Comparison of Different Purification Procedures forExtraction of Staphylococcal Enterotoxin A from Foods
AIMO NISKANEN* AND SEPPO LINDROTH
Technical Research Centre of Finland, Food Research Laboratory, SF-02150 Espoo 15, Finland
Received for publication 19 April 1976
Different procedures commonly used for extraction, purification, and concen-
tration of staphylococcal enterotoxins from foods were investigated with 13'I- and'251-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was
compared with loss of total nitrogen. The results showed that in most of thecommon procedures, such as gel filtration, ion exchange, and heat treatment,the percentage of loss of labeled enterotoxin A was greater than the loss of totalnitrogen. Chloroform extraction and acid precipitation with hydrochloric acidhad nearly the same effect on the purification of both labeled enterotoxin A andtotal nitrogen. Ammonium sulfate precipitation proved to be practical and was
successfully used for purification of enterotoxin A from sausage extract. Simul-taneous use of trypsin and Pseudomonas peptidase for treatment of food extractsconsiderably reduced food proteins capable of interfering with serological detec-tion of enterotoxins but did not essentially influence the loss of enterotoxin A.
Many different serological methods havebeen described for the detection of staphylococ-cal enterotoxins (1, 5, 8-10, 13, 14, 16, 17, 19,23). Because of the insensitivity or nonspecific-ity of the detection methods and the low level ofenterotoxins in foods capable of causing intoxi-cation in sensitive persons, it is necessary toextract and partially purify enterotoxins beforedetection. Many procedures have been used topurify the enterotoxins from competing proteinmaterial or from other impurities that can in-terfere with the serological methods (2, 4, 11,13, 16, 21, 25; I. M. Stojanow and E. G. Schenk,U. S. Patent 3,525,731, 1966).By changing the pH of the extract, it is possi-
ble to alter the solubility of different proteinswith different isoelectric points (21; Stojanowand Schenk, U. S. Patent, 1966). The adsorp-tion of enterotoxins into ion-exchange materi-als has been used in many different methods (4,13, 21, 25). Gel filtration has also been used toexclude impurities based on the molecular-sieve effect (5; Stojanow and Schenk, U. S.Patent, 1966). Because staphylococcal entero-toxins have proved to be heat stable, this prop-erty has also been used (2; Stojanow andSchenk, U. S. Patent, 1966). Chloroform ex-tractions are also necessary to remove interfer-ing contaminants (2, 21, 25). Recently, at-tempts have been made to adsorb interferingsubstances into agar (21) and to handle theconcentrated extract with proteolytic enzyme,relying on the stability of enterotoxin againstproteolytic enzymes (2, 3, 21, 25).
During the extraction procedure, it is impor-tant that the total volume of extract be keptsuitable for each step. To reduce the water con-tent of the extract, dialysis has commonly beenused against a solution of polyethylene glycol(4, 13, 21), and when the extract has been puri-fied enough lyophilization has been used as afinal concentration procedure (4, 13, 21, 25).Concentration with gels has also been pro-posed, to save time (Stojanow and Schenk,U. S. Patent, 1966).The purpose of this study was to clarify, by
using 1311- and 125I-labeled enterotoxin A, theresults of some previously published purifica-tion methods, step by step, and to separatelyinvestigate the effect of other procedures dur-ing extraction. In addition, an attempt wasmade to purify enterotoxins from food extractsby using proteolytic enzymes and to precipitateenterotoxin A from food extracts by using am-monium sulfate (AS).
MATERIALS AND METHODS
Enterotoxin reagents. Purified enterotoxin A(>99%) was obtained from Serva Feinbiochemica,Heidelberg, Germany. Enterotoxin A antiserumwas obtained from M. S. Bergdoll, Food ResearchInstitute, University of Wisconsin, Madison.
Counting equipment. The 131I radioactivity ofsamples was measured with a low-background (3cpm) Electronics (Iceland) beta counter equippedwith an anticoincidence scaler, type AST-1. Themeasurements were made from three parallel sam-
ples (0.3 ml) on aluminum plates (25.5 mm in diam-eter) fixed with Omnispray-TM (no. NEF 932, New
England Nuclear Corp.). The radioactivity of sam-ples containing '25I-labeled staphylococcal entero-toxin A (SEA) was measured using an LKB-Wallac80000 automatic gamma counter. Samples (0.5 ml)were placed into counting tubes in Eppendorf micro-tubes (no. 3810).
