International Journal of Food Microbiology, 11 (1990) 85-92 Elsevier
85
FOOD 80016
The effects of irradiation and temperature on the immunological activity of staphylococcal enterotoxin A N . K . M o d i 1, Sally A. R o s e 2 and H.S. Tranter 1 I PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, U.K., and 2 Microbiology Department, Campden Food Preservation Research Association, Chipping Campden, Gloucestershire, U.K.
The effects of irradiation and temperature on purified staphylococcal enterotoxin A were investigated using sensitive ELISA systems. Thermal inactivation of staphylococcal enterotoxin A in phosphatebuffered saline was considerably faster at temperatures of 60, 70, 115 and 121°C than at 90 and 100°C. In gelatine phosphate buffer, staphylococcal enterotoxin A was completely inactivated by irradiation at 8.0 kGy; in a 15% mince slurry, however, 27-37% of staphylococcal enterotoxin A remained at this level of irradiation. Even at a dose of 23.7 kGy, 16-26% residual staphylococcal enterotoxin A could still be detected. Generally, increasing the mince concentration increased the protection against the effect of irradiation on staphylococcal enterotoxin A. However, the protective effect of mince at a concentration of 50% was less than at a mince concentration of 30%. Both irradiation and heat processing of food should only be used in conjunction with good manufacturing practices to prevent proliferation of microorganisms and toxin productions. Key words: Temperature; Irradiation; Immunological activity; Staphylococcal enterotoxin A
Introduction I r r a d i a t i o n is a well established t e c h n o l o g y in the b i o m e d i c a l field, p a r t i c u l a r l y for the sterilisation o f p r e - p a c k e d m e d i c a l e q u i p m e n t . H o w e v e r , only a small p r o p o r t i o n (approx. 5%) of the c u r r e n t w o r l d w i d e usage is d e v o t e d to i r r a d i a t i o n o f f o o d for the c o n t r o l of m i c r o o r g a n i s m s , parasites an d insects or the i n h i b i t i o n of s p r o u t i n g in stored r o o t c r o p s (Ley, 1987). T h e i r r a d i a t i o n of f o o d is c u r r e n t l y b ei n g d e b a t e d in the U K following the r e p o r t ( A n o n y m o u s , 1986) of the A d v i s o r y C o m m i t t e e for the I r r a d i a t i o n o f N o v e l F o o d s ( A C I N F ) that such a t r e a t m e n t , if closely m o n i t o r e d , presents no special toxicological, n u t r i t i o n a l or m i c r o b i o l o g i c a l p r o b l e m s . O n e focus of a t t e n t i o n has b e e n the effects o f i r r a d i a t i o n on m i c r o b i a l toxins, such as s t a p h y l o c o c c a l e n te r o t o x in s , w h i ch m a y be p r esen t in the f o o d as a
Correspondence address: N.K. Modi, PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wilts SP4 0JG, U.K.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
86 result of poor hygienic conditions, and which can cause food poisoning (Gilbert, 1974) if ingested. Although there is little available information regarding the effects of irradiation on the staphylococcal enterotoxins, the ability of these molecules to withstand heat has been well documented. Early studies involving human volunteers (Jordan et al., 1931), monkeys (Davison et al., 1938), cats (Dolman et al., 1936) or other animals clearly demonstrated that the biological activity of the enterotoxins is extremely heat resistant. Since then, several other workers (Denny et al., 1971; Humber et al., 1975) have demonstrated the thermal stability using immunological techniques. However, accurate, quantitative studies on the thermal stability of the enterotoxins have only been possible with the advent of reliable and sensitive immunological assay systems such as radioimmunoassays (RIA) and enzyme-linked immunoassays (ELISA), together with the availability of purified toxin preparations. In this study we describe the effect of irradiation and temperature on the immunological activity of purified staphylococcal enterotoxin A (SEA) using sensitive ELISA techniques for the detection of the toxin.
Materials and Methods
Toxin Purified staphylococcal enterotoxin type A (SEA) was kindly provided by Prof. M.S. Bergdoll (Food Research Institute, University of Wisconsin, Madison, WI, U.S.A.) and Mr. D. Reynolds (Vaccine Research and Production Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.). Buffers Phosphate-buffered saline (PBS), pH 7.4, contained 140 mM NaC1, 2.7 mM KC1, 8.1 mM N a 2 H P O 4 and 1.5 mM KH2PO 4. Gelatine phosphate buffer (GPB) adjusted to p H 6.5 with phosphoric acid contained 0.2% gelatine and 70 mM N a 2 H P O 4. Heat treatment Ten heating tubes (Pierce & Warner), containing 5 ml PBS and 2.5% ( v / v ) normal rabbit serum per tube, were equilibrated to a required temperature (60, 70, 90, 100, 110 or 121 ° C) in an oil bath. 100 #1 of SEA solution was injected into each tube through the septum lid to give a final concentration of 100 n g / m l . The contents were mixed and reincubated at the same temperatures. At various intervals of up to 60 rain duphcate tubes were removed from the bath and immediately frozen in a CO2/alcohol freezing mixture. To a separate tube containing 5 ml PBS and 2.5% ( v / v ) normal rabbit serum, SEA was added to a final concentration of 100 n g / m l and immediately frozen to serve as an unheated control sample. Both the test and control samples were stored at - 1 8 ° C until assayed by ELISA.
