APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1976, Copyright (D 1976 American Society for Microbiology
p. 11-20
Vol. 31, No. 1 Printed in U.SA.
Effect of Starter Culture on Staphylococcal Enterotoxin and Thermonuclease Production in Dry Sausage AIMO NISKANEN*
AND
ESKO NURMI
Technical Research Centre of Finland, Food Research Laboratory, Biologink. 1, SF-02150 Espoo 15,* and State Veterinary Medical Institute, Hameetie 57, SF-00550 Helsinki 55, Finland Received for publication 12 August 1975
Different amounts of enterotoxin A-, B-, and C,-producing staphylococci were added to dry sausage prepared by normal processes, either alone or in conjunction with a starter culture (micrococci and lactobacilli). The sausage was examined after 0, 3, 7, 14, and 30 days for staphylococci, micrococci, and lactobacilli, and measurements were made of water activity, pH, enterotoxin, and thermostable nuclease. The results showed that in the absence of starter culture measurable amounts of entertoxin A were formed in a 200-g sample of dry sausage in 3 days, the level of Staphylococcus aureus infection being over 106 cells/g. Enterotoxin B was not found, although the total number of staphylococci was over 108 cells/g. Enterotoxin C1 was observed when the Staphylococcus count was about 8 x
107 cells/g, but
was no
longer detectable after 7 days. The starter culture
prevented the production of enterotoxin A in all cases investigated. By contrast, a very high-level inoculation of an enterotoxin Cl-producing strain gave a positive result after 3 days of incubation even in the presence of a starter culture. Heat-stable nuclease was observed in all sausages to which enterotoxinproducing staphylococci were added. The cell count determined in a sample of sausage had no definite correlation with the thermonuclease activity of the sample. Several earlier workers have shown that many microbes, when used as starter cultures, have an inhibitory effect on the growth of staph-
ylococci in experimental liquid cultures (10, 11, 19, 30). This effect has been assumed to be a result of the production of substances with antibiotic properties (13, 21, 30) and, by some Streptococcus lactis and Pediococcus cerevisiae strains, a result of the production of substances other than antibiotics (11). Competition among microorganismus for essential nutrients such as biotin and niacin has also been regarded as having an antagonistic effect (11). It has been shown that lactic acid and hydrogen peroxide produced by competing microbes can inhibit the growth of Staphylococcus aureus (11, 26). On the other hand, it has been demonstrated that certain microbes, although not significantly affecting the growth of staphylococci, do, howprevent the formation of enterotoxins (19). In addition, certain microorganisms that stimulated the production of enterotoxins without affected cell growth have been observed (19). Different lactic acid bacteria have been shown to have an inhibitory effect on growth and enterotoxin production by S. aureus in liquid cultures (31), in the order streptococci, P.
ever,
cerevisiae, and lactobacilli. Lactobacilli do not, in fact, appear to affect growth but do have a slight inhibitory effect on the production of enterotoxins (11). However, observations from experimental liquid cultures cannot be directly applied to foodstuffs, as staphylococci have been shown to behave differently in colloidal dispersions than in solutions (31). Most foods are complex colloidal systems, the physical properties of which are influenced by aeration, buffer capacity, water activity, (a,), adsorption of metabolites, availability of nutrients, etc. The properties of dry sausage vary continuously during maturation. Apart from changes in microbial populations, some varying factors are NO2, a,,, and pH. The latter has also been shown to have a significant effect on growth and enterotoxin production by S. aureus (1, 7, 14). It has been shown that by adding a suitable starter culture to sausage it is possible to influence maturation time, keeping qualities, aroma, and consistency, as well as to prevent taste and color defects (20). There are still some manufacturers of dry sausage who do not employ any addition of microorganisms and some who stabilize the process by addition of glucono11
12
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
8-lactone, either alone
or
combined with
a
starter culture.
