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Inactivation of staphylococcal enterotoxins by heat and reactivation by high pH treatment
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
<1
<1 <1
2.5 1.6 <1 16 20 25 25
1.3 1.6 50 50 79 80
2.4 <1 <1 <1 2.0 2.5
2.4 <1 1.0 1.3 8 10
<1
<1
<1 1.3 2.5
1.1 14
1.0
Beans extract
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 o C
<1 <1 3
<1 <1 11
Caulifl¢ wet extract
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 ° C
32 6 25
25 13 22
Milk
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 o C
50 4 16
100 10 25
Casein (2.5% w / v )
pH 5.0 pH 6.0
10 m i n / 8 0 ° C 10 rain/80 o C
20 25
Whey
pH 4.0 pH 6.0
10 m i n / 8 0 o C 10 m i n / 8 0 ° C
13 4
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 ° C 10 rain/80 ° C 10 rain/80 ° C
pH 4.0 pH 5.5 pH 7.0
10 rain/80 ° C 10 rain/80 ° C 10 m i n / 8 0 ° C
Bovine Serum Albumin (1% w / v )
Rice extract
11 6 29
16 <1 <1
16 8 18 4 16 40
85 20 41
37 TABLE I (continued). Medium
Treatment
Recovery of SE in percentages of added SEa SEA
SEB
SEC
Noodles extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 rain/80 o C 10 min/80 o C
25 3 13
Oat-flakes extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 °C 10 min/80 ° C 10 min/80 o C
2.5 20 50
Starch (1% w/v)
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 min/80 o C 10 min/80 ° C
20 20 20
37 45 73
Ovalbumin (1% w/v)
pH pH pH pH
10 min/80 ° C 10 min/80 ° C 10 min/80 ° C 10 rain/80 o C
4
85
3 5
23 39
4.0 5.0 5.5 7.0
81 46 53 25 38 100
<1
a 5 #g/ml of SEA and 1 #g/ml of SEB and SEC was added, respectively.
for SEA a n d 39% for SEB after heating for 10 m i n at 1 0 0 ° C a n d 8 0 ° C , respectively. I n gelatin with a p H of 4.0 the i n a c t i v a t i o n of a d d e d SE was n e a r l y c o m p l e t e a n d recoveries varied from < 1-2.5%. A t higher p H values of the g e l a t i n s o l u t i o n the heat i n a c t i v a t i o n was Tess p r o n o u n c e d a n d c o m p a r a b l e to PBS. W i t h lettuce extract, peas extracts a n d b e a n s extracts a n e a r l y c o m p l e t e i n a c t i v a t i o n of SE was observed at p H 4.0, 5.0 a n d 5.5. At p H 7.0 a very p r o n o u n c e d i n a c t i v a t i o n was observed with these extracts. W i t h cauliflower extract, milk, n o o d l e s extract a n d o v a l b u m i n it a p p e a r e d that the highest i n a c t i v a t i o n was observed at p H 5.5. W i t h casein, rice extract, oat-flakes extract a n d starch n o clear effect of the p H o n i n a c t i v a t i o n was observed a n d the recoveries of SE from b o t h h e a t - t r e a t e d p r o d u c t s varied from 4 - 1 0 0 % . F o r whey a n d b o v i n e serum a l b u m i n a p r o n o u n c e d inactivation was observed at all p H values. I n most cases SEC was m o r e heat stable t h a n SEB.
Recovery of SEB after heating in different pre-heated media D u r i n g h e a t i n g of some food a n d food p r o d u c t s it was observed that p r e c i p i t a tion occurred. T o test w h e t h e r SEB was enclosed in the precipitate or in a n e t w o r k of molecules resulting i n a decrease of the recovery of the t o x i n m e d i a were pre-heated a n d s u b s e q u e n t l y centrifuged. T o the s u p e r n a t a n t SEB was a d d e d a n d heat treated. F r o m the results p r e s e n t e d in T a b l e II it b e c a m e clear that n o such p r e c i p i t a t i o n had i n f l u e n c e d the results represented in T a b l e I. I n whey a n d cauliflower extract the recovery of SEB increased u p o n pre-heating.