87
Preparation of mince slurries Mince slurries (% w / v ) were prepared by adding appropriate amounts of lean mince beef to GPB in an electric blender (Kenwood) and homogenising at maximum speed for 5-10 min. The homogenate was autoclaved at 121°C for 15 min and 18-ml aliquots were dispensed aseptically into glass universals. Neat (100%) mince samples were prepared by weighing 18 g of the mince beef into glass bottles and autoclaving. The resulting mince aggregate was broken up using a sterile glass rod. Purified SEA was added to the mince samples to give a final concentration of 110 n g / m l . The contents of the glass bottles were gently mixed for 10 rain using a bottle roller and stored at 4 ° C until irradiation which was performed within 8 h of the toxin addition.
Irradiation of samples Samples were irradiated using 6°Co sources with dose rates of 9.4 or 12.2 k G y / h . Glass bottles containing perspex dosimeters (AERE, Harwell) were placed with the test samples to determine the actual dose received by the samples. As the temperature during irradiation was 20-25 ° C, the unirradiated control samples were kept at ambient temperature during the period of the irradiation.
Toxin extraction from mince slurries The slurry samples were centrifuged at 3000 x g for 30 min at 4 ° C and the supernatant fluid was collected. The residue was resuspended using 9 ml GPB, mixed gently for 10 rain on a bottle roller and recentrifuged. The supernatant fluids were pooled and centrifuged at 20 000 x g for 30 min at 4 ° C. The toxin concentration of the supernatant was determined using two different immunoassays.
Toxin assays SEA was determined using two different types of ELISAs.
Method 1: Wells of a microtitre plate (Dynatech) were coated (100 btl/well) using purified guinea pig IgG anti-SEA (20 ~ g / m l ) diluted in PBS, overnight at 4 ° C. The wells were emptied and washed 3 x with PBS containing 0.05% ( v / v ) Tween 20. Excess binding sites were blocked by adding ( 1 0 0 / d / w e l l ) consisting of RPMI 1640 medium (Imperial Laboratories) containing 10% (v/v) foetal calf serum and 1% ( w / v ) bovine serum albumin. The plates were shaken at room temperature for 30 min and incubated at 37 ° C for 90 min. The wells were washed as before and the test samples or standard toxin solutions diluted in GPB added at 50 #l/well. The plate was shaken at room temperature for 2 h, washed, and rabbit anti-SEA conjugated to horse radish peroxidase, diluted 1 : 200 in the blocking reagent, added at 100 #l/well. The plates were washed 4 x and substrate (TMB; 3 , 3 ' - 5 , 5 ' tetramethylbenzidine) was added (100 ffl/well). The TMB solution was prepared by dissolving 10 mg of TMB in 1 ml dimethylsulphoxide, adding this dropwise to 100 ml of 0.1 M sodium acetate/citrate buffer pH 6.0 and adding 80 /~1 of 6% ( w / v ) H202. The microtitre plate containing the substrate was shaken for 10-15 min at room temperature and the reaction was stopped by adding 2 M H2SO 4 (50 ffl/well). The absorbance was read at 450 nm using an M R 580 Plate reader (Dynatech) and
88 the toxin concentration in the test samples was estimated from calibration curve of purified SEA (0.1-1000 n g / m l ) assayed on the same plate. M e t h o d 2: The SET-EIA kit is a commercially available immunoassay system (Labor Dr. W. Bommeli, Langass-Strasse 7, CH-3012 Bern, Switzerland) for detection of Staphylococcal enterotoxins A - D and is based on a single sandwich type ELISA using polyclonal anti-enterotoxin immobilised on polystyrene beads and an alkaline phosphatase conjugated antibody as the label. The jest samples, diluted in GPB, were assayed for SEA using the SET-EIA kit according to manufacturer's instructions. The substrate (p-nitrophenyl phosphate) reaction was stopped using 2 M N a O H and the extent of the reaction was measured using a Dynatech MR 600 plate reader. The concentration of SEA in the test samples was estimated using the calibration curve obtained by using purified SEA (0.1-1.6 n g / m l GPB).