The technological advantages of the use of a starter culture are already recognized, but in addition the effects of the starter culture on the microflora of the sausage have also been studied (20). Aerobic sporulating bacteria, gramnegative bacteria, and lipolytic and proteolytic bacteria have been controlled by adding starter cultures (20). Barber et al. (1) have shown the growth and enterotoxin production by staphylococci are dependent on the available oxygen in the sausage. The biological production of an acid environment by inoculation of the sausage with large amounts of P. cerevisiae prevents anaerobic growth of staphylococci, but does not completely prevent aerobic growth (1). After addition of 1.5% glucono-&lactone and a large inoculum of P. cerevisiae, over 107 S. aureus cells per g of sausage were necessary before measurable amounts of enterotoxins were observed after 24 h of incubation at 37 C (1). In normally processed dry sausage, such quantities of staphylococci are hardly possible other than in the case of an extremely highlevel initial contamination. In practice this level of contamination is possible only if an S. aureus abcess inside the frozen meat escapes observation at the stage of meat inspection. The correlation between thermoresistent nuclease and both growth and enterotoxin production by S. aureus is considered to be very high (16). For this reason a screening test for thermonuclease has been suggested for high-risk foodstuffs as an indictor of enterotoxin (25). The main aims of the work were to clarify: (i) the level of staphylococcal infection of the sausage meat necessary for the subsequent development in the normally processed dry sausage of staphylococcal populations sufficient to produce measurable amounts of enterotoxin, (ii) the effect of starter cultures containing lactobacilli and micrococci on the growth of staphylococci and the production of enterotoxins, and (iii) the use of measurement of staphylococcal thermonuclease as an indicator of the toxicity of a sample without the necessity of the difficult and comparatively laborious determination of enterotoxin. MATERIALS AND METHODS Dry sausage. The dry sausage used was prepared according to a dry sausage recipe by the normal commercial production method. The average composition of sausage was: beef, 44%; pork, 24%; pork fat, 28%; and 3.75% of a seasoning-salt mixture (final concentrations; sodium chloride, 3.0%; glucose, 0. 6%; potassium nitrate, 0.05%; white pepper, 0.1%).
TABLE 1. Ability of staphylococcal strains to produce enterotoxins and nucleasea
.au s.strain
Source
510 530
ATCC 196 Food poisoning outbreak in Finland ATCC 14458 Food poisoning outbreak in Finland
511 754
Enterotoxin Thermonuclease Type ,g/ml (.±g/ml) A A
4 10
18 190
B
30 128
21 6
C,
a Conditions of production: sac culture technique of Donnelly et al. (5), using BHI broth as nutrient; incubation, +37 C for 24 h.
Each production lot consists of six to eight different dry sausages, each type having three parallel sausages: (i) sausage with no added microorganisms; (ii) sausage containing starter culture according to normal production procedures (Duploferment, Rudolf Muller and Co., Giessen, Germany) (the starter culture contained lactobacilli and micrococci in 1:1 proportion; lyophilized or frozen micrococci and lactobacilli were added to the sausage meat according to the manufacturer's recommendations); (iii) sausage to which was added, in addition to the starter culture, two to three different doses of an enterotoxin-producing S. aureus strain per lot (properties and inoculum strengths of the enterotoxigenic strains can be seen in Tables 1 and 2); and (iv) sausage inoculated with two to three different doses of the S. aureus strain per lot without the starter culture. The S. aureus inoculum was grown in nutrient broth (Difco) at 37 C for 18 h. The cells were washed twice with saline before inoculation of the sausage meat lots were carefully mixed and packed into artificial skins (Cutisin R, Prago Export, Czechoslovakia), 65 mm in diameter and 300 mm in length. The sausages were then incubated at 23 to 24 C and (RH) 96% relative humidity for 2 days, after which they were smoked at 21 to 22 C, RH 80 to 85%, for 5 days. Final ripening and storage was at 17 C and RH 75 to 80%. Samples were taken from each sausage of every lot 0, 3, 7, 14, and/or 30 days after mixing. The microbiological examinations and measurements of pH and a ,, were performed immediately, after which the remainder of the sample was frozen to -30 C before assay of the enterotoxin and thermonuclease. The first assays were carried out within 1 month of freezing, and the necessary duplicate assays were carried out after 6 to 12 months. Microbiological assays. The numbers of micrococci and S. aureus present at each sampling were determined from two 10-g duplicate samples, after using suitable dilution series in phosphate-buffered saline. Colony-forming units (CFU) of S. aureus were counted from both the calf blood agar and Staphylococcus Medium no. 110 (Difco). All the inoculated S. aureus strains produced hemotoxins and yellow or orange pigment on Staphylococcus medium no. 110.