38 TABLE II Recovery of SEB after heating (80 o C for 10 rain) in. pre-heated (95 ° C for 5 min) food and food extracts as determined by ELISA Medium
Recovery of SEB in percentages of added SEB (1/~g/ml) Without pre-heating
Gelatin (1% w/v) Whey Peas extract Beans extract Lettuce extract Cauliflower extract
pH pH pH pH pH pH
4.0 6.0 4.0 4.0 6.5 5.5
With pre-heating
2.5 4 < 3 < 3 <1 8
3 20 3 <3 3 16
Reactivation of heat-inactivated SE present in food and food extracts Collagenase treatment of heated SEB containing gelatin did not increase the r e c o v e r y o f SEB. I n a n e x p e r i m e n t i n w h i c h S E B w a s a d d e d t o c o l l a g e n a s e - d i g e s t e d gelatin heat included).
inactivation of SEB
was
equal
to not
digested gelatin (data
not
TABLE III Reactivation effect of high pH of heat inactivated SE present in food and food extracts as determined by EL1SA Medium
Treatment
Recovery in percentages of added SEa After heating After high pH treatment b SEA
SEB
SEA
SEB
Gelatin (5% w/v) (1% w/v)
pH 4.5 pH 4.0
10 rain/100 ° C 10 rain/80 ° C
<1
Ovalbumin (1% w/v)
pH 5.0 pH 5.5
10 min/80 ° C 10 rain/80 ° C
<1
pH 5.5 pH 5.5
10 rnin/100 ° C 10 rain/80 ° C
<1 <1
pH 4.0 pH 5.0 pH 7.0
10 min/80 ° C 10 min/80 ° C 10 min/100 o C
<1 1
Beans extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 min/80 ° C 10 rain/80 ° C
<1 2 4
32 16 13
Cauliflower extract
pH 5.5
10 min/80 ° C
8
40
Whey
pH 6.0
10 min/80 o C
4
8
Lettuce extract Peas extract
28 2.5
50 21
3 2
79 <1 2
<1
8 40
35 2
a 5 /.tg/ml of SEA and 1 p.g/ml of SEB was added. b pH treatment involved adjusting of pH to pH 11.0 using 1 N NaOH followed by adjusting of pH to pH 7.0 using 1 N HC1.
39
Other reactivation experiments carried out made clear that high p H treatment was effective in reactivation of the heated staphylococcal enterotoxins. It has to be noticed that the reactivation occurred abruptly after the p H was adjusted to 10-11. Recoveries of SE after high pH treatment are summarized in Table III. With gelatin, ovalbumin, peas extract, beans extract and cauliflower extract the reactivation effect was pronounced and recoveries up to 79% of the original added SE were observed. With lettuce extract and whey, however, no clear reactivation effect was observed upon high pH treatment.
Comparison of ELISA and monkey feeding test From the foregoing it becomes clear that after heating of certain food and food extracts amounts of SE demonstrated were quite low. Subsequent high p H treatment showed that for some products the immunological activity was largely regained. In order to test whether the heated food and food extracts were still biologically active monkey feeding tests were carried out (Table IV). The sensitivity of the monkeys was tested by administering SEA diluted in BHI to each of nine monkeys. Eight of them vomited 120-180 rain after administration of 20/~g of SEA. No emetic reactions were observed in monkeys fed with heated gelatin (5% w / v , p H 4.5), heated ovalbumin (1% w / v , p H 5.0) or heated peas extract (pH 5.0). All these
T A B L E IV Immunological and biological activity of SEA after heat treatment and after reactivation by high p H a in different media Total quantity of SEA tested (#g/monkey) 20 120
180
180
Medium
Treatment
Total immunological activity (# g / m o n k e y ) as determined with ELISA
Monkey feeding test b
Brain heart infusion broth
not treated
20
8/9
Gelatin (5% w / v p H 4.5)
(a) 100 o C for 10 rain
0.6
0/5
(b) 100 ° C for 10 rain followed by high p H treatment
38.4
2/2
(a) 80 o C for 10 min
0.4
0/2
(b) 80 ° C for 10 min followed by high p H treatment
43.2
2/2
(a) 80 ° C for 10 rain (b) 80 ° C for 10 min followed by high p H treatment
2.0 63.0
0/2 2/2
Ovalbumin (1% w / v p H 5.0)
Peas extract (pH 5.0)
a Treatment consisted of adjusting the p H to p H 11 followed by adjusting the p H to p H 7.0 b N u m b e r of monkeys showing v o m i t i n g / n u m b e r of monkeys tested.
