- Email: [email protected]
Production of staphylococcal enterotoxin A in cream-filled cake
International Journal International Journal of Food Microbiology 26 (1995) 259-263
ELSEVIER
of Food Microbiology
Short Communication
Production of staphylococcal enterotoxin A in cream-filled cake Liliana Laura Costanzo Anuncia~ao a, Walter Roberto Linardi Luiz Simeao do Carmo b, Merlin S. Bergdoll C,'
a,
a Departamento de Microbiologia, Instituto de Ciencias Biologicas da UMFG, Rua Conde Pereira Carneiro 80, Gameleira, CEP 30550, Belo Horizonte, Brazil b Fundac;.ao Ezequiel Dias, Laboratorio de Microbiologia de Alimentos, Rua Conde Pereira Carneiro 80, Gameleira, CEP 30550, Belo Horizonte, Brazif Food Research Institute, University of WISconsin, 1925 Willow Drive, Madison, WI 53706, USA
Received 13 January 1994; revision received 8 July 1994; accepted 18 August 1994
Abstract Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10 6 ) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210°C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.
Keywords: Cake; Cream filling; Staphylococci; Enterotoxin; Food poisoning
1. Introduction
Staphylococcal food poisoning is a common foodborne disease that occurs in most countries of the world. The vomiting and diarrhea that usually accompanies it last only a few hours to one day (Bergdoll, 1989), hence, most people who are affected never seek medical attention. This type of food poisoning has come to the attention of public health authorities in Brazil in recent years and a number of • Corresponding author. Tel.: 608-263-6938. Fax: 608-263-1114. 0168-1605/95/$09.50 © 1995 Elsevier Science SV. All rights reserved SSDI 0168-1605(94)00122-7
260
L.L. Costanzo Anunciaq,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
outbreaks have been investigated. One of the major items of food involved in Brazil has been cream-filled cake (Carmo and Bergdoll, 1990). Most outbreaks occurred after improper refrigeration of the cakes, probably due to lack of appreciation that this type of food is vulnerable to staphylococcal food poisoning. This research project was undertaken to demonstrate the conditions necessary for cream-filled cakes to be involved in staphylococcal food poisoning.
2. Materials and methods Staphylococcus aureus. The Staphylococcus aureus strain used in this study was FRI-lOO, a strain that produces enterotoxin A (SEA), the enterotoxin most commonly involved in staphylococcal food poisoning (Weineke, 1974). Preparation of inoculum. Strain FRI-100 was grown in brain heart infusion (BHI) broth at 37°C for 24 h. Slants of agar Lignieres were inoculated with the BHI broth culture and incubated at 37°C for 24 h. One slant was used to inoculate two tryptic soy agar slants which were incubated at 37°C for 24 h. Buffered peptone water (7 ml for each slant) was used to remove the cells from the slants. One ml of the cell suspension was diluted with 9 ml of buffered peptone water and eight subsequent lO-fold dilutions were made. One ml was used from each dilution to inoculate each of two plate count agar plates to record the cfu count. Preparation of cakes with milk. Two cakes were prepared to test the survival of enterotoxin during the baking. Each cake was made by mixing 200 g of sugar, 80 g of cornstarch, 150 g of flour, 50 g of margarine, ± 8 g of baking powder, 2 eggs, and 200 ml of milk. Six ng SEA/g of cake dough was added before the mixing of the cake dough for one cake. 100 g of this cake dough was used for SEA analysis as a control for the part that was baked. The cakes were baked at 210°C for 45 min. The second cake was used as a positive and negative control. For the positive control, 200 ng of SEA was added to 100 g of this cake (2 ng/g of cake) before homogenizing and analyzing for the SEA. As a negative control, 100 g without the addition of SEA was used. Preparation of cakes without milk. Each cake was made by mixing 3 egg yolks, 160 g of sugar, 140 g of flour, ± 10 g baking powder, and 150 ml of water. The cakes were baked at 210°C for ± 45 min. Cake with sweetened condensed. milk filling. The cake was made using the same recipe as was used for the cream-filled cakes. The sweetened condensed milk was purchased in a can and was used directly for the filling of the cake. The inoculum used was 10 4 cfu/g. Preparation of cream filling. The cream filling was prepared as follows: one can of condensed milk (395 g), 480 ml of milk, 3 egg yolks, and 15 g corn starch were mixed, heated until the mixture boiled (about 3-5 min), allowed to cool, weighed, and inoculated. After filling the cakes with the cream, one piece was put in the refrigerator at 6°C, the remainder incubated at room temperature (27-29°C). At predetermined time intervals, a piece of cake was taken to be analyzed for staphylococcal count and enterotoxin content
L.L. Costanzo Anunciac;,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
261
Detennination of staphylococcal count. 25 g of the cake filling was suspended in 225 ml of buffered peptone water; 0.1 ml was placed on Baird-Parker agar plates. Additional plates were prepared with lO-fold dilutions if necessary (Tatini et aI., 1987). The plates were incubated for 48 hours at 37°C. Enterotoxin detection in the cakes. 50 g of the middle of the cake containing the cream filling was homogenized in a Waring blender with 1-1.5 ml of 0.02 M NaHP0 4 in saline, pH 7.4, per gram of food (Freed et aI., 1982). The homogenized food was adjusted to pH 4.5 and centrifuged. The pH of the supernatant fluid was readjusted to pH 7.4 and recentrifuged if any precipitate occurred. The extract was tested for the presence of enterotoxin by use of the ELISA ball kit obtained from Dr. Bomelli AG, Stationstrasse 12, CH-3097 Libbefeld-Bern, Switzerland (Fey and Pfister, 1983). This method is sensitive to 0.5 ng/ml and is one of the more reliable sensitive methods available for checking foods for enterotoxin (Weineke, 1987, 1991).