VOL. 31, 1976
13
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE co
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14
NISKANEN AND NURMI
APPL. ENVIRON. MICROBIOL.
Counting of CFU from calf blood agar. All colo- was dissolved in 0.2 ml of distilled water. The connies, surrounded after 24 h of incubation at +37 C centrated sample obtained by this method was asby hemotoxic reaction and having typical shape, sayed for enterotoxin by the microslide method. size, and color, were considered as S. aureus. Some When it was required to assay enterotoxins from a typical colonies were investigated for coagulase pro- 200-g sample, this was done by carrying out the duction. This investigation was made more often procedure outlined above on two 100-g samples. Bewith samples from control sausages because they fore the final lyophilization, the toxin-containing fractions were concentrated to 1 to 1.5 ml, conbined, were without S. aureus inoculum. Counting of CFU from Staphylococcus medium and washed in chloroform when necessary. In addino. 110. From separate colonies, representative of tion to the method described above, some of the different types of colonies occurring on the plate, samples (Table 2, samples 6.1 through 6.6) were color, size, shape, Gram staining, catalase produc- examined by the method described by Reiser et al. tion, aerobic acid production from mannitol, gela- (23). Detection of enterotoxins by the microslide tine hydrolyzation, and anaerobic fermentation of glucose were estimated. Some colonies were exam- method. Determinations of enterotoxins from concentrated extracts were carried out by the gel diffuined for coagulase production. All mannitol-positive, gelatine-hydrolyzing, sion test of Casman et al. (2). The concentration of gram-positive, catalase-positive, yellow or orange the control toxin was 0.5 ,ug/ml. Before dying of colonies, which had typical size and shape and also microslides, these were immersed in 0.2% Haemoproduced coagulase, were considered as S. aureus. Sol solution (Merz and Dade AG, 3018 Bern) at room White or gray colonies, with size and shape typical temperature for 1 to 1.5 h. The nonspecific precipiof micrococci and caused by gram-positive, catalase- tate caused by proteins originating from the sausage positive cocci, incapable offermenting glucose anaer- was thus removed, and the line of precipitation was clarified (1). obically, were considered as micrococci. Extraction of thermonuclease. Thermonuclease CFU of lactobacilli were estimated using Rogosa was assayed from 30-g samples taken to 1 cm below SL agar (Difco). pH measurement. The pH of all samples was the surface and homogenized in 60 ml of distilled water. Otherwise the assay was carried out accordmeasured with a Beckman Zeromatic pH meter. a ,. The a,, of five different sausages from each lot ing to Tatini (26), except that the final volume of the was measured using a Sina water activity meter sample before heat treatment was 3 ml, which sim(type SMT-C). Measurement was made at 20 C in a plified pH control using a glass electrode. Assay of thermonuclease. Thermonuclease was thermostat. In addition, the a, of 80-g samples was measured at 20 C using an electric hygrometer-indi- assayed by a modification of the metachromatic cator water activity meter (Hygrodynamics Inc.) for well-agar-diffusion technique (17). Deoxyribonucleic acid agar was prepared by the method described by purposes of comparison. Enterotoxins and antisera. Staphylococcal enter- Lachica et al. (15). Amounts of 12.1 ml were poured otoxin A (SEA) and enterotoxin A antiserum were into petri dishes (diameter, 89 mm). After cooling, obtained from Serva Feinbiochemica GmbH and 18 wells (diameter, 2 mm) were made in each dish. Co., Heidelberg, Germany. The other purified enter- ITo four dishes were pipetted 5-gl aliquots of nine otoxins (Staphylococcal enterotoxin B [SEBI and different levels (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, Staphylococcal enterotoxin Cl [SECJ]) and their spe- and 10.0 Ag/ml) of micrococcal nuclease (Worthingcific antisera were obtained from M. S. Bergdoll, ton Biochemical Corp. Freehold, N.J.) for use in the Food Research Institute, University of Wisconsin, standard line. The enzyme was dissolved in 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 8.5. Madison. Extraction of enterotoxins from sausage. En- Each sample under investigation (5-,ul aliquots) was terotoxins were extracted from samples of sausage pipetted to two dishes in a random order. Incubation by a method which will be fully described in a later was for 4 h at 37 C, and the results were read simulpublication. The main features of the method were taneously by two people with a stereomicroscope