Labeling of SEA. SEA was iodinated with car-rier-free Na'251 (Radiochemical Centre, Amersham,England) or Na'31I (Technical Research Centre ofFinland, Reactor Laboratory). All chemicals for io-dination were reagent-grade quality and were pre-pared fresh on the day of iodination in 0.05 M phos-phate-buffered saline, pH 7.5 (PBS). All reagentswere stabilized to +4°C before use. Labeling wasperformed by a modification of the method of Green-wood and Hunter (10, 14). The following reagentswere then added to a small glass tube suitable forEppendorf microtubes: (i) 25 ,ul of '31I, 0.4 mCi or 10,lI of 1251, 1.0 mCi in 0.1 M sodium hydroxide; (ii) 25,ul of SEA (300 ,ug/ml); (iii) 25 ,ul of chloramine T (88,ug). After 45 s of mixing by centrifugation in anEppendorf centrifuge (type 3200), the iodination wasstopped by the addition of 240 jllg of sodium bisulfitein 50 to 100 ,lI of water. Before transferring thereaction mixture to a Sephadex G-25 column (2001lI), carrier potassium iodide (10 mg/ml) was added.The Sephadex column (1.3 by 20 cm; v0, 8.8 ml), SG-75, was saturated just before gel filtration of thelabeled mixture with 1.0 ml of 1% bovine serumalbumin in PBS and eluted with 5 ml of PBS. Theiodinated SEA was eluted with PBS. Fractions (1ml) were collected into tubes containing 0.5 ml of 1%bovine serum albumin in PBS containing 0.1% so-dium azide as preservative and were stored frozen.The release of 131I or 125I and the damage of labeledSEA were controlled from the fractions before use(submitted for publication). The specific activities ofthe fractions of label used were 8 ,Ci/,g of SEA for'3'I and 115 /XCi/,ug of SEA for 125I. '3'I was used inmethods (i) and (ii) and 1251 was used in method (iii)(see below), because the experiments were run atdifferent times and the gamma counter was notavailable at the time methods (i) and (ii) were run;the radioactivity of 131I can, however, be measuredwith sufficient efficiency also by a beta counter.
Addition of labeled or unlabeled SEA in differentexperiments. Labeled SEA was added in all experi-ments except in those with extraction methods usingonly unlabeled SEA. The addition of label was ad-justed so that the radioactivity in different stages ofthe experiments was suitable for counting, being,however, below 1.0 gg/100 g of sausage in all cases.Labeled and unlabeled SEA together were addedonly in the experiment using combined enzymetreatment and AS precipitation. The amounts oflabeled or unlabeled toxin added are given with thedescription of each experiment.
Extraction of labeled or unlabeled SEA fromfoods. The labeled SEA was extracted from fer-mented dry sausage by using method (i), the methoddescribed by Barber and Deibel (2), and the follow-ing methods (method ii).
(i) Separation of enterotoxin from insoluble foodconstituents. A 100-g sample of dry sausage wasdiced and ground at low speed in a Virtis homogeni-
zator (The Virtis Co. Inc., Gardiner, N.Y.) in 200 mlof 0.2 N NaCl, pH 7.5, at room temperature until auniform slurry was formed. The pH of the slurry wasadjusted to 7.5 with 5 N NaOH. The slurry was coldcentrifuged (Sorvall RC2-B) at 27,300 x g for 20 minat -5°C. The supernatant was collected, and theprecipitate was re-extracted with 200 ml of 0.2 NNaCl, pH 7.5, and cold centrifuged as above. Re-extraction and centrifugation were repeated oncemore, and all supernatant fluids were collected.
(ii) Chloroform washing. Chloroform (20 ml) wasadded to the combined extracts with mixing on amagnetic stirrer for 2 min. The mixture was centri-fuged as above, and chloroform was discarded byfiltering the supernatant through wetted Whatmanno. 1 filter paper. The pH of the aqueous phase wasadjusted to 4.5 using 6 N HCI with continuous stir-ring. The mixture was centrifuged, and the pH ofthe supernatant was adjusted to 7.0 with 5 N NaOH.If precipitating material was observed during thisneutralization, the mixture was recentrifuged.