Results Heat treatment
Fig. 1 shows data on the thermal inactivation of purified SEA heated at a range of temperatures from 6 0 - 1 2 1 ° C . Thermal destruction of SEA was considerably
I 70
6(}
5C ._= E 40
2C
10
0_1 60
i
70
8~}
9~) Temperature
110
1110
120l
L~o
(°C)
Fig. 1. Thermal inactivation of staphylococcal enterotoxin A in PBS buffer containing 2.5% normal rabbit serum as measured by ELISA2.
89 TABLE I E f f e c t o f i r r a d i a t i o n o n the d e t e c t i o n o f s t a p h y l o c o c c a l e n t e r o t o x i n A b y E L I S A in b u f f e r a n d m i n c e d beef Menstruum
Assay
Dose (kGy) 0
1.9
Gelatin phosphate buffer 15% M i n c e slurry
ELISA 1 a ELISA 2 "
53.2 82.2
14.0 4.5
2.7 0.2
2.2 -
ELISA 1 " ELISA 2 a
41.9 42.7
29.5 32.4
24.5 31.4
30.4 31.5
5.2
7.5
8.0 33.6 27.1
23.7 26.5 16.0
a M e a n o f t r i p l i c a t e results. F i g u r e s in the t a b l e r e f e r to the a m o u n t s o f t o x i n d e t e c t e d a f t e r i r r a d i a t i o n e x p r e s s e d as a p e r c e n t a g e o f the initial t o x i n a d d e d .
faster at 60 °, 70 °, 115 ° and 121°C (D values of 16.6, 6.9, 16.3 and 8.3, respectively) than at 90 o and 100 ° C (D values of 73.7 and 43.4, respectively). Irradiation treatment
The effect of irradiation dosage on the immunological detection of SEA is shown in Table I. Results from the two ELISA methods show that little immunological activity remained in GPB even after the lowest irradiation dose of 1.9 kGy. SEA was barely detectable in GPB using ELISA method 1 after irradiation at 7.5 k G y and could not be detected by either method after a dose of 8.0 kGy. Conversely, 1-25
i,-oof 0.75
~.~ .E .-=
0.5£
g 0.2
25
Mince
50
75
I 100
Concentration ( ° / o w / v )
Fig. 2. Effect o f m i n c e c o n c e n t r a t i o n o n the a m o u n t o f s t a p h y l o c o c c a l e n t e r o t o x i n A r e m a i n i n g a f t e r i r r a d i a t i o n at 25 k G y b y E L I S A 1 (©) a n d E L I S A 2 (O). V a l u e s in t h e figure r e f e r to the m e a n o f t r i p l i c a t e results.
90 27-34% of the toxins' initial immunological activity remained in a 15% minced beef-GPB slurry at an equivalent irradiation dose of 8.0 kGy. Even at a dose as high as 23.7 kGy, 16 27% of the initial level of SEA could still be detected in the mince. Results showing the protective effect of the mince slurry on the immunological activity of SEA following irradiation at an average dose of 23.4 k G y are given in Fig. 2. As the recovery of toxin depended on the mice concentration used, the results were expressed as the ratio of toxin recovered from irradiated to that from unirradiated samples. Generally, higher mince concentrations conferred increased protection against the effects of irradiation (Fig. 2). Regardless of the type of ELISA used to assay residual SEA, the protective effect of mince at a concentration of 50% was less than that of mince at a concentration of 30%.
