphylococcal TNase, and was then allowed to cool. Twenty five mL of the cooled extract was used to fill a 5-mm well, cut into a toluidine blue DNA agar plate (TB-DNA) which was prepared as described by Kamman and Tatini (1977). After 4 h incubation at 501C, the TB-DNA plates were observed for a pink halo around the wells. Only samples showing specific and distinctive reactions (pink haloes extending 1 mm beyond the wells) were considered positive. 2.3.3. Staphylococcal enterotoxin detection Detection analysis was performed only on TNase positive samples. The presence of staphylococcal enterotoxin C (SEC) was examined using a staphylococcal enterotoxin reversed passive latex agglutination (SETRPLA) test kit (Oxoid limited, Basing-stoke, UK). To avoid any dilution of toxin present, the supernatant from TNase testing was used as extract. 2.3.4. Microbial counts Lactococci count was obtained by pour-plating appropriate sample dilutions of milk, and homogenates of coagulum and fresh cheese in sterile quarter strength Ringer’s solution, in M17 agar (Merck, Darmstadt, Germany) plates and incubating for 48 h at 301C (Terzaghi & Sandine, 1975). S. aureus count was determined by surface plating of appropriate sample
ND ND ND ND 5.42 (0.48) 2.72 (0.32) 1.23 (0.08) 3.70 1.18 (0.03) 1.58 (0.19) 1.56 (0.17) Results are averages of a total of six samples (two samples from each of the three trials performed). Expressed as g lactic acid 100 mL1 or 100 g1 product. c TNase, thermonuclease. d Expressed as diameter in mm of the zone of growth inhibition. No nisin activity was detected in nisin () cheese. e ND, non-detectable. f Figures in brackets are standard deviations. g NtD, not determined. nnn Significantly different from the control (nisin () cheese) value at the 0.1% level. b
a
6.06 NtDg NtD 10.6 (0.14) Milk Coagulum Coagulum Cheese
0 24 72 96
6.11 4.50 (0.31)f 4.23 (0.05) 4.14 (0.21)
0.42 1.13 (0.09) 1.37 (0.07) 1.44 (0.26)
3.66 5.36 (0.32) 2.25 (0.21) NDe,nnn
NDe ND ND ND
0.46 4.38 (0.41) 4.12 (0.32) 4.25 (0.21)
TNase activity S. aureus count (log cfu mL1 or g1) Acidity Nisin activityd
pH
935
TNasec activity S. aureus count (log cfu mL1 or g1)
Data regarding the fate of S. aureus during the manufacture of Jben from recombined milk inoculated with nisin-producing lactococci or normal mesophilic lactic starters (non nisin-producing) are shown in Tables 1 and 2. Each figure shown in these tables is an average value of six samples representing two samples from each of the three trials performed. There was good fermentation in all the trials, as indicated by the reduction in milk pH which was 4.50–4.77 at 24 h after milk inoculation with the nisin (+) starter (UL730) and 4.38–4.61 after inoculation with nisin () starter (control). At an inoculation level of 103 cfu mL1, S. aureus reached maximum populations of 105.4 and 105.4 cfu mL1 at 24 h in the coagula from the nisin (+) milk and nisin () milk, respectively; thereafter, the counts of S. aureus dropped progressively in numbers in 72-h coagula and cheeses from both milks (Table 1) and TNase did not accumulate to detectable levels. Similar results were found by Tatini, Wesala, Jezeski, and Morris (1973) who studied the behaviour of S. aureus during manufacture of Mozzarella cheese from milk inoculated with 104 cfu mL1 S. aureus. Working with Lben (traditional Moroccan fermented milk), Hamama and Tatini (1991) reported an increase in the population of S. aureus, at 24 h after milk inoculation with S. aureus at a level of 103 cfu mL1. TNase was not detected in the fermented milk. The decrease in the population of S. aureus in coagula and cheeses at times >24 h differed for the nisin (+) and nisin () milks. There was a slight but nonsignificant decrease in S. aureus numbers in nisin (+)
Acidityb
3. Results and discussion
Milk with nisin () lactococci
2.3.6. Statistical analysis Analysis of variance (ANOVA; SAS software program, 1986) was used to determine significant differences in S. aureus behaviour between cheeses preinoculated with nisin-producing lactococci or non-nisinproducing lactic starters.