40 products contained approximately 120-180 ~g SEA before heating and < 2.0 /~g after heating, which is less than the EDs0 of 5 t~g (Bergdoll, 1970). The effect of high p H treatment upon recovery of heated SEA containing products was also tested by monkey feedings tests (Table IV). Due to the high pH treatment the immunological detectable quantity of SEA increased from 0.6 to 38.4 t~g for gelatin. For ovalbumin an increase from 0.4 to 43.2 was observed. For peas extract this was from 2.0 to 63.0 t~g. Using these products vomiting reactions were observed at each of the monkeys tested. Vomiting of monkeys obtaining the high-pH-treated peas extract occurred after 140 and 160 rain, respectively. With ovalbumin vomiting occurred after 95 and 100 rain respectively. For gelatin this was 202 and 255 min, respectively.
Discussion
Several authors have described the effect of heating on the stability of SE. Also kinetics of heat inactivation of SE in terms of D-values and z-values have been determined (Humber et al., 1975). However, it has become clear that heat inactivation of SE depends on several factors and that the effect of heat treatment is hardly predictable. The results presented in this paper (Table I) clearly demonstrate that in a number of food and food extracts a rapid loss of toxin activity occurs upon heating. On the other hand foods exist in which the toxin is relatively stable. The loss of toxin activity is indeed pH dependent as shown by Satterlee and Kraft (1969), Humber et al. (1975) and Tatini (1976). The effect of p H on heat inactivation is clearly demonstrated with gelatin as menstruum. At p H 4.0 the SE added lost its activity almost completely. At pH 5.5 or higher the loss of activity was much less. With extracts of peas and beans the loss of toxin upon heating was practically complete at pH 5.5 or lower. Initially we thought that the low recovery in certain extracts was caused by enclosing of SE in particles which occurred during heating. In order to test this hypothesis some food extracts were pre-heated and centrifuged. Following this treatment heat inactivation experiments were carried out with the supernatant obtained. However, no clear effects of this pre-heating treatment was observed (Table II). That enclosing of SE is not likely to be the mechanism of inactivation is underlined by the experiments in which gelatin was digested by collagenase. No SE was recovered upon collagenase digestion of heated SE containing gelatin (pH 4.0). Heating of SE in collagenase-digested gelatin at pH 4.0 resulted in a loss of activity identical to non-digested gelatin. Low recoveries of SEA by ELISA showed a parallel in loss of biological activity of SEA (Table IV). These results are in accordance to those observed by Tatini (1976) and Humber et al. (1975) for SEA and by Jamlang et al. (1971) for SEB. However, it was observed by Tatini (1976) that in the monkey feeding test heated toxin at a serologically measured activity of 5/~g appeared to be more toxic than the corresponding unheated toxin. An explanation for this might be that the partially denatured enterotoxin regained its native state in the intestinal tract of the monkey. It may be expected that in case of heating of SE containing food extracts the toxin
41
is coupled in a more or less reversible way to certain components of the extract. This theory is underlined by our findings that SE that lost its activity completely upon heating could be reactivated (Table III). It was found that a high pH treatment was very suited for reactivation of the toxic activity, Both a serological and biological reactivation was observed (Table IV). It might be expected that due to this high pH treatment the SE is dissociated from its accompanying molecule(s) and that it regains its complete activity. The above-mentioned theory is supported by the findings of Hugo et al. (1988). In their study it was demonstrated that staphylococcal c~-toxin became unreactive in the ELISA after heat aggregation with solubilized erythrocyte membranes. Following dissociation by boiling in sodium dodecyl sulphate the ELISA reactivity was fully restored. From our results it became clear that SE lost its activity upon heating in certain food and food extracts. However, the toxin is still present as a biologically complete molecule. Activity can be regained by e.g. high pH treatment. More research is needed to solve the exact mechanism of heat inactivation and the subsequent reactivation of SE. As long as the exact mechanism of heat inactivation and reactivation of SE is not solved heat treatment of SE-containing foods is not the way to improve food safety.