Discussion The staphylococcal enterotoxins are generally appreciated to be resistant to thermal inactivation (Tatini, 1976). Numerous studies have documented their stability to heat and its dependence upon p H ( H u m b e r et al., 1975), toxin concentration and the nature of the heating menstruum (Bennett et al., 1977; Lee et al., 1977). Most of the results reported are based on the toxins' loss of reaction with antibodies using relatively insensitive techniques such as single gel immunodiffusion (Oudin, 1952) which require large amounts of toxin for quantitative estimation. Care should be taken in interpreting m a n y of these early studies as m a n y workers used crude or partially pure forms of the toxin which are noticeably more thermostable than the purified forms (Tatini, 1976). In addition, the antibody preparations used may not have been raised against highly purified toxin. Using highly sensitive and specific ELISA techniques we can confirm that the staphylococcal enterotoxins are remarkably heat stable. In this study, longer heating times were required to inactivate purified SEA at 90 and 100 ° C than at lower and higher temperatures (Fig. 1). A similar observation was made by Jamlang et al. (1971) who reported that SEB lost its immunological activity more rapidly at 70-80 ° C than at 90-100 ° C when heated in acetate or phosphate buffer. Similarly, Soo et al. (1974) found that SEA showed a more rapid loss of immunological activity when heated at 7 0 ° C than at 8 0 - 9 0 ° C . Such variation in the loss of immunological activity observed during heating of the enterotoxins also appears when the toxins are inactivated by irradiation. Fig. 2 shows that the residual amount of SEA detected by ELISA was less at a mince concentration of 50% than at a concentration of 30%. It has been suggested (Jamlang et al., 1971) that the rapid denaturation of SEB at 70-80 ° C was the result of enterotoxin aggregation which might be reversible at higher temperatures. Such aggregates occurring between toxin molecules may mask some of the toxins' antigenic sites and so protect against denaturation. This phenomenon could explain the shape of the heat inactivation curve. A more complicated situation probably exists when toxin is irradiated in a complex menstruum such as meat. Here toxin/toxin and t o x i n / m e a t interactions
91 m a y occur to varying degrees, thus making the biphasic nature of Fig. 2 more difficult to explain. In the only report on the effect of irradiation on the staphylococcal enterotoxins, Read and Bradshaw (1967) found that a dose of 5 Mrad (50 kGy) was required to reduce the concentration of SEB in 0.04 M Veronal buffer, p H 7.2, from 31 ~ g / m l to less than 0 . 7 / t g / m l . In milk, the same workers showed that a dose of 20 Mrad (200 kGy) was needed to achieve an equivalent reduction of SEB. In this study, a far smaller dose (8 kGy) was required to inactivate purified SEA in gelatin phosphate buffer, p H 6.8 (Table I), however, some SEA could still be detected in a 3% mince slurry even at the highest dose of irradiation (23 kGy) used. Although factors such as buffer composition and toxin concentration and purity may play some role in determining the heat sensitivity of the toxin to irradiation, the results appear to suggest that SEA is less resistant to irradiation than SEB. A similar conclusion has also been made after comparing the heat inactivation of SEA to SEB in Veronal buffer (Tatini, 1976). In the study of irradiation of SEB (Read and Bradshaw, 1967), the loss of immunlogical activity paralleled the loss of biological activity as determined by emesis in cats. Some workers have noticed a similar loss in both immunological and biological activities when studying heat inactivation of the staphylococcal enterotoxins (Jamlang et al., 1971; H u m b e r et al., 1975); others report a difference in the inactivation rates of immunological activity and toxicity with a suggestion of increased toxicity following heat treatment (data of Dangerfield, 1973, cited in Tatini, 1976; Tatini, 1976). Although we did not examine the effects of irradiation on the biological activity of SEA, similar studies (Tranter et al., 1987) on the irradiation of Clostridium botulinurn type A neurotoxin revealed a more rapid loss of toxicity compared to immunological activity. In view of the enhanced toxicities reported by some workers in SEA following heating, the effects of irradiation on the biological activity of this toxin should also be closely examined. The results of this study suggest an analogy between the stability of the staphylococcal enterotoxins in foods to irradiation and heat processing. It therefore follows that neither can be relied on solely to eliminate the hazards that may arise from mishandling food prior to processing. The food industry's good record with heat processing has been achieved by the application of good manufacturing practices and the same approach must also be used if irradiation is to be used for food processing.
Acknowledgements The authors acknowledge the financial assistance provided by Porton Products Ltd. to N.K.M. and H.S.T. and thank Mr. F.J. Ley of Isotron Ltd. for the use of irradiation facilities.