Milk with nisin (+) lactococci
2.3.5. Nisin activity determination Nisin activity in Jben was determined using the agar diffusion method of Tramer and Fowler (1964) with S. aureus 196E ATCC as a sensitive indicator organism. The assay was performed as described by Rodriguez et al. (1998). The diameter of the zone of growth inhibition on All Purpose Tween agar (Difco Laboratories, Detroit, MI, USA) was measured after incubation of plates for 24 h at 371C.
Sampling time (h) pH
dilutions on Baird-Parker agar (Difco Laboratories, Detroit, MI, USA) plates and incubating for 48 h at 371C (Tatini et al., 1984).
Table 1 Growth and thermonuclease production by Staphylococcus aureus during the manufacture of Jben from milk inoculated with 103 cfu mL1 S. aureus and nisin positive [nisin (+)] and nisin negative [nisin ()] Lactococcus starter culturesa
A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
72 96 120 168 192
Coagulum Cheese
4.62 4.38 4.73 4.62 4.73
(0.22) (0.13) (0.31) (0.42) (0.06)
6.26 4.77 (0.01)g
pH
1.52 1.60 1.78 1.89 1.92
(0.03) (0.09) (0.45) (0.21) (0.01)
0.39 1.08 (0.08)
Acidityb
5.38 (1.16) 4.14 (0.62) 3.63 (0.13) 2.29nn (0.14) 2.42 (0.06)
5.61 7.11 (0.28)
S.aureus count (log cfu mL1 or g1)
Milk with nisin (+) lactococci
+ + + + +
NDf +
TNasec activity
+ + + + +
+
SECd
NtDh4.61 (0.22) NtD 10.6 (0.14) 10.6 (0.07) 10.4 (0.07) 10.4 (0.07)
Nisin activitye
b
Results are averages of a total of 6 samples (2 samples from each of the 3 trials performed). Expressed as g lactic acid 100 mL1 or 100 g1 product. c TNase, thermonuclease. d SEC, staphylococcal enterotoxin type C. e Expressed as diameter in mm of the zone of growth inhibition. Non nisin activity was detected in nisin () cheese. f ND, non-detectable. g Figures in brackets are standard deviations. h NtD, not determined. nn Significantly different from the control (nisin () cheese) value at the 1% level. n Significantly different from the 168 h-nisin () cheese value at the 5% level.
a
0 24
Milk Coagulum
Sampling time (h)
4.42 4.29 4.54 4.60 4.62
(0.31) (0.58) (0.25) (0.41) (0.09)
6.14 1.12 (0.14)
pH
1.54 1.76 1.82 1.92 1.94
(0.21) (0.09) (0.33) (0.21) (0.05)
0.37 7.41 (0.43)e
Acidity
5.44 (0.74) 4.58 (0.23) 3.83 (0.16) 3.69 (0.14) 2.75n (0.26)
5.52 +
S.aureus count (log cfu mL1 or g1)
Milk with nisin () lactococci
+ + + + +
ND +
TNase activity
+ + + + +
SEC
Table 2 Growth and thermonuclease and enterotoxin production by Staphylococcus aureus during the manufacture and storage of Jben from recombined milk inoculated with 105 cfu mL1 S.aureus and nisin positive[nisin(+)] and nisin negative [nisin()] Lactococcus starter culturesa
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A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
coagula compared to the nisin () coagula at 72 h (about 0.5 log unit difference). However, at 96 h, the decrease in S. aureus numbers was highly significant (po0:001) in nisin (+) samples (1.2 log units difference; Table 1). At an inoculation level of 105 cfu mL1, S. aureus reached maximum populations of 107.1 and 107.4 cfu mL1 at 24 h in the coagula from nisin (+) and nisin () milks, respectively; moreover, both coagula tested positive for TNase and SEC at 24 h (Table 2). Staphylococcal enterotoxin was then produced despite the presence of large numbers of lactococci (109.3 and 109.7 cfu mL1 at 24 h in the coagula from nisin (+) and nisin () milks, respectively). The nisin () commercial starter was expected to grow very fast in the conditions of this experiment since similar large numbers of lactococci were previously obtained in Jben made from pasteurized recombined milk using the same starter culture (Hamama, Zahar, Mahfoud, & Loudiyi, 1998). The behaviour of S. aureus in nisin (+) and in nisin () was similar during the manufacture of Jben. However, at 168 h, there was a significant difference (po0:01) between the numbers of S. aureus in the nisin (+) cheese (B102.3 cfu g1) and the nisin () cheese (B103.7 cfu g1). As the level of acidity was similar in both cheeses, this accentuated decrease of