(iii) Concentration. The supernatant was placedin dialysis sacs (Arthur H. Thomas Co., no. 3787-D52) and immersed in polyethylene glycol (30%, wt/vol) overnight at +4°C. The concentrate was rehy-drated, if necessary, to 10 to 15 ml with 0.01 Mphosphate buffer, pH 7.5 (PB), and removed fromthe sacs. The sacs were washed twice with 2 ml ofPB, and the concentrate was transferred to a Sepha-dex G-25 column (2.5 by 35 cm; v0, 70 ml) at +40C.The sample concentrate was eluted with PB at aflow rate of 1 ml/min. Ultraviolet (UV) absorbance(280 nm) of the eluate was measured, and 2.5-mlfractions were collected. Radioactive fractions, to-taling approximately 90 ml, were pooled and concen-trated to 10 to 15 ml with dialysis tubing as above.The concentrate was washed with 2 ml of chloroformand cold centrifuged. The aqueous supernatant wastransferred to a Bio-Gel P-60 column (1.5 by 85 cm;vo, 27 ml) and eluted with PB at a flow rate of 0.2 ml/min. The UV absorbance of the eluate and radioac-tivity of the fractions (2.5 ml) were measured. Fivelabeled SEA-containing fractions were pooled, con-centrated to 2 ml in dialysis tubing against polyeth-ylene glycol, and washed once if necessary with 0.5ml of CHC13. After centrifugation the concentratewas lyophilized.The labeled SEA was extracted from milk sam-
ples using method (iii), the method described byReiser et al. (21). The label was added to the milk(80-ml sample) just before starting the procedure. Inaddition, unlabeled enterotoxin A (0.5, 1.0, and 2.0,ug of SEA per 100 g) was added to dry sausageextract in the absence of labeled toxin (duplicatesamples) and recovered by the methods describedabove. Experiments were also made by adding unla-beled toxin (0.5, 1.0, and 2.0 ,g of SEA per 80 ml) toduplicate milk samples and recovering the entero-toxin by method (iii) (Reiser et al. [21]).
Detection of total protein. Total nitrogen in thesamples was determined by using the Kjeldahlmethod. Nitrogen values were multiplied by 6.25 togive corresponding protein content. Protein contentin AS precipitation (Fig. 2) and heat treatment (Fig.4) experiments was measured by UV absorbance at
280 nm with a Beckman DB spectrophotometer. Ab-sorbance values were converted into protein concen-tration by a standard curve obtained with 12 knownsolutions of bovine serum albumin.
Precipitation of SEA with AS. Labeled SEA wasadded to the 0.2 N NaCl, pH 7.5, extract of drysausage (100 g) made as described above. Afterbeing carefully mixed, the extract was divided into95-ml portions and cooled to +4°C. Crystalline ASwas added into the labeled SEA-containing extractwith constant magnetic stirring to give 20, 40, 50,60, 70, and 80% saturation. After mixing the suspen-sion overnight at +4°C, the mixture was cold centri-fuged at 4,080 x g for 10 min. The precipitate (A)and the supernatant (B) were separated. The precip-itate (A) was dissolved in the original volume (95ml) of PB and centrifuged at 27,300 x g for 20 min.The supernatant (C) from this centrifugation indi-cated the solubility of protein material in the precip-itate (A). Radioactivity and protein content of thesamples were measured.
Precipitation with HCI and Esbach reagent. Pre-cipitation experiments with HCI and Esbach re-agent were performed using dry sausage extractcontaining labeled SEA according to methods de-scribed in the literature (2, 21; Stojanow andSchenk, U. S. Patent, 1966).Treatment of food extract with proteolytic en-
zymes. The following amounts of reagents per 10 mlof extract were added to the soluble extract of drysausage: 20 ,l of 1251-labeled SEA (1 ,g/ml), 50 ,ul ofSEA (200 jg/ml), 1.0 ml of trypsin (50 mg of trypsin[Difco; 1:250] in 100 ml of PB), and 0.1 ml of Pseu-domonas peptidase (prepared in this laboratory, 168U/ml in PB). The respective controls were made asabove, but without enzyme addition. Then, 10-mlportions of both mixtures were incubated at +37°Cfor 2 or 22 h or at + 18°C for 19 h. After incubationthe samples were cooled in an ice bath to +4°C andproteins were precipitated using a 60% final satura-tion of AS. After mixing the liquids for 15 min with amagnetic stirrer, they were centrifuged at 4,080 x gfor 10 min. The precipitate and supernatant wereseparated. The precipitate was dissolved in 25 ml ofPB. The total protein and labeled and unlabeledSEA content of the samples were measured.