3. Results and discussion Two possibilities existed to explain the presence of enterotoxin in the cakes. One was that it could be in the ingredients used to prepare the cake. This was tested by adding SEA to the cake dough made with milk at the time of preparation and before the baking. Milk was used in these cakes to include all possible ingredients used in making cakes. Although milk can be used in making cakes, water is more commonly used, the reason for using it in the other cakes. However, no SEA could be detected in the baked cake showing that the ingredients were not a likely source of the enterotoxin in the cakes. The other source was the cream filling, and to test this possibility, the cream filling was contaminated with different size inocula. The cakes were incubated at room temperature for different periods of time to determine whether adequate growth could occur to produce enterotoxin. As is shown in Table 1, SEA was not detectable before the staphylococcal count reached approximately 10 6 cfu/g. The larger the inoculum the shorter the time required to reach this count. As could be seen, with an inoculum size of 10 3 cfu/g, SEA was detectable within 18 h at room temperature. This is the approximate amount of time that a cream-filled cake, involved in a food poisoning outbreak that occurred during the course of this investigation, was out of the refrigerator. The cake was bought in a bakery and remained out of the refrigerator overnight. It was put in the refrigerator the following morning and was eaten at 16:30 by 10 people. Seven of the people became ill with vomiting and diarrhea 3.5 h after eating the cake. Three persons were hospitalized. Staphylococci, that produced SEA, and SEA were isolated from the cake. The results from this research show that the cream-filling is the most likely source of the enterotoxin causing the food poisoning. Although the cream-filling is heated before placing it in the cake it can easily be contaminated during the filling operation. The cream filling provides an excellent medium for the growth of
262
L.L. Costanzo Anuncialiao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
Table 1 Staphylococcal growth and production of enterotoxin in cream-filled cake at room temperature (27-29°C) Inoculum (cfu/g)
Time (h)
S. aureus (cfu/g)
10 2
0 21 23 25 27
10 2 2.0x10 5 5.0X 10 5 1.4 X 10 6 2.0X 10 6
0 11 15 18 21
10 3 1.1 X 10 5 5.0x10 5 1.5 x 10 6 5.3x 10 6
0 8
10 4 5.3x105 3.0X10 5 9.3x10 5
10 3
10 4
lOa
12
3.1 x 10 6
SEA
Inoculum (cfu/g) 10 5
+ + 10 6
+ +
Time (h)
SEA
0 1 4.5 6.5
S. aureus (cfu/g) 10 5 8.7x 10 5
1.2 X 10 6 3.0x 10 6
+ +
0 1.5 3 4
10 6 1.7 X 10 6 4.0x 10 6 5.0x10 6
+ +
+ +
a The first value is from cake without cream.
staphylococci. In contrast, the sweetened milk used as a filling did not support growth of the staphylococci, as the count decreased from 4.0 X 10 3 at 0 time to 8.0 x 10 2 at 15 h, probably because the water activity is too low to promote growth (Troller and Stinson, 1975). Essentially all staphylococcal food poisoning outbreaks are a result of human contamination during the preparation of the food. Many people are colonized with enterotoxigenic staphylococci and many infections on the hands and arms are caused by staphylococci (Bergdoll, 1989). It is quite difficult to prevent the contamination of food with staphylococci. The best control of this type of food poisoning is refrigeration as staphylococci do not grow well in the cold (Bergdoll, 1979), as was demonstrated by the cakes that were refrigerated; no growth occurred even at an inoculum of 10 6 nor was SEA detectable in the cake. The major problem is that the average food handler does not realize that a number of foods should be refrigerated at all times except when being prepared and served. This can be a particular problem in the developing countries, particularly if adequate refrigeration is not available.