92
References Anonymous (1986) Report on the safety and wholesomeness of irradiated foods. Advisory Committee on Irradiated and Novel Foods, DHSS. Bennett, R.W., Bradshaw, J.G. and Amos, W.T. (1977) Biological and serological activities of heated staphylococcal enterotoxin A. Abstr. Ann. Meet. Am. Soc. Microbiol. p. 257. Davison, E., Dack, G.M. and Cary, W.E. (1938) Attempts to assay the enterotoxic substance produced by staphylococci by parenteral injection of monkeys and kittens. J. Infect. Dis. 62, 219-223. Denny, C.B., Humber, J.Y. and Bohrer, C.W. (1971) Effect of toxin concentration on the heat inactivation of staphylococcal enterotoxin A in beef bouillon and in phosphate buffer. Appl. Microbiol. 21, 1064-1066. Dolman, C.E., Wilson, R.J. and Cockcroft, W.H. (1936) A new method of detecting staphylococcus enterotoxin. Can. Publ. Health J. 27, 489-493. Gilbert, R.J. (1974) Staphylococcal food poisoning and botulism. Postgrad. Med. J. 50, 603-611. Humber, J.Y., Denny, C.B. and Bohrer, C.W. (1975) Influence of pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assays. 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. Prev. Med. 5, 383-386. Lee, I.C., Stevenson, K.E. and Harmon, L.G. (1977) Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B. Appl. Environ. Microbioi. 33, 341-344. Ley, F.J. (1987) Applying radiation technology to food. In: Food Technology International Europe (A. Turner, Ed.), pp. 72-75. Sterling Publishers Ltd., London. Oudin, J. (1952) Specific precipitation in gels. Meth. Med. Res. 5, 335-378. Read, R.B. and Bradshaw, J.G. (1967) Gamma radiation of staphylococcal enterotoxin B. Appl. Microbiol. 15,603-605. Tatini, S.R. (1976) Thermal stability of enterotoxins in food. J. Milk Food Technol. 39, 432-438. Tranter, H.S., Modi, N.K., Hambleton, P., Melling, J., Rose, S. and Stringer, M.F., (1987) Food irradiation and bacterial toxins. Lancet ii, 48.
85
FOOD 80016
The effects of irradiation and temperature on the immunological activity of staphylococcal enterotoxin A N . K . M o d i 1, Sally A. R o s e 2 and H.S. Tranter 1 I PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, U.K., and 2 Microbiology Department, Campden Food Preservation Research Association, Chipping Campden, Gloucestershire, U.K.
The effects of irradiation and temperature on purified staphylococcal enterotoxin A were investigated using sensitive ELISA systems. Thermal inactivation of staphylococcal enterotoxin A in phosphatebuffered saline was considerably faster at temperatures of 60, 70, 115 and 121°C than at 90 and 100°C. In gelatine phosphate buffer, staphylococcal enterotoxin A was completely inactivated by irradiation at 8.0 kGy; in a 15% mince slurry, however, 27-37% of staphylococcal enterotoxin A remained at this level of irradiation. Even at a dose of 23.7 kGy, 16-26% residual staphylococcal enterotoxin A could still be detected. Generally, increasing the mince concentration increased the protection against the effect of irradiation on staphylococcal enterotoxin A. However, the protective effect of mince at a concentration of 50% was less than at a mince concentration of 30%. Both irradiation and heat processing of food should only be used in conjunction with good manufacturing practices to prevent proliferation of microorganisms and toxin productions. Key words: Temperature; Irradiation; Immunological activity; Staphylococcal enterotoxin A
Introduction I r r a d i a t i o n is a well established t e c h n o l o g y in the b i o m e d i c a l field, p a r t i c u l a r l y for the sterilisation o f p r e - p a c k e d m e d i c a l e q u i p m e n t . H o w e v e r , only a small p r o p o r t i o n (approx. 5%) of the c u r r e n t w o r l d w i d e usage is d e v o t e d to i r r a d i a t i o n o f f o o d for the c o n t r o l of m i c r o o r g a n i s m s , parasites an d insects or the i n h i b i t i o n of s p r o u t i n g in stored r o o t c r o p s (Ley, 1987). T h e i r r a d i a t i o n of f o o d is c u r r e n t l y b ei n g d e b a t e d in the U K following the r e p o r t ( A n o n y m o u s , 1986) of the A d v i s o r y C o m m i t t e e for the I r r a d i a t i o n o f N o v e l F o o d s ( A C I N F ) that such a t r e a t m e n t , if closely m o n i t o r e d , presents no special toxicological, n u t r i t i o n a l or m i c r o b i o l o g i c a l p r o b l e m s . O n e focus of a t t e n t i o n has b e e n the effects o f i r r a d i a t i o n on m i c r o b i a l toxins, such as s t a p h y l o c o c c a l e n te r o t o x in s , w h i ch m a y be p r esen t in the f o o d as a
Correspondence address: N.K. Modi, PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wilts SP4 0JG, U.K.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
86 result of poor hygienic conditions, and which can cause food poisoning (Gilbert, 1974) if ingested. Although there is little available information regarding the effects of irradiation on the staphylococcal enterotoxins, the ability of these molecules to withstand heat has been well documented. Early studies involving human volunteers (Jordan et al., 1931), monkeys (Davison et al., 1938), cats (Dolman et al., 1936) or other animals clearly demonstrated that the biological activity of the enterotoxins is extremely heat resistant. Since then, several other workers (Denny et al., 1971; Humber et al., 1975) have demonstrated the thermal stability using immunological techniques. However, accurate, quantitative studies on the thermal stability of the enterotoxins have only been possible with the advent of reliable and sensitive immunological assay systems such as radioimmunoassays (RIA) and enzyme-linked immunoassays (ELISA), together with the availability of purified toxin preparations. In this study we describe the effect of irradiation and temperature on the immunological activity of purified staphylococcal enterotoxin A (SEA) using sensitive ELISA techniques for the detection of the toxin.