Concentration of simulated food extract usingdifferent methods. Stock solution containing 10.0mg of bovine serum albumin and 52.5 mg of gelatine(Difco) in 100 ml of PB was mixed thoroughly andcooled to +4°C. This solution was diluted 1:10, 1:102,and 1:103, and 0.02 ,ug of labeled SEA was added toeach dilution (150 ml). The stock solution and dilu-tions were divided into 12 10-ml portions. Threeportions were concentrated with Sephadex G-25,three with Bio-Gel P-10, and three with dialysistubing. Three unconcentrated portions served ascontrols. For concentration with Sephadex G-25 andBio-Gel P-10, dry Sephadex G-25 (3 g) was placed ina chromatograph column (1.8 cm in diameter). Theprotein solutions described above were each pipettedinto their own columns on the surface of the gelmaterial. After wetting the column for 20 min, itwas placed in a rubber-capped vacuum flask con-taining an ampoule. A 2-ml portion of concentrated
solution was sucked by vacuum into the ampoule.The gel material was washed once with 1.0 ml of PB.After the washing solution was sucked into the am-poule, the concentrated solution in the ampoule wasdiluted to the initial volume (10 ml), and the radio-activity of the samples was measured. Concentra-tion with Bio-Gel P-10 was performed as describedabove, but 1.5 g of dry gel material was used.
Concentration with dialysis tubing was per-formed as described above, but the inside of thetubing was washed three times with 1.0 ml of PB.The concentrate was diluted to 10 ml, and the radio-activity of the samples was measured.
Heat treatment of food extract. A 0.05-,ugamount of labeled SEA was added to the 0.2 N NaClextract (600 ml) of dry sausage (100 g), made asdescribed above. The solution was mixed and di-'vided into 30-ml portions in 50-ml beakers. Heattreatment was performed in a steam bath at 50, 55,58, 60, and 63°C for 10 min. Warm samples werecooled in an ice bath to +4°C and centrifuged at27,000 x g for 10 min. Radioactivity and proteincontent of the supernatants were measured.
RESULTSThe amount of label present at each stage of
the different extraction procedures was mea-sured using 13II-labeled SEA. In methods (i)and (ii) the 13'I-labeled SEA was added to anextract of dry sausage. In method (iii) the 125I1labeled SEA was added to homogenized pas-teurized milk. The aim was to determine, withthe aid of the labeled SEA, the stages at whichthe loss of enterotoxin was greatest. From Ta-ble 1 it can be seen that at least some lossoccurs in each stage. In method (i) the mostcritical stages are 6, 8, and 18 (Table 1); inmethod (ii), stages 6, 7, 13, and 17; and inmethod (iii), stages 2, 4, 16, and 18. Criticalhandling stages for enterotoxin recovery werechloroform extraction, acid precipitation, ionexchange, gel filtration, heat treatment, andconcentration. The final '311-labeled SEA recov-eries from the initial additions were, on theaverage, under 1% from dry sausage extract formethods (i) and (ii) and 3.6% from milk formethod (iii).Comparing the effects of the critical stages on
both the content of '311-labeled SEA and thetotal nitrogen (TN) of the extract (Table 2), itwas found that chloroform extraction caused anaverage 31% loss of 1311-labeled SEA and a 35%loss of extract TN. The hydrochloric acid precip-itation at pH 4.5 in stage 4 (Table 1) caused a26% reduction in both '311-labeled SEA contentand TN. CMC-22 treatment resulted in 1311Ilabeled SEA and TN decreases of about 65 and57%, respectively. Further investigation re-vealed that slightly over one-half of this losswas a result of incomplete fixing on CMC-22,and the remainder was caused by adsorption on
a Method (i) (2), method (ii) (see text), and method(iii) (21).
b A 0.05- to 0.1-,ug amount of labeled SEA wasadded at step 1.
c Average of n estimations with methods (i) and(ii) from dry sausage and with method (iii) frommilk.
-, Step not included in the method.e ND, Not detected.
the CMC-22. The gel filtrations used in method(ii) caused a 40% reduction in SEA content, butthe reduction of TN was less than 20%. Theheat treatment in method (i) (heating to 56°C)reduced the content of SEA by 65% but that ofTN by only 44%. Extract concentration by di-alysis against polyethylene glycol decreasedthe amount of 13'I-labeled SEA by an average of25% but decreased TN by 77%.More detailed investigation of the effect of
different precipitation methods on the contentof 13'I-labeled SEA and TN in dry sausage ex-tract (Table 3) revealed that the solubility ofenteroxin using hydrochloric acid precipitationwas lowest at pH 4.5. The amounts of SEA andTN remaining in the supernatant were 82 and78%, respectively. When using Esbach reagentfor precipitation, '311-labeled SEA co-precipi-
tated with other proteins noticeably more com-pletely than when using hydrochloric acid.When the amount of Esbach reagent in theextract was increased from 20 to 43%, theamount of '311-labeled SEA in the supernatantdecreased correspondingly from 40 to 4%. Re-covery of 131I-labeled SEA from the Esbach pre-cipitate was impossible for all practical pur-poses.