as follows. The analysis unit (100 g) was taken to (American Optical Co., model 570) at x 10 magnificadepth of about 10 mm and ground in 200 ml of 0.2 N tion, using the internal scale of measurement in NaCl until a uniform broth was obtained. This sus- each dish and bottom illumination. A semilogarithpension was cold centrifuged at 25,000 x g for 15 mic plot of concentration of nuclease in standard min. The precipitate was washed twice in 0.2 N solutions versus the diameters of zones of hydrolysis saline, and the insoluble fraction was discarded. The of deoxyribonucleic acid were drawn. With the aid of soluble fraction was washed in chloroform at pH 7.5, this standard line the thermonuclease activity of after which proteins were acid precipitated at pH each sausage was determined. The activities thus 4.5. After precipitation the pH of the solution was obtained were assigned to four groups (Table 4). Enterotoxigenicity and production of thermorestored to 7.0, and a 20x concentration was affected by dialysis against Carbowax CM20. The concen- nuclease by the S. aureus strains mixed in dry trate was subjected to gel filtration on Sephadex G- sausage mass. These investigations were carried 25 gel. Fractions containing toxin were collected and out on the buffer solution of dialysis sac cultures further concentrated, after which they were treated incubated for 24 h at 37 C in brain heart infusion on Bio-Gel P-60. Toxin-containing fractions were (BHI; Difco), as described by Donnelly et al. (5). washed in chloroform and concentrated to 2 ml. The Enterotoxin and thermonuclease determinations concentrate was freeze dried, and the dry material were carried out as described above.
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
15
RESULTS Growth of S. aureus in different types of dry sausage. Details of the bacteriological 7 changes taking place in the sausages from each lot, which were inoculated either with different amounts of S. aureus (sausage type A) or with 6 S. aureus and starter culture (type B), are given in Table 2. In Fig. 1 the average bacteriological changes as a function of maturation 5 time are given for types A and B and also for controls C and D (see footnote to Table 2). From these results it can be seen that the starter 3 culture has inhibited the growth of S. aureus throughout the maturation. Nearly as great an effect can be seen both in sausages into which 4 S. aureus has been inoculated (type B) and in respective control sausages (type C). However, even in sausages to which starter culture has been added, some increase in S. aureus is found 7 during the first 3 days, after which the level 4 decreases continuously throughout the maturation. The initial level in type A sausage is LL 6 attained after 10 days (Fig. 1) and in type B 0 after 7 days. The number of viable staphylococci remains high, however, even after 30 days 5 of maturation, in type A over 105 cells/g and in type B 104 cells/g. In controls C and D the numbers of staphylococci are lower than the z 4 values shown in Fig. 1, because the method used is not quantitative in all cases, where CFU, in fact, have been below 102, 103, or 104/g. 0-I Therefore, in calculating averages the corresponding logarithms of the limits 102, 103, or 104 have been used. 7 Changes in the starter culture microbes in dry sausage during ripening. The relative rates of additions of micrococci and lactobacilli 6 are approximately in the ratio 1:1, except in the case of lot 4 in which the relation is about 103: 1 (Table 2). The number of micrococci in type B 5 and C sausages increases only slightly after 3 days of maturation, after which a steady decrease is observable throughout storage. Simi4 lar changes occur also in types A and D. It appears that the presence of S. aureus does not affect the starter culture micrococci (type B), but by contrast the growth of micrococci entering sausage type A by natural contamination is 14 30 0 3 7 reduced by addition of S. aureus. In the sausages with starter culture (B and DAYS AFTER MIXING C) the maximum number of lactobacilli is found after 3 days of incubation, whereas in sausage 1. Changes in bacterial count (mean values) types A and D the maximum number, after in FIG. different types of dry sausage during ripening. growth from natural contamination, occurs Symbols: 0, With S. aureus inoculum (sausage type after 7 days (Fig. 1). A); *, with S. aureus and starter culture (type B); Ol, The level of lactobacilli falls to approxi- without S. aureus inoculum and with starter culture mately its initial level after about 14 days in inoculum (type C); *, without S. aureus inoculum the sausages with starter culture. Thereafter and without starter culture inoculum (type D).