Acknowledgements
We wish to acknowledge the technical assistance of the personnel of the Food Microbiological Laboratory. This research was supported in part by Fundacsao de Amparo a Pesquisa de Minas Gerais (FAPEMIG). The bacterial culture media were supplied by Biobras.
L.L. Costanzo Anunciaq,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
263
References Bergdoll, M.S. (1979) Staphylococcal intoxications. In: H. Reimann and F.L. Bryan, F.L. (editors), Foodborne Infections and Intoxications. Academic Press, Inc., New York, pp. 443-494. Bergdoll, M.S. (1989) Staphylococcus aureus. In: M.P. Doyle (editor), Foodborne Bacterial Pathogens. Marcel Dekker, Inc., New York, pp. 463-523. Carmo, L.S. do and Bergdoll, M.S. (1990) Staphylococcal food poisoning in Belo Horizonte (Brazil). Rev. Microbiol. 21, 320-323. Fey, H. and Pfister, H. (1983) A diagnostic kit for the detection of staphylococcal enterotoxins (SET) A, B, C, and D (SEA, SEB, SEC, SED). In: S. Arameas (editor), Immunoenzyrnatic Techniques. Elsevier/North-Holland Publ. Co., Amsterdam, pp. 345-348. Freed, R.c., Evenson, M.L., Reiser, R.F. and Bergdoll, M.S. (1982) Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44, 1349-1355. Tatini, S.R., Hoover, D.G. and Lachica, R.V.F. (1984) Methods for the isolation and enumeration of Staphylococcus aureus. In: M.L. Speck (editor), Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association. Inc., Washington, pp. 411-427. Troller, J.A. and Stinson, J.V. (1975) Influence of water activity of growth and enterotoxin formation by Staphylococcus aureus in foods. J. Food Sci. 40, 802-804. Wieneke, AA. (1974) Enterotoxin production by strains of Staphylococcus aureus. J. Hyg. Camb. 73, 255-262. Wieneke, AA. (1987) Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 4, 135-143. Weineke, AA. (1991) Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 14, 305-312.
ELSEVIER
of Food Microbiology
Short Communication
Production of staphylococcal enterotoxin A in cream-filled cake Liliana Laura Costanzo Anuncia~ao a, Walter Roberto Linardi Luiz Simeao do Carmo b, Merlin S. Bergdoll C,'
a,
a Departamento de Microbiologia, Instituto de Ciencias Biologicas da UMFG, Rua Conde Pereira Carneiro 80, Gameleira, CEP 30550, Belo Horizonte, Brazil b Fundac;.ao Ezequiel Dias, Laboratorio de Microbiologia de Alimentos, Rua Conde Pereira Carneiro 80, Gameleira, CEP 30550, Belo Horizonte, Brazif Food Research Institute, University of WISconsin, 1925 Willow Drive, Madison, WI 53706, USA
Received 13 January 1994; revision received 8 July 1994; accepted 18 August 1994
Abstract Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10 6 ) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210°C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.
Keywords: Cake; Cream filling; Staphylococci; Enterotoxin; Food poisoning
1. Introduction
Staphylococcal food poisoning is a common foodborne disease that occurs in most countries of the world. The vomiting and diarrhea that usually accompanies it last only a few hours to one day (Bergdoll, 1989), hence, most people who are affected never seek medical attention. This type of food poisoning has come to the attention of public health authorities in Brazil in recent years and a number of • Corresponding author. Tel.: 608-263-6938. Fax: 608-263-1114. 0168-1605/95/$09.50 © 1995 Elsevier Science SV. All rights reserved SSDI 0168-1605(94)00122-7
260
L.L. Costanzo Anunciaq,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
outbreaks have been investigated. One of the major items of food involved in Brazil has been cream-filled cake (Carmo and Bergdoll, 1990). Most outbreaks occurred after improper refrigeration of the cakes, probably due to lack of appreciation that this type of food is vulnerable to staphylococcal food poisoning. This research project was undertaken to demonstrate the conditions necessary for cream-filled cakes to be involved in staphylococcal food poisoning.
