International Journal of Food Microbiology, 10 (1990) 33-42 Elsevier
33
FOOD 00293
Inactivation of staphylococcal enterotoxins by heat and reactivation by high pH treatment M. Schwabe 2, S. N o t e r m a n s ~, R. Boot 1, S.R. Tatini 3 and J. Kr~imer 2 I National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, 2 Department of Agricultural and Food Microbiology, Institute of Microbiology, University of Bonn, Bonn; FR.G., and 3 University of Minnesota, St. Paul, MN, U.S.A. (Received 8 May 1989; accepted 24 July 1989)
Inactivation of staphylococcal enterotoxins (SE) added to different media upon heat treatment (80 o C or 100 ° C for 10 min) and reactivation of inactivated SE was studied. In gelatin (pH 4.0), lettuce extract, peas and beans extracts and ovalbumin (pH 5.0) the immunological activity of SE was lost almost completely upon heating. The loss of immunological activity of SEA was accompanied by a concomitant loss of biological activity of this toxin (monkey feeding test). A high pH treatment (pH 11) of the different preparations restored both the immunological and biological activity in most samples tested. Heating at 80 o C or 100 ° C for 10 rain of SE containing gelatin (pH 7.0), cauliflower extract (pH 4.0 or pH 7.0), milk (pH 4.0), casein (pH 6.0), rice extract (pH 7.0), noodles extract (pH 4.0) and oat-flakes extract (pH 7.0) had a much lower effect on the immunological activity of the SE (activity > 25%). Key words: Staphylococcal enterotoxin; Heat inactivation; Reactivation
Introduction
Staphylococcal food poisoning results from the ingestion of enterotoxins (SE) formed in foods by certain strains of S t a p h y l o c o c c u s aureus (Bergdoll, 1970). It was recognized early by Jordan et al. (1931), by feeding human volunteers, that sterile filtrates of an enterotoxigenic strain of S. aureus could be boiled for 30 rain without complete loss of toxic activity. In subsequent studies of Bergdoll et al. (1951) it was demonstrated that SE are indeed extremely heat resistant, but that their activity decreased with prolonged heating. Although SE are generally considered to be heat stable, evidence has been obtained that heat resistance depends on several factors. Satterlee and Kraft (1969) showed that SEB was more easily inactivated in a beef slurry, pH 6.6, than in 0.013 M phosphate buffer, p H 7.4, at 100°C. The same authors reported that addition of either myosin or metmyoglobin to a phosphateCorrespondence address: S. Notermans, Laboratory of Water and Food Microbiology, National Institute of Public Health and Environmental Protection, 3720 BA Bilthoven, The Netherlands. 0168o1605/90/$03.50
© 1990 Elsevier Science Publishers B.V. (Biomedical Division)
34 saline buffer resulted in a rapid loss of the SE upon heating. Also the p H of the menstruum in which the toxin is heated influenced the heat resistance. It was demonstrated that SEA in beef bouillon was inactivated faster at p H 5.3 than at p H 6.2 (Humber et al., 1975). Also experiments cited by Tatini (1976) showed that SE is relatively more stable to heat at p H 6.0 or higher than at p H 4.5-5.5, although the opposite was true for SED! Different inactivation experiments have shown that a more rapid loss of immunological activity at 70 ° C to 80 ° C occurs than at 90 ° C to 1 0 0 ° C (Jamlang et al., 1971; Fung et al., 1973; N o t e r m a n s et al., 1987). Satterlee and Kraft (1969) showed, however, that only the initial thermal inactivation at 8 0 ° C was faster than at 1 0 0 ° C or 110°C. By using small quantities of SE (100 n g / m l ) Tibana et al. (1987) confirmed these observations with SEB. However, the total time required for complete inactivation of SEB was much longer at 80 o C than at 100 ° C. In fact a complete inactivation of SEA, SEB and SEC present in 0.05 M phosphate buffer, p H 7.4, was not obtained during heating at 80 ° C for 180 rain. Reactivation of SE after heat treatment has been noted by Fung et al. (1973). In their experiments SEC was heated at 8 0 ° C until about 50% of the toxin was inactivated. Incubation of the heated toxin for over 24 h at 25 ° C resulted in a 100% regain of activity. It was shown by Tatini (1976) that treatment of heated SEA or SED with urea also increased the recovery of both toxins almost four-fold. H o w ever, reactivation of SEB present in heat treated frankfurter-type sausages was not observed when the sausages were stored at 4 ° C and 25 ° C for more than 24 h ( H o r n et al., 1986). In this study we have tested the effect of p H of different food and food extracts on the heat inactivation of SE in more detail. Also the possibility of reactivation by high p H treatment and addition of collagenase were tested. The SE was detected by two methods: monkey feeding and serological assay. Material and Methods
Enterotoxins The SEA used in recovery experiments and in the monkey feeding tests were a gift of Dr. Anna Johnson-Winegar, U.S. A r m y Medical Research Institute, Fort Detrick, MD, U.S.A. SEB and SEC were produced and purified by the method described by Shinagawa et al. (1975).