S. aureus in the nisin (+) Jben might only be due to the effect of the accumulated nisin in the cheese. Nisin activity was detected only in cheese made with nisin (+) starter (Table 2). On the other hand, a noticeable decrease (po0:05) in S. aureus population was observed in nisin () cheese samples between times 168 and 192. This might be explained by the inhibitory effect of the relatively high levels of lactic acid (1.92–1.94 g 100 g1) in the nisin () cheese on the S. aureus population during this period (Table 2). The data presented in this study concur with those of Hamama and Tatini (1991) who reported the presence of staphylococcal enterotoxin in Lben at 8–12 h after inoculation of raw milk with S. aureus (at a level of 105 cfu mL1) despite the presence of a relatively high population of native lactococci (B106–107 cfu mL1). Other studies conducted on different types of cheese (i.e., Domiati, Brazilian Minas, and Burgos) suggested that the presence of relatively high enterotoxigenic S. aureus counts (106–108 cfu mL1) in milk led frequently to detectable levels of enterotoxin in cheese (Ahmed, Mustafa, & Marth, 1983; Dos Santos & Genigeorgis, 1981; Otero, Garcia, Prieto, & Moreno, 1988). The data from this experiment suggest that initial levels of S. aureus p103 cfu mL1 in milk are not expected to result in enterotoxin in Jben mainly because of the rapid drop in pH due to the active lactic
937
fermentation. The low pH prevents S. aureus from reaching numbers which are capable of producing enterotoxin at detectable levels. Moreover, if nisinproducing starters were used for fermentation, there would probably be a lower likelihood of S. aureus being found in the final product (Table 1). In contrast, when higher S. aureus counts (105 cfu mL1) are present in the initial milk, enterotoxin is rapidly formed in the product despite the use of a nisin-producing lactic culture for fermentation. Hence, when raw milk is to be used for Jben making, attention should be paid to its quality as well as to the conditions of manufacture so as to prevent any contamination of milk with S. aureus. However, because of the high incidence of mastitis among Moroccan dairy cattle and the relative poor hygienic conditions during milk handling and traditional Jben preparation, it is highly recommended to use pasteurized milk and very active lactic starter cultures (similar to those used in this study) which are capable of lowering the initial milk pH by more than one unit over a 24-h period. The nisin-producing lactococci are of little help, as shown in this study, to prevent S. aureus growth and subsequent formation of enterotoxin particularly when the initial milk contamination with S. aureus is relatively high (e.g. 105 cfu mL1).
Acknowledgements This research work was supported by the Fond International de Coope! ration Universitaire (FICU) of Agence Universitaire de la Francophonie (AUPELF/ URU). The authors are grateful to Pr. Ronald Simard (University Laval, Que! bec, Canada) for providing the nisin-producing starter used in this work, and to the Dairy Cooperative ‘‘Extra-Lait’’, Ke! nitra, Morocco, for providing the raw materials.
References Ahmed, A. A. H., Mustafa, M. K., & Marth, E. H. (1983). Growth and enterotoxin production by Staphylococcus aureus in whey from the manufacture of Domiati cheese. Journal of Food Protection, 46(3), 235–237. Dos Santos, E. C., & Genigeorgis, C. (1981). Potential for presence and growth of staphylococci in Brazilian Minas cheese whey. Journal of Food Protection, 44(3), 185–188. El Marrakchi, A., Hamama, A., & El Othmani, F. (1993). Occurrence of Listeria monocytogenes in milk and dairy products produced or imported into Morocco. Journal of Food Protection, 56(3), 256–259. Ferrera, M. A. S. S., & Lund, B. M. (1996). The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese. Letters in Applied Microbiology, 22(6), 433–438. Hamama, A. (1989). Studies on the hygienic quality of Moroccan traditional dairy products. Ph.D. thesis. University of Minnesota, St. Paul, MN, USA.