Materials and Methods
Toxin Purified staphylococcal enterotoxin type A (SEA) was kindly provided by Prof. M.S. Bergdoll (Food Research Institute, University of Wisconsin, Madison, WI, U.S.A.) and Mr. D. Reynolds (Vaccine Research and Production Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.). Buffers Phosphate-buffered saline (PBS), pH 7.4, contained 140 mM NaC1, 2.7 mM KC1, 8.1 mM N a 2 H P O 4 and 1.5 mM KH2PO 4. Gelatine phosphate buffer (GPB) adjusted to p H 6.5 with phosphoric acid contained 0.2% gelatine and 70 mM N a 2 H P O 4. Heat treatment Ten heating tubes (Pierce & Warner), containing 5 ml PBS and 2.5% ( v / v ) normal rabbit serum per tube, were equilibrated to a required temperature (60, 70, 90, 100, 110 or 121 ° C) in an oil bath. 100 #1 of SEA solution was injected into each tube through the septum lid to give a final concentration of 100 n g / m l . The contents were mixed and reincubated at the same temperatures. At various intervals of up to 60 rain duphcate tubes were removed from the bath and immediately frozen in a CO2/alcohol freezing mixture. To a separate tube containing 5 ml PBS and 2.5% ( v / v ) normal rabbit serum, SEA was added to a final concentration of 100 n g / m l and immediately frozen to serve as an unheated control sample. Both the test and control samples were stored at - 1 8 ° C until assayed by ELISA.
87
Preparation of mince slurries Mince slurries (% w / v ) were prepared by adding appropriate amounts of lean mince beef to GPB in an electric blender (Kenwood) and homogenising at maximum speed for 5-10 min. The homogenate was autoclaved at 121°C for 15 min and 18-ml aliquots were dispensed aseptically into glass universals. Neat (100%) mince samples were prepared by weighing 18 g of the mince beef into glass bottles and autoclaving. The resulting mince aggregate was broken up using a sterile glass rod. Purified SEA was added to the mince samples to give a final concentration of 110 n g / m l . The contents of the glass bottles were gently mixed for 10 rain using a bottle roller and stored at 4 ° C until irradiation which was performed within 8 h of the toxin addition.
Irradiation of samples Samples were irradiated using 6°Co sources with dose rates of 9.4 or 12.2 k G y / h . Glass bottles containing perspex dosimeters (AERE, Harwell) were placed with the test samples to determine the actual dose received by the samples. As the temperature during irradiation was 20-25 ° C, the unirradiated control samples were kept at ambient temperature during the period of the irradiation.
Toxin extraction from mince slurries The slurry samples were centrifuged at 3000 x g for 30 min at 4 ° C and the supernatant fluid was collected. The residue was resuspended using 9 ml GPB, mixed gently for 10 rain on a bottle roller and recentrifuged. The supernatant fluids were pooled and centrifuged at 20 000 x g for 30 min at 4 ° C. The toxin concentration of the supernatant was determined using two different immunoassays.
Toxin assays SEA was determined using two different types of ELISAs.
Method 1: Wells of a microtitre plate (Dynatech) were coated (100 btl/well) using purified guinea pig IgG anti-SEA (20 ~ g / m l ) diluted in PBS, overnight at 4 ° C. The wells were emptied and washed 3 x with PBS containing 0.05% ( v / v ) Tween 20. Excess binding sites were blocked by adding ( 1 0 0 / d / w e l l ) consisting of RPMI 1640 medium (Imperial Laboratories) containing 10% (v/v) foetal calf serum and 1% ( w / v ) bovine serum albumin. The plates were shaken at room temperature for 30 min and incubated at 37 ° C for 90 min. The wells were washed as before and the test samples or standard toxin solutions diluted in GPB added at 50 #l/well. The plate was shaken at room temperature for 2 h, washed, and rabbit anti-SEA conjugated to horse radish peroxidase, diluted 1 : 200 in the blocking reagent, added at 100 #l/well. The plates were washed 4 x and substrate (TMB; 3 , 3 ' - 5 , 5 ' tetramethylbenzidine) was added (100 ffl/well). The TMB solution was prepared by dissolving 10 mg of TMB in 1 ml dimethylsulphoxide, adding this dropwise to 100 ml of 0.1 M sodium acetate/citrate buffer pH 6.0 and adding 80 /~1 of 6% ( w / v ) H202. The microtitre plate containing the substrate was shaken for 10-15 min at room temperature and the reaction was stopped by adding 2 M H2SO 4 (50 ffl/well). The absorbance was read at 450 nm using an M R 580 Plate reader (Dynatech) and
88 the toxin concentration in the test samples was estimated from calibration curve of purified SEA (0.1-1000 n g / m l ) assayed on the same plate. M e t h o d 2: The SET-EIA kit is a commercially available immunoassay system (Labor Dr. W. Bommeli, Langass-Strasse 7, CH-3012 Bern, Switzerland) for detection of Staphylococcal enterotoxins A - D and is based on a single sandwich type ELISA using polyclonal anti-enterotoxin immobilised on polystyrene beads and an alkaline phosphatase conjugated antibody as the label. The jest samples, diluted in GPB, were assayed for SEA using the SET-EIA kit according to manufacturer's instructions. The substrate (p-nitrophenyl phosphate) reaction was stopped using 2 M N a O H and the extent of the reaction was measured using a Dynatech MR 600 plate reader. The concentration of SEA in the test samples was estimated using the calibration curve obtained by using purified SEA (0.1-1.6 n g / m l GPB).