References Bergdoll, M.S. (1970) Enterotoxins. In: T.C. Montie, S. Kadis and S.J. Ajl (Eds.), Microbial Toxins, Vol. 3, Academic Press, New York, NY, pp. 265-326. Bergdoll, M.S., Kadavy, J., Surgalla, M. and Dack, G.M. (1951) Partial purification of staphylococcal enterotoxin. Arch. Biochem. 33, 259-262. Fung, D.Y.C., Steinberg, D.H., Miller, R.D., Kurantnick, M.J. and Murphy, T.F. (1973) Thermal inactivation of staphylococcal enterotoxins B and C. Appl. Microbiol. 26, 938-942. Horn, D., Hebel, R.D. and Kreuzer, W. (1986) Influence of frankfurter-type sausage manufacture and composition on the serological inactivation of staphylococcal enterotoxin B. Fleischwirtschaft 66, 584-591. Hugo, F., Sinner, A., Reichwein, J. and Bhakdi, S. (1988) Quantification of monomeric and oligomeric forms of membrane-bound staphylococcal a-toxin by ELISA using a neutralizing monoclonal antibody. Zentralbl. Bakteriol. Mikrobiol. Hyg. SER. A 268, 519. Humber, J.Y., Denny, C.B. and Bohner, W. (1975) Influence on pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assay. Appl. Microbiol. 30, 755-758. Jamlang, E.M., Bartlett, M.L. and Snyder, H.E. (1971) Effect of pH, protein concentration, and ionic strength on heat inactivation of staphylococcal enterotoxin B. Appl. Microbiol. 22, 1034-1040. Jordan, E.O., Dack, G.M. and Woolpert, O. (1931) The effect of heat, storage and chlorination on the toxicity of staphylococcus filtrates. J. Prevent. Med. 5, 383-386. Notermans, S., Bout, R., Tips, P.D. and de Nooy, M.P. (1983) Extraction of staphylococcal enterotoxins (SE) from minced meat and subsequent detection of SE with enzyme linked immunosorbent assay (ELISA). J. Food Protect. 46, 238-241. Notermans, S., Boot, R. and Tatini, S.R. (1987) Selection of monoclonal antibodies for detection of staphylococcal enterotoxins in heat processed foods. Int. J. Food Microbiol. 5, 49-55. Satterlee, L.D. and Kraft, A.A. (1969) Effect of meat and isolated meat proteins on the thermal inactivation of staphylococcal enterotoxin B. Appl. Microbiol. 17, 906-909.
42 Shinagawa, K.N., Kunita, N. and Sakaguchi, G. (1975) Simplified methods for purification of staphylococcal enterotoxins A and C and preparation of anti-enterotoxin sera. Jpn. J. Bacteriol. 30, 683-692. Tatini, S.R. (1976) Thermal stability of enterotoxins in food. J. Milk Food Technol. 39, 432-438. Tibana, A., Rayman, K,, Akhtar, M. and Szabo, R. (1987) Thermal stability of staphylococcal enterotoxin A, B and C in a buffered system. J. Food Protect. 50, 293-242.