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Hamama, A., & Bayi, M. (1991). Composition and microbiological profile of two Moroccan traditional dairy products: raib and jben. Journal of the Society of Dairy Technology, 44(4), 118–120. Hamama, A., El Marrakchi, A., & El Othmani, F. (1992). Occurrence of Yersinia enterocolitica in milk and dairy products in Morocco. International Journal of Food Microbiology, 16(1), 69–77. Hamama, A., & Tatini, S. R. (1991). Behavior of an enterotoxigenic Staphylococcus aureus strain during manufacture of the Moroccan fermented milk ‘‘lben’’. Le Lait, 71(5), 589–596. Hamama, A., Zahar, M., Mahfoud, Y., & Loudiyi, M. (1998). Essais industriels de fabrication du fromage frais ‘‘Jben’’ a" partir de lait recombin!e. Microbiologie-Aliments-Nutrition, 16(3), 163–169. Hirsh, A., Grinsted, E., Chapman, H. R., & Mattick, A. T. R. (1951). A note on the inhibition of an anaerobic spore former in Swiss-type cheese by a nisin-producing Streptococcus. Journal of Dairy Research, 18, 205–206. Kamman, J. F., & Tatini, S. R. (1977). Optimal conditions for essai of staphylococcal nuclease in cheese. Journal of Food Science, 42(2), 421–424. Maisnier-Patin, S., Deschamps, N., Tatini, S. R., & Richard, J. (1992). Inhibition of Listeria monocytogenes in Camembert cheese made with a nisin-producing starter. Le Lait, 72(3), 249–263. Otero, A., Garcia, M. C., Prieto, M., & Moreno, B. (1988). Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese. Journal of Applied Bacteriology, 64(2), 117–122. Roberts, R. F., Zottola, A., & Mc Kay, L. L. (1992). Use of a nisinproducing starter culture suitable for Cheddar cheese manufacture. Journal of Dairy Science, 75(9), 2353–2363.
Rodriguez, E., Gaya, P., Nunez, M., & Medina, M. (1998). Inhibitory activity of a nisin-producing startert culture on Listeria innocua in raw ewes milk Manchego cheese. International Journal of Food Microbiology, 39(2), 129–132. Serres, L., Amariglio, S., & Petransxiene, D. (1973). Analyse physique # de la qualit!e des produits laitiers. France: et chimique. In Controle Direction des Services V!et!erinaires, Minist"ere de l’Agriculture. Sulzer, G., & Busse, M. (1991). Growth inhibition of Listeria spp. on Camembert cheese by bacteria producing inhibitory substances. International Journal of Food Microbiology, 14(4), 287–296. Tatini, S. R. (1981). Thermonuclease as an indicator of staphylococcal enterotoxins in food. In R. L. Ory (Ed.), Antinutrients and natural toxicants in foods (pp. 53–75). Westport: Food and Nutrition Press, Inc. Tatini, S. R., Cords, B. R., & Gramoli, J. (1976). Screening for staphylococcal enterotoxin in food. Food Technology, 40(4), 64–76. Tatini, S. R., Hoover, D. G., & Lachica, R. V. F. (1984). Methods for the isolation and enumeration of Staphylococcus aureus. In M. L. Speck (Ed.), The compendium of methods for the microbiological examination of foods (pp. 411–427). Washington, DC: American Public Health Association. Tatini, S. R., Wesala, W. D., Jezeski, J. J., & Morris, J. A. (1973). Production of staphylococcal enterotoxin A in blue, brick, Mozzarella and Swiss cheeses. Journal of Dairy Science, 56(4), 429–435. Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Applied and Environmental Microbiology, 29(4), 807–813. Tramer, J., & Fowler, G. G. (1964). Estimation of nisin in foods. Journal of the Science of Food and Agriculture, 15, 522–528.
Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben, a Moroccan traditional fresh cheese Abed Hamama*, Nisrine El Hankouri, Mohamed El Ayadi Institut Agronomique et V!et!erinaire Hassan II, B.P. 6202 Rabat-Instituts, Rabat, Morocco Received 19 November 2001; accepted 23 July 2002
Abstract The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated. With an inoculum level of 103 cfu mL1, S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 103 cfu mL1. With a higher inoculum of 105 cfu mL1, S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 105 cfu mL1. Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Fresh cheese; Staphylococcus aureus; Lactic starters; Nisin
1. Introduction Jben is a Moroccan fresh cheese (unripened), traditionally made from raw milk and consumable within 10 or 15 days after manufacture. It contains about 37.5 g 100 g1 total solids and 16.5 g 100 g1 fat (Hamama & Bayi, 1991). Jben has been found to present a doubtful hygienic quality. Several investigations have reported the detection of pathogens such as Listeria monocytogenes (El Marrakchi, Hamama, & El Othmani, 1993), Salmonella (Hamama, 1989), Yersinia enterocolitica (Hamama, El Marrakchi, & El Othmani, 1992) and S. aureus (Hamama, 1989) in this product. Although data regarding the incidence of staphylococcal intoxications from Jben are not available, type C enterotoxins have been detected from two of 30 Jben samples (Hamama, 1989). *Corresponding author. E-mail address: [email protected] (A. Hamama).