Results Heat treatment
Fig. 1 shows data on the thermal inactivation of purified SEA heated at a range of temperatures from 6 0 - 1 2 1 ° C . Thermal destruction of SEA was considerably
I 70
6(}
5C ._= E 40
2C
10
0_1 60
i
70
8~}
9~) Temperature
110
1110
120l
L~o
(°C)
Fig. 1. Thermal inactivation of staphylococcal enterotoxin A in PBS buffer containing 2.5% normal rabbit serum as measured by ELISA2.
89 TABLE I E f f e c t o f i r r a d i a t i o n o n the d e t e c t i o n o f s t a p h y l o c o c c a l e n t e r o t o x i n A b y E L I S A in b u f f e r a n d m i n c e d beef Menstruum
Assay
Dose (kGy) 0
1.9
Gelatin phosphate buffer 15% M i n c e slurry
ELISA 1 a ELISA 2 "
53.2 82.2
14.0 4.5
2.7 0.2
2.2 -
ELISA 1 " ELISA 2 a
41.9 42.7
29.5 32.4
24.5 31.4
30.4 31.5
5.2
7.5
8.0 33.6 27.1
23.7 26.5 16.0
a M e a n o f t r i p l i c a t e results. F i g u r e s in the t a b l e r e f e r to the a m o u n t s o f t o x i n d e t e c t e d a f t e r i r r a d i a t i o n e x p r e s s e d as a p e r c e n t a g e o f the initial t o x i n a d d e d .
faster at 60 °, 70 °, 115 ° and 121°C (D values of 16.6, 6.9, 16.3 and 8.3, respectively) than at 90 o and 100 ° C (D values of 73.7 and 43.4, respectively). Irradiation treatment
The effect of irradiation dosage on the immunological detection of SEA is shown in Table I. Results from the two ELISA methods show that little immunological activity remained in GPB even after the lowest irradiation dose of 1.9 kGy. SEA was barely detectable in GPB using ELISA method 1 after irradiation at 7.5 k G y and could not be detected by either method after a dose of 8.0 kGy. Conversely, 1-25
i,-oof 0.75
~.~ .E .-=
0.5£
g 0.2
25
Mince
50
75
I 100
Concentration ( ° / o w / v )
Fig. 2. Effect o f m i n c e c o n c e n t r a t i o n o n the a m o u n t o f s t a p h y l o c o c c a l e n t e r o t o x i n A r e m a i n i n g a f t e r i r r a d i a t i o n at 25 k G y b y E L I S A 1 (©) a n d E L I S A 2 (O). V a l u e s in t h e figure r e f e r to the m e a n o f t r i p l i c a t e results.
90 27-34% of the toxins' initial immunological activity remained in a 15% minced beef-GPB slurry at an equivalent irradiation dose of 8.0 kGy. Even at a dose as high as 23.7 kGy, 16 27% of the initial level of SEA could still be detected in the mince. Results showing the protective effect of the mince slurry on the immunological activity of SEA following irradiation at an average dose of 23.4 k G y are given in Fig. 2. As the recovery of toxin depended on the mice concentration used, the results were expressed as the ratio of toxin recovered from irradiated to that from unirradiated samples. Generally, higher mince concentrations conferred increased protection against the effects of irradiation (Fig. 2). Regardless of the type of ELISA used to assay residual SEA, the protective effect of mince at a concentration of 50% was less than that of mince at a concentration of 30%.