2. Materials and methods Staphylococcus aureus. The Staphylococcus aureus strain used in this study was FRI-lOO, a strain that produces enterotoxin A (SEA), the enterotoxin most commonly involved in staphylococcal food poisoning (Weineke, 1974). Preparation of inoculum. Strain FRI-100 was grown in brain heart infusion (BHI) broth at 37°C for 24 h. Slants of agar Lignieres were inoculated with the BHI broth culture and incubated at 37°C for 24 h. One slant was used to inoculate two tryptic soy agar slants which were incubated at 37°C for 24 h. Buffered peptone water (7 ml for each slant) was used to remove the cells from the slants. One ml of the cell suspension was diluted with 9 ml of buffered peptone water and eight subsequent lO-fold dilutions were made. One ml was used from each dilution to inoculate each of two plate count agar plates to record the cfu count. Preparation of cakes with milk. Two cakes were prepared to test the survival of enterotoxin during the baking. Each cake was made by mixing 200 g of sugar, 80 g of cornstarch, 150 g of flour, 50 g of margarine, ± 8 g of baking powder, 2 eggs, and 200 ml of milk. Six ng SEA/g of cake dough was added before the mixing of the cake dough for one cake. 100 g of this cake dough was used for SEA analysis as a control for the part that was baked. The cakes were baked at 210°C for 45 min. The second cake was used as a positive and negative control. For the positive control, 200 ng of SEA was added to 100 g of this cake (2 ng/g of cake) before homogenizing and analyzing for the SEA. As a negative control, 100 g without the addition of SEA was used. Preparation of cakes without milk. Each cake was made by mixing 3 egg yolks, 160 g of sugar, 140 g of flour, ± 10 g baking powder, and 150 ml of water. The cakes were baked at 210°C for ± 45 min. Cake with sweetened condensed. milk filling. The cake was made using the same recipe as was used for the cream-filled cakes. The sweetened condensed milk was purchased in a can and was used directly for the filling of the cake. The inoculum used was 10 4 cfu/g. Preparation of cream filling. The cream filling was prepared as follows: one can of condensed milk (395 g), 480 ml of milk, 3 egg yolks, and 15 g corn starch were mixed, heated until the mixture boiled (about 3-5 min), allowed to cool, weighed, and inoculated. After filling the cakes with the cream, one piece was put in the refrigerator at 6°C, the remainder incubated at room temperature (27-29°C). At predetermined time intervals, a piece of cake was taken to be analyzed for staphylococcal count and enterotoxin content
L.L. Costanzo Anunciac;,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
261
Detennination of staphylococcal count. 25 g of the cake filling was suspended in 225 ml of buffered peptone water; 0.1 ml was placed on Baird-Parker agar plates. Additional plates were prepared with lO-fold dilutions if necessary (Tatini et aI., 1987). The plates were incubated for 48 hours at 37°C. Enterotoxin detection in the cakes. 50 g of the middle of the cake containing the cream filling was homogenized in a Waring blender with 1-1.5 ml of 0.02 M NaHP0 4 in saline, pH 7.4, per gram of food (Freed et aI., 1982). The homogenized food was adjusted to pH 4.5 and centrifuged. The pH of the supernatant fluid was readjusted to pH 7.4 and recentrifuged if any precipitate occurred. The extract was tested for the presence of enterotoxin by use of the ELISA ball kit obtained from Dr. Bomelli AG, Stationstrasse 12, CH-3097 Libbefeld-Bern, Switzerland (Fey and Pfister, 1983). This method is sensitive to 0.5 ng/ml and is one of the more reliable sensitive methods available for checking foods for enterotoxin (Weineke, 1987, 1991).