Experiments using AS precipitation showedthat with a concentration of50% saturation, theamounts of 1311-labeled SEA and TN in the su-
TABLE 2. Effect of different proceduresa onextraction of labeled SEA from foods
AS (% of saturation)50 6.9 22.060 1.9 16.770 1.5 11.1a Conditions of precipitation: 40 ml of dry sausage
extract (TN, 0.5 mg/ml, and '3II-labeled SEA, 0.001jig/ml) was precipitated with HCl, as mentioned byBarber and Deibel (2), with Esbach reagent, accord-ing to Stojanow and Schenk (U. S. Patent, 1966) andwith AS (see text).
pernatant were 7 and 22%, respectively, of theoriginal. With a concentration of 60% satura-tion, enterotoxin precipitated almost quantita-tively and other proteins precipitated about85%. Further investigation of the effect of ASprecipitation on the solubility of 131I-labeledSEA (Fig. 1) showed that the enterotoxin redis-solved quite well in PB, corresponding to theoriginal volume, up to 60% saturation of AS.With increasing percentage of saturation, thesolubility of the 13II-labeled SEA in the precipi-tate decreased. The same pattern was observedfor other proteins (Fig. 2), but their solubilitywas, overall, much lower than that of the enter-otoxin, with a maximum of 30% saturation ofthe original protein from the extract.Experiments to determine the effect of heat
treatment on the precipitation of labeled SEAand of other proteins demonstrated that thesolubility of '3II-labeled SEA decreased as afunction of the amount of heat applied (Fig. 3).After 10 min of heat treatment at 60, 58, and55°C, residual amounts of SEA were 45, 50, and85%, respectively (Fig. 4). After 10 min at+550C, residual TN was approximately 75% asmeasured by UV absorbance and 85% as mea-sured by the Kjeldahl method. After 10 min at+58°C, the corresponding values were 82% byUV absorbance but only 62% by the Kjeldahlmethod.
Investigation of concentration using dialysistubing and gel materials revealed that the TNof the solution did not appreciably affect entero-toxin recovery when using dialysis tubing (Fig.
60
rL0I-40
2Lu'J 20ccwUa.
20 40 60 8o(NH4)2SO4 SATURATION %
FIG. 1. Precipitation of '3'I-labeled SEA with AS,at +4°C, from food extract. Symbols: ; supernatant;0, precipitate;0, soluble '3'I-labeled SEA in precipi-tate.
_2804
0 .1
n_j
UA.Oz
2I-
O 1020
'La
20 40 60 80(NH4)2S04 SATURATION %
FIG. 2. Precipitation of soluble protein from foodextract with AS, at +40C. Symbols: ,supernatant;0, precipitate; ct,soluble protein in precipitate.
11.0 60
40
0 10 20
TIME OF HEATI NG/minFIG. 3. Effectofheattreatmentonprecipitation of
13o-labeled SEA from food extract. Symbols: 0,500C; 0, 55-C; EJ, 580C; M, 600C.
5). However, when using Sephadex G-25 or Bio-Gel P-10, the recovery was dependent on theamount of other proteins in the solution. Ad-sorption of 3e11-labeled SEA to the gel increaseswith decreasing concentration of soluble pro-teins. Recovery with gels was in all cases lowerthan with dialysis, and different gels had con-siderably different adsorption properties.
Further tests were performed to determinethe loss of '31I-labeled SEA during concentra-tion. The results show that with increasingconcentration labeled toxin becomes morefirmly attached to the wall of the dialysis tube
FIG. 4. Effect of heat treatment (10 min) on pre-cipitation of total protein from food extract. Symbols:0, UV absorbance method; *, macro-Kjeldahlmethod.
ui
0 80
cc60
w
w 0C I
I" 40z U.uJ 00
20
C-
10.0 1.0 0.1 0,01TOTAL N I TROGEN mg/ml
FIG. 5. Dependence of '3'1-labeled SEA recovery
on TN (initial concentration) of food extract usingdifferent concentration methods; approximately five-fold concentration. Symbols: 0, dialysis tubing; El,
Sephadex G-25; A, Bio-Gel P-10.