Moroccan raw milk used for making Jben usually has high total bacterial counts (106–107 cfu mL1) and fermentation takes place at room temperature (15–251C). This may not favour the growth of S. aureus and production of enterotoxins. Nevertheless, S. aureus behaviour depends upon other factors such as its initial population, the nature of the competing microflora and conditions of fermentation. In the present work, we investigated the fate of an enterotoxigenic S. aureus during making of the Moroccan fresh cheese, Jben, in the presence of either a nisin-producing Lactococcus lactis strain or a commercial lactic starter. Nisin-producing lactococci have been already used to prevent the growth of Gram-positive pathogens in many dairy fermented products, especially in cheeses (Ferrera & Lund, 1996; Hirsh, Grinsted, Chapman, & Mattick, 1951; Maisnier-Patin, Deschamps, Tatini, & Richard, 1992; Roberts, Zottola, & Mc Kay, 1992; Rodriguez, Gaya, Nunez, & Medina, 1998; Sulzer & Busse, 1991).
0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 2 ) 0 0 1 1 3 - 9
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A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
2. Materials and methods
Cheeses were then surface salted with 1 g 100 g1 NaCl before storing at 41C.
2.1. Microorganisms 2.3. Sampling and analyses An enterotoxin type C-producing strain of S. aureus (J10) previously isolated from Jben (Hamama, 1989), was used in this study. A 24 h culture of this organism, grown in brain heart infusion (BHI; Difco Laboratories, Detroit, MI, USA) broth at 371C, was obtained and used to make different dilutions. The optical density (OD 340 nm) was measured with a spectrophotometer (Spectronic 21, Milton Roy Company, USA) in order to prepare a standard curve of the type: Y ¼ aX þ b; where Y refers to the population of S. aureus and X to the corresponding OD. The equation obtained (Y ¼ 80:9X þ 0:26) was used to calculate the appropriate dilutions of S. aureus to obtain the desired inocula of 1 103 and 1 105 S. aureus cfu mL1 of milk. A nisin Z-producing strain of L. lactis subsp. lactis (UL730), kindly offered by Pr. Ronald Simard (University of Laval, Que! bec, Canada) was subcultured twice in sterile reconstituted milk for 24 h at 301C before use as a culture for the manufacture of Jben. 2.2. Cheese manufacture Jben was prepared in three trials from pasteurized (751C, 1 min) recombined milk (3.5 g fat 100 mL1 milk) made from low-heat non-fat milk powder and industrial butter, both produced by the dairy cooperative ‘‘ExtraLait’’ of Ke! nitra, Morocco. For each trial, the milk was divided into four portions of 4 L each. Milk portion 1 was inoculated with 103 S. aureus cfu mL1 and L. lactis UL730 (2 mL 100 mL1) starter culture, portion 2 with 103 S. aureus cfu mL1 and a commercial mesophilic lactic starter (Ezal, Lacto-Labo, Dange! Saint-Romain, France), portion 3 with 105 S. aureus cfu mL1 and L. lactis UL730 (2 mL 100 mL1) culture, and portion 4 with 105 S. aureus cfu mL1 and a commercial mesophilic lactic starter. Portions 2 and 4 were used as controls and were designated as nisin () milks and portions 1 and 3 as nisin (+) milks. Inoculated milks were left at room temperature (18–221C) for 20 h to allow lactic fermentation. At a milk pH of 6.1–6.2, commercial rennet (1/10,000 strength) and calcium chloride (Merck, Darmstadt, Germany) were then added at levels of 0.1 mL L1 and 0.2 g L1, respectively, and coagulation occurred in about 30 min at room temperature. The pH was monitored hourly until a value of 4.4–4.6 was reached. Whey drainage was initiated by pouring the coagulum into 1 kg-capacity cylindrical moulds using a scoop, and was continued for at least 48 h at room temperature until the appropriate texture of Jben was obtained.