3. Results and discussion Two possibilities existed to explain the presence of enterotoxin in the cakes. One was that it could be in the ingredients used to prepare the cake. This was tested by adding SEA to the cake dough made with milk at the time of preparation and before the baking. Milk was used in these cakes to include all possible ingredients used in making cakes. Although milk can be used in making cakes, water is more commonly used, the reason for using it in the other cakes. However, no SEA could be detected in the baked cake showing that the ingredients were not a likely source of the enterotoxin in the cakes. The other source was the cream filling, and to test this possibility, the cream filling was contaminated with different size inocula. The cakes were incubated at room temperature for different periods of time to determine whether adequate growth could occur to produce enterotoxin. As is shown in Table 1, SEA was not detectable before the staphylococcal count reached approximately 10 6 cfu/g. The larger the inoculum the shorter the time required to reach this count. As could be seen, with an inoculum size of 10 3 cfu/g, SEA was detectable within 18 h at room temperature. This is the approximate amount of time that a cream-filled cake, involved in a food poisoning outbreak that occurred during the course of this investigation, was out of the refrigerator. The cake was bought in a bakery and remained out of the refrigerator overnight. It was put in the refrigerator the following morning and was eaten at 16:30 by 10 people. Seven of the people became ill with vomiting and diarrhea 3.5 h after eating the cake. Three persons were hospitalized. Staphylococci, that produced SEA, and SEA were isolated from the cake. The results from this research show that the cream-filling is the most likely source of the enterotoxin causing the food poisoning. Although the cream-filling is heated before placing it in the cake it can easily be contaminated during the filling operation. The cream filling provides an excellent medium for the growth of
262
L.L. Costanzo Anuncialiao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
Table 1 Staphylococcal growth and production of enterotoxin in cream-filled cake at room temperature (27-29°C) Inoculum (cfu/g)
Time (h)
S. aureus (cfu/g)
10 2
0 21 23 25 27
10 2 2.0x10 5 5.0X 10 5 1.4 X 10 6 2.0X 10 6
0 11 15 18 21
10 3 1.1 X 10 5 5.0x10 5 1.5 x 10 6 5.3x 10 6
0 8
10 4 5.3x105 3.0X10 5 9.3x10 5
10 3
10 4
lOa
12
3.1 x 10 6
SEA
Inoculum (cfu/g) 10 5
+ + 10 6
+ +
Time (h)
SEA
0 1 4.5 6.5
S. aureus (cfu/g) 10 5 8.7x 10 5
1.2 X 10 6 3.0x 10 6
+ +
0 1.5 3 4
10 6 1.7 X 10 6 4.0x 10 6 5.0x10 6
+ +
+ +
a The first value is from cake without cream.
staphylococci. In contrast, the sweetened milk used as a filling did not support growth of the staphylococci, as the count decreased from 4.0 X 10 3 at 0 time to 8.0 x 10 2 at 15 h, probably because the water activity is too low to promote growth (Troller and Stinson, 1975). Essentially all staphylococcal food poisoning outbreaks are a result of human contamination during the preparation of the food. Many people are colonized with enterotoxigenic staphylococci and many infections on the hands and arms are caused by staphylococci (Bergdoll, 1989). It is quite difficult to prevent the contamination of food with staphylococci. The best control of this type of food poisoning is refrigeration as staphylococci do not grow well in the cold (Bergdoll, 1979), as was demonstrated by the cakes that were refrigerated; no growth occurred even at an inoculum of 10 6 nor was SEA detectable in the cake. The major problem is that the average food handler does not realize that a number of foods should be refrigerated at all times except when being prepared and served. This can be a particular problem in the developing countries, particularly if adequate refrigeration is not available.
Acknowledgements
We wish to acknowledge the technical assistance of the personnel of the Food Microbiological Laboratory. This research was supported in part by Fundacsao de Amparo a Pesquisa de Minas Gerais (FAPEMIG). The bacterial culture media were supplied by Biobras.
L.L. Costanzo Anunciaq,ao et al. / Int. 1. Food Microbiology 26 (1995) 259-263
263
References Bergdoll, M.S. (1979) Staphylococcal intoxications. In: H. Reimann and F.L. Bryan, F.L. (editors), Foodborne Infections and Intoxications. Academic Press, Inc., New York, pp. 443-494. Bergdoll, M.S. (1989) Staphylococcus aureus. In: M.P. Doyle (editor), Foodborne Bacterial Pathogens. Marcel Dekker, Inc., New York, pp. 463-523. Carmo, L.S. do and Bergdoll, M.S. (1990) Staphylococcal food poisoning in Belo Horizonte (Brazil). Rev. Microbiol. 21, 320-323. Fey, H. and Pfister, H. (1983) A diagnostic kit for the detection of staphylococcal enterotoxins (SET) A, B, C, and D (SEA, SEB, SEC, SED). In: S. Arameas (editor), Immunoenzyrnatic Techniques. Elsevier/North-Holland Publ. Co., Amsterdam, pp. 345-348. Freed, R.c., Evenson, M.L., Reiser, R.F. and Bergdoll, M.S. (1982) Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44, 1349-1355. Tatini, S.R., Hoover, D.G. and Lachica, R.V.F. (1984) Methods for the isolation and enumeration of Staphylococcus aureus. In: M.L. Speck (editor), Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association. Inc., Washington, pp. 411-427. Troller, J.A. and Stinson, J.V. (1975) Influence of water activity of growth and enterotoxin formation by Staphylococcus aureus in foods. J. Food Sci. 40, 802-804. Wieneke, AA. (1974) Enterotoxin production by strains of Staphylococcus aureus. J. Hyg. Camb. 73, 255-262. Wieneke, AA. (1987) Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 4, 135-143. Weineke, AA. (1991) Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 14, 305-312.