16
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
there is no significant decrease in the numbers of lactobacilli. In the sausages without starter culture the behavior of lactobacilli follows a similar pattern, but the final level of lactobacilli is in fact higher than that in the sausages with starter culture. This is probably because the micrococci of the starter culture have an
inhibitory effect on lactobacilli. A similar inhibition of natural lactobacilli flora by S. aureus can be observed in sausage type A. Changes in pH and aw in different types of dry sausage during ripening. In dry sausages with starter culture the pH changes during maturation are dominated by the lactic acid produced by lactobacilli. The sausage meat used in these experiments had a pH of 5.95 + 0.40 immediately after mixing (Fig. 2). The initial pH of lot 4 was 6.4, indicating that the meat used was unsatisfactory. In sausage types B and C the pH fell after 7 days of maturation to an average of 5.3, which is considered desirable in normal production (20). The lowest average pH recorded was 5.1, after 14 days. In sausages without starter culture the pH did not fall below 5.5 in general at any time during maturation, and in lot 4 the pH remained above 5.75 in all cases, probably because the starter culture inoculum was too small and the quality of the meat too
poor.
The average a w changes occurring in the control sausage (type D) as a function of maturation time are shown in Fig. 2. The other types of sausage showed no significant deviation from this curve. No significant change in aw was observed during the first 3 days. This is reasonable since the first stage (2 days) took place under conditions of 96% RH. During the next stage (smoking), under conditions of 80 to 85% RH, the sausages began to desorb water so that
the final a, reached an average value of 0.948. During storage (75 to 80% RH) the sausages lost water continuously by evaporation, the a, falling below 0.880 by day 30 after mixing. This value is quite near the lower limit for multiplication of S. aureus (22, 24). Capability of the inoculated S. aureus strains to secrete enterotoxins and thermonuclease in BHI broth. The different S. aureus strains added to different lots are shown in Table 2. Strain 510 was added to four different lots, strain 530 to three, and both 511 and 754 to one lot only. The ability of these strains to produce enterotoxins and thermonuclease in 24 h at 37 C in BHI broth is shown in Table 1. From these results it can be seen that strains 510 and 530 produced comparable levels of enterotoxin and thermonuclease. In the case of strains 511 and 754, no correlation exists. S. aureus and detectable SEA in different types of dry sausage. In this investigation two SEA-producing S. aureus strains were used (Table 1). These strains were added to the sausage meat during production with different inoculum sizes (Table 2), either separately or in combination. After 3 days of maturation, a weak positive reaction in two duplicates was observed in sausage 1. CFU of S. aureus was determined as 2.2 x 106/g. In the control sample 1.2 the level of CFU was 6.9 x 105/g, and no enterotoxin was detected. At lower inoculation levels strain 510 did not produce measurable levels of SEA. Large inocula of strain 510 were added to the sausage meat of lot 4. SEA was detected in all sausages without starter culture after 3 days of maturation. In the sausages with starter culture enterotoxin was not detected, although the number of S. aureus cells exceeded that in sausage 1.1. Another interesting WATE R
_1-6
0,96 ,
ACT I V ITYY 5,7
0,94
5,5
0.92
5,3
0.90 0.88
0
3
DAYS
7
14
AFTER
30 MIXING
.
t
.