For each of the three replicate cheese-making trials, two samples of milk, 24-h coagulum, 72-h coagulum and cheese were examined for the following analyses: pH, titratable acidity, lactococci count, S. aureus count, presence of staphylococcal thermonuclease (TNase) (screening test for potential presence of enterotoxin) and enterotoxin in TNase-positive samples only (since TNase-negative samples are not expected to contain enterotoxin (Tatini, 1981), and nisin activity. 2.3.1. pH and titratable acidity determinations These parameters were determined according to the methods recommended by Serres, Amariglio, and Petransxiene (1973) for milk and fresh cheese. 2.3.2. TNase testing TNase was extracted using a sample of 40 mL or 40 g of milk, coagulum, or cheese adjusted to pH 4.5 (Tatini, Cords, & Gramoli, 1976), where necessary with a solution of 3 n HCl. Samples were then centrifuged at 5635g for 30 min at 51C using a refrigerated centrifuge (Dupont Instruments, USA). The resultant supernatant was heated for 60 min at 1001C (Tatini, Hoover, & Lachica, 1984) to eliminate non-staphylococcal TNase, and was then allowed to cool. Twenty five mL of the cooled extract was used to fill a 5-mm well, cut into a toluidine blue DNA agar plate (TB-DNA) which was prepared as described by Kamman and Tatini (1977). After 4 h incubation at 501C, the TB-DNA plates were observed for a pink halo around the wells. Only samples showing specific and distinctive reactions (pink haloes extending 1 mm beyond the wells) were considered positive. 2.3.3. Staphylococcal enterotoxin detection Detection analysis was performed only on TNase positive samples. The presence of staphylococcal enterotoxin C (SEC) was examined using a staphylococcal enterotoxin reversed passive latex agglutination (SETRPLA) test kit (Oxoid limited, Basing-stoke, UK). To avoid any dilution of toxin present, the supernatant from TNase testing was used as extract. 2.3.4. Microbial counts Lactococci count was obtained by pour-plating appropriate sample dilutions of milk, and homogenates of coagulum and fresh cheese in sterile quarter strength Ringer’s solution, in M17 agar (Merck, Darmstadt, Germany) plates and incubating for 48 h at 301C (Terzaghi & Sandine, 1975). S. aureus count was determined by surface plating of appropriate sample
ND ND ND ND 5.42 (0.48) 2.72 (0.32) 1.23 (0.08) 3.70 1.18 (0.03) 1.58 (0.19) 1.56 (0.17) Results are averages of a total of six samples (two samples from each of the three trials performed). Expressed as g lactic acid 100 mL1 or 100 g1 product. c TNase, thermonuclease. d Expressed as diameter in mm of the zone of growth inhibition. No nisin activity was detected in nisin () cheese. e ND, non-detectable. f Figures in brackets are standard deviations. g NtD, not determined. nnn Significantly different from the control (nisin () cheese) value at the 0.1% level. b
a
6.06 NtDg NtD 10.6 (0.14) Milk Coagulum Coagulum Cheese
0 24 72 96
6.11 4.50 (0.31)f 4.23 (0.05) 4.14 (0.21)
0.42 1.13 (0.09) 1.37 (0.07) 1.44 (0.26)
3.66 5.36 (0.32) 2.25 (0.21) NDe,nnn
NDe ND ND ND
0.46 4.38 (0.41) 4.12 (0.32) 4.25 (0.21)
TNase activity S. aureus count (log cfu mL1 or g1) Acidity Nisin activityd
pH
935
TNasec activity S. aureus count (log cfu mL1 or g1)
Data regarding the fate of S. aureus during the manufacture of Jben from recombined milk inoculated with nisin-producing lactococci or normal mesophilic lactic starters (non nisin-producing) are shown in Tables 1 and 2. Each figure shown in these tables is an average value of six samples representing two samples from each of the three trials performed. There was good fermentation in all the trials, as indicated by the reduction in milk pH which was 4.50–4.77 at 24 h after milk inoculation with the nisin (+) starter (UL730) and 4.38–4.61 after inoculation with nisin () starter (control). At an inoculation level of 103 cfu mL1, S. aureus reached maximum populations of 105.4 and 105.4 cfu mL1 at 24 h in the coagula from the nisin (+) milk and nisin () milk, respectively; thereafter, the counts of S. aureus dropped progressively in numbers in 72-h coagula and cheeses from both milks (Table 1) and TNase did not accumulate to detectable levels. Similar results were found by Tatini, Wesala, Jezeski, and Morris (1973) who studied the behaviour of S. aureus during manufacture of Mozzarella cheese from milk inoculated with 104 cfu mL1 S. aureus. Working with Lben (traditional Moroccan fermented milk), Hamama and Tatini (1991) reported an increase in the population of S. aureus, at 24 h after milk inoculation with S. aureus at a level of 103 cfu mL1. TNase was not detected in the fermented milk. The decrease in the population of S. aureus in coagula and cheeses at times >24 h differed for the nisin (+) and nisin () milks. There was a slight but nonsignificant decrease in S. aureus numbers in nisin (+)
Acidityb
3. Results and discussion
Milk with nisin () lactococci
2.3.6. Statistical analysis Analysis of variance (ANOVA; SAS software program, 1986) was used to determine significant differences in S. aureus behaviour between cheeses preinoculated with nisin-producing lactococci or non-nisinproducing lactic starters.