Preparation of food and food extracts Food extracts were prepared by blending equal amounts of food and destilled water and subsequent centrifuging (10000 × g for 10 min) of the mixtures. Foods were obtained from a local store. Peas and beans were dried products and were soaked in distilled water for 5 h before extraction. Isolated proteins and starch were soluted in distilled water in specified concentrations. Pre-heated food and food extracts were prepared as described above. However, after preparation they were heated at 95 ° C for 5 min and subsequently centrifuged (10000 × g for 10 min). The p H of the food and food extracts were adjusted with either 1 N N a O H or with 1 N HC1.
35
Recovery experiments SE was added to phosphate buffered saline solution, different food products and food extracts and was subsequently heated at either 1 0 0 ° C or 8 0 ° C for 10 rain in a water bath. After this treatment the quantity of SE present was detected by ELISA and by the monkey feeding test. Recovery is expressed as percentage of added SE.
Reactioation experiments SE was added to food and food extracts and was heated at either 100 ° C or 80 o C for 10 min. After cooling the pH was adjusted to p H 11.0 using 1 N N a O H immediately followed by adjustment of the p H to p H 7.0 using 1 N HC1. Reactivation of SE heat-inactivated in gelatin was also carried out by digestion of the gelatin by collagenase. For this collagenase (Sigma C-9891) was added at a final concentration of 0.1% ( w / v ) at a pH 7.4 of the test solution. The mixture was incubated at 37 ° C for 24 h. The effect of reactivation was measured by detecting the SE by ELISA and by the monkey feeding test.
ELISA procedure For quantitative determination of SE the sandwich E L I S A technique was used as described earlier (Notermans et al., 1983). Wells of polystyrene trays (Cooke, Dynatech. cat. no. 1-220-25) were coated with anti SE-IgG from sheep. After sample incubation test samples were diluted in 0.07 M phosphate buffered saline solution (PBS), p H 7.1, and the amount of adsorbed SE was detected using sheep anti SE-IgG conjugated to peroxidase. The enzyme reaction was determined spectrophotometrically at 450 nm after addition of 5-amino salicylic acid and H202. The IgG used was prepared by immunizing sheep with purified SEA, SEB and SEC, respectively (Notermans et al., 1983).
Monkey feeding tests For testing whether SEA added to different extracts had lost its biological activity upon heating or regained its activity upon high p H treatment monkey feeding tests were carried out. From the samples monkeys (Macaca fascicularis) with a body weight of approximately 2-2.5 kg received 20-ml portions. Administration was done using a nasal tube. The monkeys were observed for vomiting during a period of 6 h. Monkeys were only used once in the experiments. However, if vomiting was not observed after administering the samples, the same monkey received 20 ml Brain Heart Infusion Broth (Difco 0037-01-6) with SEA alone after an interval of at least 14 days.
Results
Recovery of SE from different media after heat treatment SE were added to PBS, food and food extracts and subsequently heat treated. The effect of the heat treatment on immunological recovery of SE is presented in Table I. In PBS the SE was only inactivated partially and recovery amounted to 65%
36 TABLE I Recovery of SE from phosphate buffered saline (PBS), food and food extracts after different treatment, as determined by ELISA Medium
Treatment
Recovery of SE in percentages of added S E a SEA
PBS
Gelatin (1% (5% (5% (5% (5% (5% (5%
pH 7.2
w/v) w/v) w/v) w/v) w/v) w/v) w/v)
Lettuce extract
Peas extract
10 m i n / 1 0 0 o C 10 m i n / 8 0 ° C
pH pH pH pH pH pH pH
4.0 4.0 4.0 5.5 5.5 7.0 7.0
10 10 10 10 10 10 10
min/80 ° C min/80 o C min/100 ° C min/80 o C min/100 ° C min/80 ° C min/100 ° C
pH pH pH pH pH pH
4.0 4.0 5.5 5.5 7.0 7.0
10 10 10 10 10 10
min/80 ° C min/100 ° C min/100 o C rain/80 ° C rain/100 ° C
pH pH pH pH pH
4.0 5.0 5.5 7.0 7.0
10 10 10 10 10
min/80 o C min/80 ° C min/80 o C min/80 o C min/100 ° C
rain~80 o C
SEB
SEC
65 39
33
FOOD 00293
Inactivation of staphylococcal enterotoxins by heat and reactivation by high pH treatment M. Schwabe 2, S. N o t e r m a n s ~, R. Boot 1, S.R. Tatini 3 and J. Kr~imer 2 I National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, 2 Department of Agricultural and Food Microbiology, Institute of Microbiology, University of Bonn, Bonn; FR.G., and 3 University of Minnesota, St. Paul, MN, U.S.A. (Received 8 May 1989; accepted 24 July 1989)