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
<1
<1 <1
2.5 1.6 <1 16 20 25 25
1.3 1.6 50 50 79 80
2.4 <1 <1 <1 2.0 2.5
2.4 <1 1.0 1.3 8 10
<1
<1
<1 1.3 2.5
1.1 14
1.0
Beans extract
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 o C
<1 <1 3
<1 <1 11
Caulifl¢ wet extract
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 ° C
32 6 25
25 13 22
Milk
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 o C 10 m i n / 8 0 o C 10 m i n / 8 0 o C
50 4 16
100 10 25
Casein (2.5% w / v )
pH 5.0 pH 6.0
10 m i n / 8 0 ° C 10 rain/80 o C
20 25
Whey
pH 4.0 pH 6.0
10 m i n / 8 0 o C 10 m i n / 8 0 ° C
13 4
pH 4.0 pH 5.5 pH 7.0
10 m i n / 8 0 ° C 10 rain/80 ° C 10 rain/80 ° C
pH 4.0 pH 5.5 pH 7.0
10 rain/80 ° C 10 rain/80 ° C 10 m i n / 8 0 ° C
Bovine Serum Albumin (1% w / v )
Rice extract
11 6 29
16 <1 <1
16 8 18 4 16 40
85 20 41
37 TABLE I (continued). Medium
Treatment
Recovery of SE in percentages of added SEa SEA
SEB
SEC
Noodles extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 rain/80 o C 10 min/80 o C
25 3 13
Oat-flakes extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 °C 10 min/80 ° C 10 min/80 o C
2.5 20 50
Starch (1% w/v)
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 min/80 o C 10 min/80 ° C
20 20 20
37 45 73
Ovalbumin (1% w/v)
pH pH pH pH
10 min/80 ° C 10 min/80 ° C 10 min/80 ° C 10 rain/80 o C
4
85
3 5
23 39
4.0 5.0 5.5 7.0
81 46 53 25 38 100
<1
a 5 #g/ml of SEA and 1 #g/ml of SEB and SEC was added, respectively.
for SEA a n d 39% for SEB after heating for 10 m i n at 1 0 0 ° C a n d 8 0 ° C , respectively. I n gelatin with a p H of 4.0 the i n a c t i v a t i o n of a d d e d SE was n e a r l y c o m p l e t e a n d recoveries varied from < 1-2.5%. A t higher p H values of the g e l a t i n s o l u t i o n the heat i n a c t i v a t i o n was Tess p r o n o u n c e d a n d c o m p a r a b l e to PBS. W i t h lettuce extract, peas extracts a n d b e a n s extracts a n e a r l y c o m p l e t e i n a c t i v a t i o n of SE was observed at p H 4.0, 5.0 a n d 5.5. At p H 7.0 a very p r o n o u n c e d i n a c t i v a t i o n was observed with these extracts. W i t h cauliflower extract, milk, n o o d l e s extract a n d o v a l b u m i n it a p p e a r e d that the highest i n a c t i v a t i o n was observed at p H 5.5. W i t h casein, rice extract, oat-flakes extract a n d starch n o clear effect of the p H o n i n a c t i v a t i o n was observed a n d the recoveries of SE from b o t h h e a t - t r e a t e d p r o d u c t s varied from 4 - 1 0 0 % . F o r whey a n d b o v i n e serum a l b u m i n a p r o n o u n c e d inactivation was observed at all p H values. I n most cases SEC was m o r e heat stable t h a n SEB.
Recovery of SEB after heating in different pre-heated media D u r i n g h e a t i n g of some food a n d food p r o d u c t s it was observed that p r e c i p i t a tion occurred. T o test w h e t h e r SEB was enclosed in the precipitate or in a n e t w o r k of molecules resulting i n a decrease of the recovery of the t o x i n m e d i a were pre-heated a n d s u b s e q u e n t l y centrifuged. T o the s u p e r n a t a n t SEB was a d d e d a n d heat treated. F r o m the results p r e s e n t e d in T a b l e II it b e c a m e clear that n o such p r e c i p i t a t i o n had i n f l u e n c e d the results represented in T a b l e I. I n whey a n d cauliflower extract the recovery of SEB increased u p o n pre-heating.