Milk with nisin (+) lactococci
2.3.5. Nisin activity determination Nisin activity in Jben was determined using the agar diffusion method of Tramer and Fowler (1964) with S. aureus 196E ATCC as a sensitive indicator organism. The assay was performed as described by Rodriguez et al. (1998). The diameter of the zone of growth inhibition on All Purpose Tween agar (Difco Laboratories, Detroit, MI, USA) was measured after incubation of plates for 24 h at 371C.
Sampling time (h) pH
dilutions on Baird-Parker agar (Difco Laboratories, Detroit, MI, USA) plates and incubating for 48 h at 371C (Tatini et al., 1984).
Table 1 Growth and thermonuclease production by Staphylococcus aureus during the manufacture of Jben from milk inoculated with 103 cfu mL1 S. aureus and nisin positive [nisin (+)] and nisin negative [nisin ()] Lactococcus starter culturesa
A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
72 96 120 168 192
Coagulum Cheese
4.62 4.38 4.73 4.62 4.73
(0.22) (0.13) (0.31) (0.42) (0.06)
6.26 4.77 (0.01)g
pH
1.52 1.60 1.78 1.89 1.92
(0.03) (0.09) (0.45) (0.21) (0.01)
0.39 1.08 (0.08)
Acidityb
5.38 (1.16) 4.14 (0.62) 3.63 (0.13) 2.29nn (0.14) 2.42 (0.06)
5.61 7.11 (0.28)
S.aureus count (log cfu mL1 or g1)
Milk with nisin (+) lactococci
+ + + + +
NDf +
TNasec activity
+ + + + +
+
SECd
NtDh4.61 (0.22) NtD 10.6 (0.14) 10.6 (0.07) 10.4 (0.07) 10.4 (0.07)
Nisin activitye
b
Results are averages of a total of 6 samples (2 samples from each of the 3 trials performed). Expressed as g lactic acid 100 mL1 or 100 g1 product. c TNase, thermonuclease. d SEC, staphylococcal enterotoxin type C. e Expressed as diameter in mm of the zone of growth inhibition. Non nisin activity was detected in nisin () cheese. f ND, non-detectable. g Figures in brackets are standard deviations. h NtD, not determined. nn Significantly different from the control (nisin () cheese) value at the 1% level. n Significantly different from the 168 h-nisin () cheese value at the 5% level.