(Table 4). A fraction of the '3'I-labeled SEApasses through the wall of the dialysis tube.This fraction is also dependent on the level ofconcentration.
Table 5 shows the results of experiments per-
formed to improve enterotoxin recovery usingproteolytic enzymes. Simultaneous application
of trypsin and Pseudomonas peptidase reducedthe amount of proteins precipitating with am-monium sulfate by about 40%. 125'-labeled SEAprecipitation was, however, reduced by below10%. Incubation for 2 h at +37°C was sufficient.Percentages of cold toxin measured by the se-miquantitative microslide method were foundto be practically equal in both enzyme-treatedand control samples. Percentages of SEA wereover 80% after 2 h of enzyme treatment at+37°C and 18 h at 19°C, and over 70% after 22 hof treatment at 22°C.
DISCUSSIONThe dose of enterotoxin required to cause food
poisoning symptoms in humans has been esti-mated at 10 to 13 ,ug (11). For particularlysensitive individuals, however, the dose may beless than 1 ,g (3). For this reason the detectionmethods presently in use for determination ofenterotoxins in suspected foodstuffs cannot reli-ably be used without extraction, partial purifi-cation, and concentration of the enterotoxin.Almost all the published methods contain thefollowing stages: (i) separation of the entero-toxin from insoluble constituents; (ii) separa-tion from soluble extractives; and (iii) concen-tration prior to detection by means of gel diffu-sion or some other suitable serological method.
In our research we attempted to determinethe role of stages (ii) and (iii), above, in the lossof SEA, using 13 I- and '25I-labeled SEA. Twomajor assumptions were that the labeled SEAhad chemical and antigenic properties suffi-
TABLE 4. Effect of concentration with dialysistubing on the yield of labeled SEA
% of 1311- % of 3_I-
Concn labeled % of 1311 labeledConcn coeffi- SEA in labeled SEA pass-
Conditions of concentration: 40 ml of dry sau-sage extract, three parallel samples in each concen-tration (TN, 0.5 mg/ml, and "'I-labeled SEA, 0.001,Lg/ml), was concentrated with dialysis tubingagainst polyethylene glycol (30%, wt/vol) at +4°C.Concentrated extract was pooled with 2 x 2-ml por-tions of PB used for washing adhering material in-side the dialysis tubing. Before measurements ofradioactivity, PB was added to give the initial vol-ume (40 ml).
b Concentration coefficient = initial volume pervolume after concentration.
a For conditions of enzyme treatment and precipitation by AS, see text.b Reduction percent = 100 - (100 x [TNenzyme/TNcontroI]).
ciently similar to those of native SEA, and thatrelease of '3'I and 125I was below 5% during theresearch.The '31I-labeled SEA extraction from dry sau-
sage using methods (i) and (ii) yielded 0.7 and0.8% recovery of added toxin (Table 1). Usingmethod (iii) (SEA extraction from milk), theresults were somewhat better, with a recoveryof 3.6%. Reports published earlier, although nothaving identical experimental conditions, haveclaimed significantly higher recovery percent-ages (4, 13, 16, 22), indicating either that thelabeled toxins in our study did not behave in amanner similar to that of unlabeled toxins orthat the earlier reports were too optimistic. Aportion of the loss probably also results frompartial detachment of the label, which takesplace as a function of temperature and time(18). On the other hand, in the previous publi-cations concerning percentage of recovery,much larger additions of enterotoxin were used,which in itself renders a higher percentage ofrecovery more probable (13, 21). Reiser et al.(21) have developed a method by which it ispossible to detect 0.125 ,ug ofSEA added to 100 gof food. Since the sensitivity of the microslidetechnique can be considered as 0.1 ,ug/ml (5),and since ths SEA has been concentrated to 0.2ml, this indicates a recovery of at least 15%.The minimum amount of added unlabeled SEAthat we were able to detect with the methodsused was approximately 1 ,ug/100 g of dry sau-sage, indicating 1.9% recovery. This also meansthat, with the equipment and materials availa-ble in this laboratory, it was not possible toreproduce the results of Reiser et al. (21). Fromthese observations it must be supposed that therecovery percentage using labeled SEA is a bitlower than that obtained when using coldtoxin.