Discussion The staphylococcal enterotoxins are generally appreciated to be resistant to thermal inactivation (Tatini, 1976). Numerous studies have documented their stability to heat and its dependence upon p H ( H u m b e r et al., 1975), toxin concentration and the nature of the heating menstruum (Bennett et al., 1977; Lee et al., 1977). Most of the results reported are based on the toxins' loss of reaction with antibodies using relatively insensitive techniques such as single gel immunodiffusion (Oudin, 1952) which require large amounts of toxin for quantitative estimation. Care should be taken in interpreting m a n y of these early studies as m a n y workers used crude or partially pure forms of the toxin which are noticeably more thermostable than the purified forms (Tatini, 1976). In addition, the antibody preparations used may not have been raised against highly purified toxin. Using highly sensitive and specific ELISA techniques we can confirm that the staphylococcal enterotoxins are remarkably heat stable. In this study, longer heating times were required to inactivate purified SEA at 90 and 100 ° C than at lower and higher temperatures (Fig. 1). A similar observation was made by Jamlang et al. (1971) who reported that SEB lost its immunological activity more rapidly at 70-80 ° C than at 90-100 ° C when heated in acetate or phosphate buffer. Similarly, Soo et al. (1974) found that SEA showed a more rapid loss of immunological activity when heated at 7 0 ° C than at 8 0 - 9 0 ° C . Such variation in the loss of immunological activity observed during heating of the enterotoxins also appears when the toxins are inactivated by irradiation. Fig. 2 shows that the residual amount of SEA detected by ELISA was less at a mince concentration of 50% than at a concentration of 30%. It has been suggested (Jamlang et al., 1971) that the rapid denaturation of SEB at 70-80 ° C was the result of enterotoxin aggregation which might be reversible at higher temperatures. Such aggregates occurring between toxin molecules may mask some of the toxins' antigenic sites and so protect against denaturation. This phenomenon could explain the shape of the heat inactivation curve. A more complicated situation probably exists when toxin is irradiated in a complex menstruum such as meat. Here toxin/toxin and t o x i n / m e a t interactions
91 m a y occur to varying degrees, thus making the biphasic nature of Fig. 2 more difficult to explain. In the only report on the effect of irradiation on the staphylococcal enterotoxins, Read and Bradshaw (1967) found that a dose of 5 Mrad (50 kGy) was required to reduce the concentration of SEB in 0.04 M Veronal buffer, p H 7.2, from 31 ~ g / m l to less than 0 . 7 / t g / m l . In milk, the same workers showed that a dose of 20 Mrad (200 kGy) was needed to achieve an equivalent reduction of SEB. In this study, a far smaller dose (8 kGy) was required to inactivate purified SEA in gelatin phosphate buffer, p H 6.8 (Table I), however, some SEA could still be detected in a 3% mince slurry even at the highest dose of irradiation (23 kGy) used. Although factors such as buffer composition and toxin concentration and purity may play some role in determining the heat sensitivity of the toxin to irradiation, the results appear to suggest that SEA is less resistant to irradiation than SEB. A similar conclusion has also been made after comparing the heat inactivation of SEA to SEB in Veronal buffer (Tatini, 1976). In the study of irradiation of SEB (Read and Bradshaw, 1967), the loss of immunlogical activity paralleled the loss of biological activity as determined by emesis in cats. Some workers have noticed a similar loss in both immunological and biological activities when studying heat inactivation of the staphylococcal enterotoxins (Jamlang et al., 1971; H u m b e r et al., 1975); others report a difference in the inactivation rates of immunological activity and toxicity with a suggestion of increased toxicity following heat treatment (data of Dangerfield, 1973, cited in Tatini, 1976; Tatini, 1976). Although we did not examine the effects of irradiation on the biological activity of SEA, similar studies (Tranter et al., 1987) on the irradiation of Clostridium botulinurn type A neurotoxin revealed a more rapid loss of toxicity compared to immunological activity. In view of the enhanced toxicities reported by some workers in SEA following heating, the effects of irradiation on the biological activity of this toxin should also be closely examined. The results of this study suggest an analogy between the stability of the staphylococcal enterotoxins in foods to irradiation and heat processing. It therefore follows that neither can be relied on solely to eliminate the hazards that may arise from mishandling food prior to processing. The food industry's good record with heat processing has been achieved by the application of good manufacturing practices and the same approach must also be used if irradiation is to be used for food processing.
Acknowledgements The authors acknowledge the financial assistance provided by Porton Products Ltd. to N.K.M. and H.S.T. and thank Mr. F.J. Ley of Isotron Ltd. for the use of irradiation facilities.
92
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