a
0 24
Milk Coagulum
Sampling time (h)
4.42 4.29 4.54 4.60 4.62
(0.31) (0.58) (0.25) (0.41) (0.09)
6.14 1.12 (0.14)
pH
1.54 1.76 1.82 1.92 1.94
(0.21) (0.09) (0.33) (0.21) (0.05)
0.37 7.41 (0.43)e
Acidity
5.44 (0.74) 4.58 (0.23) 3.83 (0.16) 3.69 (0.14) 2.75n (0.26)
5.52 +
S.aureus count (log cfu mL1 or g1)
Milk with nisin () lactococci
+ + + + +
ND +
TNase activity
+ + + + +
SEC
Table 2 Growth and thermonuclease and enterotoxin production by Staphylococcus aureus during the manufacture and storage of Jben from recombined milk inoculated with 105 cfu mL1 S.aureus and nisin positive[nisin(+)] and nisin negative [nisin()] Lactococcus starter culturesa
936 A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
A. Hamama et al. / International Dairy Journal 12 (2002) 933–938
coagula compared to the nisin () coagula at 72 h (about 0.5 log unit difference). However, at 96 h, the decrease in S. aureus numbers was highly significant (po0:001) in nisin (+) samples (1.2 log units difference; Table 1). At an inoculation level of 105 cfu mL1, S. aureus reached maximum populations of 107.1 and 107.4 cfu mL1 at 24 h in the coagula from nisin (+) and nisin () milks, respectively; moreover, both coagula tested positive for TNase and SEC at 24 h (Table 2). Staphylococcal enterotoxin was then produced despite the presence of large numbers of lactococci (109.3 and 109.7 cfu mL1 at 24 h in the coagula from nisin (+) and nisin () milks, respectively). The nisin () commercial starter was expected to grow very fast in the conditions of this experiment since similar large numbers of lactococci were previously obtained in Jben made from pasteurized recombined milk using the same starter culture (Hamama, Zahar, Mahfoud, & Loudiyi, 1998). The behaviour of S. aureus in nisin (+) and in nisin () was similar during the manufacture of Jben. However, at 168 h, there was a significant difference (po0:01) between the numbers of S. aureus in the nisin (+) cheese (B102.3 cfu g1) and the nisin () cheese (B103.7 cfu g1). As the level of acidity was similar in both cheeses, this accentuated decrease of S. aureus in the nisin (+) Jben might only be due to the effect of the accumulated nisin in the cheese. Nisin activity was detected only in cheese made with nisin (+) starter (Table 2). On the other hand, a noticeable decrease (po0:05) in S. aureus population was observed in nisin () cheese samples between times 168 and 192. This might be explained by the inhibitory effect of the relatively high levels of lactic acid (1.92–1.94 g 100 g1) in the nisin () cheese on the S. aureus population during this period (Table 2). The data presented in this study concur with those of Hamama and Tatini (1991) who reported the presence of staphylococcal enterotoxin in Lben at 8–12 h after inoculation of raw milk with S. aureus (at a level of 105 cfu mL1) despite the presence of a relatively high population of native lactococci (B106–107 cfu mL1). Other studies conducted on different types of cheese (i.e., Domiati, Brazilian Minas, and Burgos) suggested that the presence of relatively high enterotoxigenic S. aureus counts (106–108 cfu mL1) in milk led frequently to detectable levels of enterotoxin in cheese (Ahmed, Mustafa, & Marth, 1983; Dos Santos & Genigeorgis, 1981; Otero, Garcia, Prieto, & Moreno, 1988). The data from this experiment suggest that initial levels of S. aureus p103 cfu mL1 in milk are not expected to result in enterotoxin in Jben mainly because of the rapid drop in pH due to the active lactic
937
fermentation. The low pH prevents S. aureus from reaching numbers which are capable of producing enterotoxin at detectable levels. Moreover, if nisinproducing starters were used for fermentation, there would probably be a lower likelihood of S. aureus being found in the final product (Table 1). In contrast, when higher S. aureus counts (105 cfu mL1) are present in the initial milk, enterotoxin is rapidly formed in the product despite the use of a nisin-producing lactic culture for fermentation. Hence, when raw milk is to be used for Jben making, attention should be paid to its quality as well as to the conditions of manufacture so as to prevent any contamination of milk with S. aureus. However, because of the high incidence of mastitis among Moroccan dairy cattle and the relative poor hygienic conditions during milk handling and traditional Jben preparation, it is highly recommended to use pasteurized milk and very active lactic starter cultures (similar to those used in this study) which are capable of lowering the initial milk pH by more than one unit over a 24-h period. The nisin-producing lactococci are of little help, as shown in this study, to prevent S. aureus growth and subsequent formation of enterotoxin particularly when the initial milk contamination with S. aureus is relatively high (e.g. 105 cfu mL1).
Acknowledgements This research work was supported by the Fond International de Coope! ration Universitaire (FICU) of Agence Universitaire de la Francophonie (AUPELF/ URU). The authors are grateful to Pr. Ronald Simard (University Laval, Que! bec, Canada) for providing the nisin-producing starter used in this work, and to the Dairy Cooperative ‘‘Extra-Lait’’, Ke! nitra, Morocco, for providing the raw materials.
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