In regard to the extraction of enterotoxin, itis, of course, important that components in thefoodstuff capable of interfering with the sero-logical determination be removed without sig-nificantly affecting the enterotoxins them-selves. Every stage of extraction that decreasesTN also decreases enterotoxins, and this de-
crease is sometimes even greater for the toxinsthan for TN, particularly in the case of gelfiltration (Table 2). The gel materials and col-umns used did not have sufficient resolvingpower. Although change of columns and gelmaterial, and process optimization, would al-most certainly result in improved results, gelfiltration is also too time consuming for seriousconsideration.Heat treatment has been used in some ex-
traction methods (2, 24) and has been consid-ered to have a positive effect in reducing thelevel of components interfering with the micro-slide method. The high loss of SEA (50%) andcorresponding low loss of TN (15%) observed inour research indicate that this method is ratherunsound. Although enterotoxin A is compara-tively heat labile compared with enterotoxin B(6, 22), the loss during heat treatment canhardly be a result of denaturation alone. Part ofthe loss probably results from co-precipitation,which can be eliminated by washing the precip-itate, a labor-consuming step. Another factorinfluencing the high loss of SEA in this re-search may be the low initial concentrationused. The heat stability of SEA increases withincreasing concentration (6). The minimum sol-ubility of TN from dry sausage extract was at58°C when measured by the macro-Kjeldahlmethod and at 55°C when measured by UVabsorbance. The former method is probablymore reliable for the purposes of our research.The divergence of these results stems from thefact that the content of UV-absorbing aminoacids varies among different proteins, and asingle protein, bovine serum albumin, wasused in the preparation of the standard.
Chloroform extraction caused an equal per-centage of loss of 13II-labeled SEA and TN.Chloroform extraction probably also has theeffect ofremoving other, nonprotein impurities.Results from this laboratory support the obser-vation of other workers (2, 4, 21, 24) that chloro-form extraction tends to reduce the proportionof materials causing nonspecific precipitationwith the microslide method.With hydrochloric acid precipitation, the
minimum solubility of 13'I-labeled SEA was atpH 4.5, as was that of the dry sausage proteins,although somewhat less markedly. If minimiz-ing the loss of enterotoxins is required, pH 4.5would not be the correct precipitation pH. Sincethe protein solubility of different food extractsdepends on the isoelectric points of the maincomponents, and since co-precipitation and sol-ubility affect the loss of SEA, the optimum pHfor acid precipitation would have to be deter-mined separately for every type of food.Stojanow and Schenk (U.S. Patent, 1966)
have recommended the use of Esbach reagentfor the precipitation of materials interferingwith the determination of enterotoxins. Al-though Esbach reagent does precipitate inter-fering proteins from the extract very effec-tively, it also precipitates the enterotoxins.Since the subsequent redissolving of these pre-
cipitated enterotoxins in our experiments was
practically impossible, the use of Esbach re-
agent for purification of enterotoxins cannot beconsidered feasible.The use of AS was, on the basis of this re-
search, a very useful method for purification ofenterotoxins from foodstuffs. When the toxicsolution was made 60% saturated with respectto AS, 131I-labeled SEA precipitated almostquantitatively, and TN precipitated about 85%.Thus, the toxin was freed from 15% of contami-nating proteins. When the AS precipitate was
redissolved in a corresponding volume of PB,80% of the original enterotoxins redissolved,but apparently only 25% of the TN redissolved.However, the latter result must be regardedwith caution, because the measurements were
made by UV absorbance, and purines and py-
rimidines present in foods cause interference atthe wavelength used (280 nm).
Since both radioactive label and cold SEAantigenic properties were well preserved dur-ing the overall process, the use of AS purifica-tion in the detection of enterotoxins from food-stuffs can be recommended on the basis of thegood recovery obtained. AS treatment shouldtake place at the beginning of the method andmust, of course, be followed by dialysis.Experiments with ion-exchange resins re-
vealed that by using CMC-22 the loss of entero-toxin was slightly greater than the loss of TN.The recovery we obtained using CMC-22 was
comparable to that obtained by Casman andBennett (4). Using Amberlite CG-50, recovery
was similar to that reported by Hall et al. (13).On this basis it can be presumed that the 1311_and 125I-labeled SEA behaved in ion-exhangeresin in a manner similar to that of the unla-beled toxin. The use of ion-exhange resin for
purification of enterotoxins from food extractswould appear to be a definite possibility afterfurther optimization of the processes.