38 TABLE II Recovery of SEB after heating (80 o C for 10 rain) in. pre-heated (95 ° C for 5 min) food and food extracts as determined by ELISA Medium
Recovery of SEB in percentages of added SEB (1/~g/ml) Without pre-heating
Gelatin (1% w/v) Whey Peas extract Beans extract Lettuce extract Cauliflower extract
pH pH pH pH pH pH
4.0 6.0 4.0 4.0 6.5 5.5
With pre-heating
2.5 4 < 3 < 3 <1 8
3 20 3 <3 3 16
Reactivation of heat-inactivated SE present in food and food extracts Collagenase treatment of heated SEB containing gelatin did not increase the r e c o v e r y o f SEB. I n a n e x p e r i m e n t i n w h i c h S E B w a s a d d e d t o c o l l a g e n a s e - d i g e s t e d gelatin heat included).
inactivation of SEB
was
equal
to not
digested gelatin (data
not
TABLE III Reactivation effect of high pH of heat inactivated SE present in food and food extracts as determined by EL1SA Medium
Treatment
Recovery in percentages of added SEa After heating After high pH treatment b SEA
SEB
SEA
SEB
Gelatin (5% w/v) (1% w/v)
pH 4.5 pH 4.0
10 rain/100 ° C 10 rain/80 ° C
<1
Ovalbumin (1% w/v)
pH 5.0 pH 5.5
10 min/80 ° C 10 rain/80 ° C
<1
pH 5.5 pH 5.5
10 rnin/100 ° C 10 rain/80 ° C
<1 <1
pH 4.0 pH 5.0 pH 7.0
10 min/80 ° C 10 min/80 ° C 10 min/100 o C
<1 1
Beans extract
pH 4.0 pH 5.5 pH 7.0
10 min/80 ° C 10 min/80 ° C 10 rain/80 ° C
<1 2 4
32 16 13
Cauliflower extract
pH 5.5
10 min/80 ° C
8
40
Whey
pH 6.0
10 min/80 o C
4
8
Lettuce extract Peas extract
28 2.5
50 21
3 2
79 <1 2
<1
8 40
35 2
a 5 /.tg/ml of SEA and 1 p.g/ml of SEB was added. b pH treatment involved adjusting of pH to pH 11.0 using 1 N NaOH followed by adjusting of pH to pH 7.0 using 1 N HC1.
39
Other reactivation experiments carried out made clear that high p H treatment was effective in reactivation of the heated staphylococcal enterotoxins. It has to be noticed that the reactivation occurred abruptly after the p H was adjusted to 10-11. Recoveries of SE after high pH treatment are summarized in Table III. With gelatin, ovalbumin, peas extract, beans extract and cauliflower extract the reactivation effect was pronounced and recoveries up to 79% of the original added SE were observed. With lettuce extract and whey, however, no clear reactivation effect was observed upon high pH treatment.
Comparison of ELISA and monkey feeding test From the foregoing it becomes clear that after heating of certain food and food extracts amounts of SE demonstrated were quite low. Subsequent high p H treatment showed that for some products the immunological activity was largely regained. In order to test whether the heated food and food extracts were still biologically active monkey feeding tests were carried out (Table IV). The sensitivity of the monkeys was tested by administering SEA diluted in BHI to each of nine monkeys. Eight of them vomited 120-180 rain after administration of 20/~g of SEA. No emetic reactions were observed in monkeys fed with heated gelatin (5% w / v , p H 4.5), heated ovalbumin (1% w / v , p H 5.0) or heated peas extract (pH 5.0). All these
T A B L E IV Immunological and biological activity of SEA after heat treatment and after reactivation by high p H a in different media Total quantity of SEA tested (#g/monkey) 20 120
180
180
Medium
Treatment
Total immunological activity (# g / m o n k e y ) as determined with ELISA
Monkey feeding test b
Brain heart infusion broth
not treated
20
8/9
Gelatin (5% w / v p H 4.5)
(a) 100 o C for 10 rain
0.6
0/5
(b) 100 ° C for 10 rain followed by high p H treatment
38.4
2/2
(a) 80 o C for 10 min
0.4
0/2
(b) 80 ° C for 10 min followed by high p H treatment
43.2
2/2
(a) 80 ° C for 10 rain (b) 80 ° C for 10 min followed by high p H treatment
2.0 63.0
0/2 2/2
Ovalbumin (1% w / v p H 5.0)
Peas extract (pH 5.0)
a Treatment consisted of adjusting the p H to p H 11 followed by adjusting the p H to p H 7.0 b N u m b e r of monkeys showing v o m i t i n g / n u m b e r of monkeys tested.