Inactivation of staphylococcal enterotoxins (SE) added to different media upon heat treatment (80 o C or 100 ° C for 10 min) and reactivation of inactivated SE was studied. In gelatin (pH 4.0), lettuce extract, peas and beans extracts and ovalbumin (pH 5.0) the immunological activity of SE was lost almost completely upon heating. The loss of immunological activity of SEA was accompanied by a concomitant loss of biological activity of this toxin (monkey feeding test). A high pH treatment (pH 11) of the different preparations restored both the immunological and biological activity in most samples tested. Heating at 80 o C or 100 ° C for 10 rain of SE containing gelatin (pH 7.0), cauliflower extract (pH 4.0 or pH 7.0), milk (pH 4.0), casein (pH 6.0), rice extract (pH 7.0), noodles extract (pH 4.0) and oat-flakes extract (pH 7.0) had a much lower effect on the immunological activity of the SE (activity > 25%). Key words: Staphylococcal enterotoxin; Heat inactivation; Reactivation
Introduction
Staphylococcal food poisoning results from the ingestion of enterotoxins (SE) formed in foods by certain strains of S t a p h y l o c o c c u s aureus (Bergdoll, 1970). It was recognized early by Jordan et al. (1931), by feeding human volunteers, that sterile filtrates of an enterotoxigenic strain of S. aureus could be boiled for 30 rain without complete loss of toxic activity. In subsequent studies of Bergdoll et al. (1951) it was demonstrated that SE are indeed extremely heat resistant, but that their activity decreased with prolonged heating. Although SE are generally considered to be heat stable, evidence has been obtained that heat resistance depends on several factors. Satterlee and Kraft (1969) showed that SEB was more easily inactivated in a beef slurry, pH 6.6, than in 0.013 M phosphate buffer, p H 7.4, at 100°C. The same authors reported that addition of either myosin or metmyoglobin to a phosphateCorrespondence address: S. Notermans, Laboratory of Water and Food Microbiology, National Institute of Public Health and Environmental Protection, 3720 BA Bilthoven, The Netherlands. 0168o1605/90/$03.50
© 1990 Elsevier Science Publishers B.V. (Biomedical Division)
34 saline buffer resulted in a rapid loss of the SE upon heating. Also the p H of the menstruum in which the toxin is heated influenced the heat resistance. It was demonstrated that SEA in beef bouillon was inactivated faster at p H 5.3 than at p H 6.2 (Humber et al., 1975). Also experiments cited by Tatini (1976) showed that SE is relatively more stable to heat at p H 6.0 or higher than at p H 4.5-5.5, although the opposite was true for SED! Different inactivation experiments have shown that a more rapid loss of immunological activity at 70 ° C to 80 ° C occurs than at 90 ° C to 1 0 0 ° C (Jamlang et al., 1971; Fung et al., 1973; N o t e r m a n s et al., 1987). Satterlee and Kraft (1969) showed, however, that only the initial thermal inactivation at 8 0 ° C was faster than at 1 0 0 ° C or 110°C. By using small quantities of SE (100 n g / m l ) Tibana et al. (1987) confirmed these observations with SEB. However, the total time required for complete inactivation of SEB was much longer at 80 o C than at 100 ° C. In fact a complete inactivation of SEA, SEB and SEC present in 0.05 M phosphate buffer, p H 7.4, was not obtained during heating at 80 ° C for 180 rain. Reactivation of SE after heat treatment has been noted by Fung et al. (1973). In their experiments SEC was heated at 8 0 ° C until about 50% of the toxin was inactivated. Incubation of the heated toxin for over 24 h at 25 ° C resulted in a 100% regain of activity. It was shown by Tatini (1976) that treatment of heated SEA or SED with urea also increased the recovery of both toxins almost four-fold. H o w ever, reactivation of SEB present in heat treated frankfurter-type sausages was not observed when the sausages were stored at 4 ° C and 25 ° C for more than 24 h ( H o r n et al., 1986). In this study we have tested the effect of p H of different food and food extracts on the heat inactivation of SE in more detail. Also the possibility of reactivation by high p H treatment and addition of collagenase were tested. The SE was detected by two methods: monkey feeding and serological assay. Material and Methods
Enterotoxins The SEA used in recovery experiments and in the monkey feeding tests were a gift of Dr. Anna Johnson-Winegar, U.S. A r m y Medical Research Institute, Fort Detrick, MD, U.S.A. SEB and SEC were produced and purified by the method described by Shinagawa et al. (1975).