Concentration of solutions is a very impor-tant stage in the detection of enterotoxins fromfoodstuffs. The results of the present researchshow that concentration by dialysis (more pre-cisely, osmodialysis) gave a better recovery ofenterotoxin than did gel concentration. In ouropinion, the essential information obtainedfrom the results in Fig. 5 is that concentrationwith dialysis is better suited to all concentra-tion stages of the extraction procedure thanconcentration with gels, because concentrationwith dialysis tubing seems to be less dependentthat does gel concentration on the total proteinconcentration of the solution. The great loss of1251-labeled SEA in the concentration experi-ments presented in Fig. 5 is apparently due toexperimental conditions, adsorption onto gels,and incomplete recovery of the water layerfrom gels. As can be seen from the results inTable 4, the almost equally concentrated drysausage extract has suffered only a 0.9% loss ofSEA. Thus, in this experiment labeled and un-labeled SEA appear to behave in the same man-ner. One positive feature of gel concentration,however, is its speed, which is equaled only byultrafiltration.The results in Table 4 indicate that a fraction
of the enterotoxins may pass through the di-alysis tube during concentration, and somemay become attached to the wall of the tube sothat it cannot be returned to the concentrate.Some of the label detected passing through thedialysis tube may be due to the release of radio-active iodine from the SEA. However, takinginto account the experimental conditions, all ofthe activity observed on the outside of the di-alysis tube cannot be explained in this way.
Enterotoxins have been shown to be resistantto proteolytic enzymes (3). On this basis thecombined effect of trypsin and Pseudomonaspeptidase was examined on both dry sausageextract and added labeled and unlabeled SEA.The effect was measured as the precipitate ob-tained using AS precipitation. The resultsshowed that with a 2-h enzyme treatment, 40%of the TN protein precipitated by AS was ren-dered nonprecipitating. The amount ofTN pre-cipitating from the enzyme-treated extract wasaround 28% of the original; i.e., 72% remainedin the rejected supernatant. Of the 125I-labeledSEA, 81% precipitated from the enzyme-treatedextract and 89% from the untreated control. A19-h treatment at + 18°C gave a somewhat bet-ter result, particularly in terms of precipitatingTN. This treatment time, however, is so long
that the method could only be used with thisstage as an overnight procedure. A 22-h treat-ment at +37°C cannot be recommended becauseresults indicate some proteolysis of the controlextract. Loss of enterotoxin has occurred, pre-sumably because of the ability of the microbialenzymes to hydrolyze SEA. This result mayalso be affected by reduction in co-precipitationas the amount of other precipitating proteinsdecreases. Similarity of the recovery of labeledand unlabeled SEA after precipitation in boththe enzyme-treated and control extracts indi-cates that labeled and unlabeled SEA have be-haved practically in the same way. The resultsin Table 5, in comparison to those expressed inFig. 1 and 2, show that 15 min of precipitationwith 60% saturated AS gave a better resultthan overnight AS precipitation. The precipita-tion of enterotoxin was not affected, but otherproteins precipitated noticeably less. Thus, it ispossible to remove an appreciable amount ofthe proteins interfering with enterotoxin detec-tion quickly and with less handling.
Enterotoxin A is considered to be a morecommon cause of food poisoning than other en-terotoxins (3). Because of its chemical proper-ties it is more difficult to extract from foodstuffsthan is, for example, enterotoxin B. The detec-tion is laborious and takes several days. It istherefore necessary to continue development ofimproved methods for the extraction, purifica-tion, and concentration of enterotoxins. Onlythe reserved passive hemagglutination inhibi-tion and radioimmunoassay methods are suffi-ciently sensitive to detect the presence of staph-ylococcal enterotoxin in foodstuffs without con-centration. However, both of these methodshave inherent problems, so that the microslidetechnique is still often used in both qualitycontrol and research work. Both the reservedpassive hemagglutination inhibition and ra-dioimmunoassay methods, and also the newenzyme-linked immunosorbent assay (7) sys-tem, require the removal of interfering compo-nents from the food extract for their reliablefunctioning. Enzyme treatment and AS precipi-tation in conjunction with cold centrifugationand dialysis, and possibly in the future alsowith affinity chromatography, are pretreat-ments that could guarantee rapid and morereliable detection of staphylococcal enterotox-ins from foodstuffs by sensitive serologicalmethods.
ACKNOWLEDGMENTS
We thank M. S. Bergdoll of the Food Research Institute,University of Wisconsin, Madison, for supplying the entero-toxin A antiserum. We are indebted to Lea Kallio andAulikki Koskimaki for technical assistance, and to MichaelBailey for inspecting the English text.
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