40 products contained approximately 120-180 ~g SEA before heating and < 2.0 /~g after heating, which is less than the EDs0 of 5 t~g (Bergdoll, 1970). The effect of high p H treatment upon recovery of heated SEA containing products was also tested by monkey feedings tests (Table IV). Due to the high pH treatment the immunological detectable quantity of SEA increased from 0.6 to 38.4 t~g for gelatin. For ovalbumin an increase from 0.4 to 43.2 was observed. For peas extract this was from 2.0 to 63.0 t~g. Using these products vomiting reactions were observed at each of the monkeys tested. Vomiting of monkeys obtaining the high-pH-treated peas extract occurred after 140 and 160 rain, respectively. With ovalbumin vomiting occurred after 95 and 100 rain respectively. For gelatin this was 202 and 255 min, respectively.
Discussion
Several authors have described the effect of heating on the stability of SE. Also kinetics of heat inactivation of SE in terms of D-values and z-values have been determined (Humber et al., 1975). However, it has become clear that heat inactivation of SE depends on several factors and that the effect of heat treatment is hardly predictable. The results presented in this paper (Table I) clearly demonstrate that in a number of food and food extracts a rapid loss of toxin activity occurs upon heating. On the other hand foods exist in which the toxin is relatively stable. The loss of toxin activity is indeed pH dependent as shown by Satterlee and Kraft (1969), Humber et al. (1975) and Tatini (1976). The effect of p H on heat inactivation is clearly demonstrated with gelatin as menstruum. At p H 4.0 the SE added lost its activity almost completely. At pH 5.5 or higher the loss of activity was much less. With extracts of peas and beans the loss of toxin upon heating was practically complete at pH 5.5 or lower. Initially we thought that the low recovery in certain extracts was caused by enclosing of SE in particles which occurred during heating. In order to test this hypothesis some food extracts were pre-heated and centrifuged. Following this treatment heat inactivation experiments were carried out with the supernatant obtained. However, no clear effects of this pre-heating treatment was observed (Table II). That enclosing of SE is not likely to be the mechanism of inactivation is underlined by the experiments in which gelatin was digested by collagenase. No SE was recovered upon collagenase digestion of heated SE containing gelatin (pH 4.0). Heating of SE in collagenase-digested gelatin at pH 4.0 resulted in a loss of activity identical to non-digested gelatin. Low recoveries of SEA by ELISA showed a parallel in loss of biological activity of SEA (Table IV). These results are in accordance to those observed by Tatini (1976) and Humber et al. (1975) for SEA and by Jamlang et al. (1971) for SEB. However, it was observed by Tatini (1976) that in the monkey feeding test heated toxin at a serologically measured activity of 5/~g appeared to be more toxic than the corresponding unheated toxin. An explanation for this might be that the partially denatured enterotoxin regained its native state in the intestinal tract of the monkey. It may be expected that in case of heating of SE containing food extracts the toxin
41
is coupled in a more or less reversible way to certain components of the extract. This theory is underlined by our findings that SE that lost its activity completely upon heating could be reactivated (Table III). It was found that a high pH treatment was very suited for reactivation of the toxic activity, Both a serological and biological reactivation was observed (Table IV). It might be expected that due to this high pH treatment the SE is dissociated from its accompanying molecule(s) and that it regains its complete activity. The above-mentioned theory is supported by the findings of Hugo et al. (1988). In their study it was demonstrated that staphylococcal c~-toxin became unreactive in the ELISA after heat aggregation with solubilized erythrocyte membranes. Following dissociation by boiling in sodium dodecyl sulphate the ELISA reactivity was fully restored. From our results it became clear that SE lost its activity upon heating in certain food and food extracts. However, the toxin is still present as a biologically complete molecule. Activity can be regained by e.g. high pH treatment. More research is needed to solve the exact mechanism of heat inactivation and the subsequent reactivation of SE. As long as the exact mechanism of heat inactivation and reactivation of SE is not solved heat treatment of SE-containing foods is not the way to improve food safety.