Preparation of food and food extracts Food extracts were prepared by blending equal amounts of food and destilled water and subsequent centrifuging (10000 × g for 10 min) of the mixtures. Foods were obtained from a local store. Peas and beans were dried products and were soaked in distilled water for 5 h before extraction. Isolated proteins and starch were soluted in distilled water in specified concentrations. Pre-heated food and food extracts were prepared as described above. However, after preparation they were heated at 95 ° C for 5 min and subsequently centrifuged (10000 × g for 10 min). The p H of the food and food extracts were adjusted with either 1 N N a O H or with 1 N HC1.
35
Recovery experiments SE was added to phosphate buffered saline solution, different food products and food extracts and was subsequently heated at either 1 0 0 ° C or 8 0 ° C for 10 rain in a water bath. After this treatment the quantity of SE present was detected by ELISA and by the monkey feeding test. Recovery is expressed as percentage of added SE.
Reactioation experiments SE was added to food and food extracts and was heated at either 100 ° C or 80 o C for 10 min. After cooling the pH was adjusted to p H 11.0 using 1 N N a O H immediately followed by adjustment of the p H to p H 7.0 using 1 N HC1. Reactivation of SE heat-inactivated in gelatin was also carried out by digestion of the gelatin by collagenase. For this collagenase (Sigma C-9891) was added at a final concentration of 0.1% ( w / v ) at a pH 7.4 of the test solution. The mixture was incubated at 37 ° C for 24 h. The effect of reactivation was measured by detecting the SE by ELISA and by the monkey feeding test.
ELISA procedure For quantitative determination of SE the sandwich E L I S A technique was used as described earlier (Notermans et al., 1983). Wells of polystyrene trays (Cooke, Dynatech. cat. no. 1-220-25) were coated with anti SE-IgG from sheep. After sample incubation test samples were diluted in 0.07 M phosphate buffered saline solution (PBS), p H 7.1, and the amount of adsorbed SE was detected using sheep anti SE-IgG conjugated to peroxidase. The enzyme reaction was determined spectrophotometrically at 450 nm after addition of 5-amino salicylic acid and H202. The IgG used was prepared by immunizing sheep with purified SEA, SEB and SEC, respectively (Notermans et al., 1983).
Monkey feeding tests For testing whether SEA added to different extracts had lost its biological activity upon heating or regained its activity upon high p H treatment monkey feeding tests were carried out. From the samples monkeys (Macaca fascicularis) with a body weight of approximately 2-2.5 kg received 20-ml portions. Administration was done using a nasal tube. The monkeys were observed for vomiting during a period of 6 h. Monkeys were only used once in the experiments. However, if vomiting was not observed after administering the samples, the same monkey received 20 ml Brain Heart Infusion Broth (Difco 0037-01-6) with SEA alone after an interval of at least 14 days.
Results
Recovery of SE from different media after heat treatment SE were added to PBS, food and food extracts and subsequently heat treated. The effect of the heat treatment on immunological recovery of SE is presented in Table I. In PBS the SE was only inactivated partially and recovery amounted to 65%
36 TABLE I Recovery of SE from phosphate buffered saline (PBS), food and food extracts after different treatment, as determined by ELISA Medium
Treatment
Recovery of SE in percentages of added S E a SEA
PBS
Gelatin (1% (5% (5% (5% (5% (5% (5%
pH 7.2
w/v) w/v) w/v) w/v) w/v) w/v) w/v)
Lettuce extract
Peas extract
10 m i n / 1 0 0 o C 10 m i n / 8 0 ° C
pH pH pH pH pH pH pH
4.0 4.0 4.0 5.5 5.5 7.0 7.0
10 10 10 10 10 10 10
min/80 ° C min/80 o C min/100 ° C min/80 o C min/100 ° C min/80 ° C min/100 ° C
pH pH pH pH pH pH
4.0 4.0 5.5 5.5 7.0 7.0
10 10 10 10 10 10
min/80 ° C min/100 ° C min/100 o C rain/80 ° C rain/100 ° C
pH pH pH pH pH
4.0 5.0 5.5 7.0 7.0
10 10 10 10 10
min/80 o C min/80 ° C min/80 o C min/80 o C min/100 ° C
rain~80 o C
SEB
SEC
65 39