References Bergdoll, M.S. (1970) Enterotoxins. In: T.C. Montie, S. Kadis and S.J. Ajl (Eds.), Microbial Toxins, Vol. 3, Academic Press, New York, NY, pp. 265-326. Bergdoll, M.S., Kadavy, J., Surgalla, M. and Dack, G.M. (1951) Partial purification of staphylococcal enterotoxin. Arch. Biochem. 33, 259-262. Fung, D.Y.C., Steinberg, D.H., Miller, R.D., Kurantnick, M.J. and Murphy, T.F. (1973) Thermal inactivation of staphylococcal enterotoxins B and C. Appl. Microbiol. 26, 938-942. Horn, D., Hebel, R.D. and Kreuzer, W. (1986) Influence of frankfurter-type sausage manufacture and composition on the serological inactivation of staphylococcal enterotoxin B. Fleischwirtschaft 66, 584-591. Hugo, F., Sinner, A., Reichwein, J. and Bhakdi, S. (1988) Quantification of monomeric and oligomeric forms of membrane-bound staphylococcal a-toxin by ELISA using a neutralizing monoclonal antibody. Zentralbl. Bakteriol. Mikrobiol. Hyg. SER. A 268, 519. Humber, J.Y., Denny, C.B. and Bohner, W. (1975) Influence on pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assay. Appl. Microbiol. 30, 755-758. Jamlang, E.M., Bartlett, M.L. and Snyder, H.E. (1971) Effect of pH, protein concentration, and ionic strength on heat inactivation of staphylococcal enterotoxin B. Appl. Microbiol. 22, 1034-1040. Jordan, E.O., Dack, G.M. and Woolpert, O. (1931) The effect of heat, storage and chlorination on the toxicity of staphylococcus filtrates. J. Prevent. Med. 5, 383-386. Notermans, S., Bout, R., Tips, P.D. and de Nooy, M.P. (1983) Extraction of staphylococcal enterotoxins (SE) from minced meat and subsequent detection of SE with enzyme linked immunosorbent assay (ELISA). J. Food Protect. 46, 238-241. Notermans, S., Boot, R. and Tatini, S.R. (1987) Selection of monoclonal antibodies for detection of staphylococcal enterotoxins in heat processed foods. Int. J. Food Microbiol. 5, 49-55. Satterlee, L.D. and Kraft, A.A. (1969) Effect of meat and isolated meat proteins on the thermal inactivation of staphylococcal enterotoxin B. Appl. Microbiol. 17, 906-909.
42 Shinagawa, K.N., Kunita, N. and Sakaguchi, G. (1975) Simplified methods for purification of staphylococcal enterotoxins A and C and preparation of anti-enterotoxin sera. Jpn. J. Bacteriol. 30, 683-692. Tatini, S.R. (1976) Thermal stability of enterotoxins in food. J. Milk Food Technol. 39, 432-438. Tibana, A., Rayman, K,, Akhtar, M. and Szabo, R. (1987) Thermal stability of staphylococcal enterotoxin A, B and C in a buffered system. J. Food Protect. 50, 293-242.