0
3
7
DAYS
14
AFTER
30 MIXING
FIG. 2. Changes in pH and a,, (mean values) in different types of dry sausage during ripening. Symbols: 0, With S. aureus inoculum; 0, with S. aureus and starter culture inoculum; O, without S. aureus inoculum and with starter culture inoculum; *, without S. aureus inoculum and without starter culture inoculum.
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
result from the experiments with lot 4 is that the formation of SEA is apparently prevented by the presence of the micrococci, although the numbers of staphylococci and micrococci are almost the same. On the other hand, the number oflactobacilli present after 3 days ofmaturation are relatively small, with the result that the fall in pH did not take place normally. Strain 530 did not produce appreciably more enterotoxin than strain 510 in dry sausage, although in BHI broth it was a more efficient producer. SEB and SEC, production. Three levels of SEB-producing strain 511 were inoculated into sausages of lot 5. The samples taken were negative for enterotoxin B, in all cases, although high levels of staphylococci were present (Table 2). Strain 754, producing SEC,, was added to lot 6. After 3 days, SEC, was found in sausages 6.3, 6.5, and 6.6. The presence of enterotoxin in sausage 6.6 is of interest because in other cases the presence of a starter culture prevented the formation of enterotoxins, even though the numbers of S. aureus in samples 4.6 and 5.6 were in excess of those in sample 6.6. After 7 days, on the other hand, no SEC, was detectable in any of the sausages, whereas SEA was detectable after 7, 14, and 30 days in all sausages in which it was detectable after 3 days, with the exception of sausage 1.1. In some sausages containing SEA, toxin could still be detected after 1 year at -30 C, despite organoleptic changes taking place during this time. Relation of staphylococcal thermonuclease to numbers of S. aureus and enterotoxin production. In this research it was shown that, with a few exceptions, thermonuclease was formed more in sausage of type A than in the corresponding type B sausage prepared with starter culture. The results are presented in Table 3, using the groupings in Table 4. The greatest amounts of thermonuclease, over 0.45 ,ug/ml of extract, were found in lot 4, in which pH values remained exceptionally high throughout the maturation time. Despite high cell counts in sausages 6.3 and 6.5, the levels of thermonuclease were low. On the other hand, as can be seen from the results in lot 4, the nuclease was detectable in the absence ofdetectable enterotoxin. Although strains 510 and 511 produced nearly identical amounts of nuclease in BHI, strain 510 seems to be a better producer in normally fermented sausage (compare samples 5.5 and 5.6 and 6.1 and 6.2 after 3 days) and much better in sausages of low lactobacilli level (compare samples 4.5 and 4.6 and 5.5 and 5.6). From these results it can be seen that the numbers of staphylococcal cells present in the
17
sausage did not correlate with the thermonuclease level detected in the 30-g sample. No significant changes were observed in the thermonuclease content of one sausage as a function of maturation time.
DISCUSSION The results obtained showed that contaminations of over 105 cells/g are required in the production stage, and over 2 x 106 cells/g in the final product, before enterotoxin is produced in quantities detectable from a 200-g sample of dry sausage. These results are in agreement with those obtained by Barber et al. (1) for fermented sausage, by Donnelly et al. (4) for milk, and by Genigeorgis et al. (8) for salted meat. In examining the results, however, it should be noted that dry sausage is an extremely difficult material for the isolation of enterotoxin, making precise estimation impossible for any strain. An interesting observation was that, although the starter culture had a definite inhibitory effect on the growth ofStaphylococcus only when the Staphylococcus inoculum was smaller than the starter culture inoculum, the starter culture without exception prevented the formation of detectable levels of enterotoxin. This result is apparently dependent on the presence of micrococci in the starter culture used. A similar effect has been determined by McCoy et al. (19) for Serratia marcescens and two Escherichia coli strains. On the other hand the lactobacilli of starter cultures have, at comparable levels of micrococci, an inhibitory effect on staphylococcal growth, as can be seen in samples 4.2 and 6.2. In this way the lactobacilli apparently decrease enterotoxin formation. Thus the number of staphylococcal cells present cannot be used as a measure of toxicity. The inability of the starter culture to prevent the formation of SEC, indicates a difference either in production mechanism or quantity compared to SEA. SEC, was observed after 3 days but not after 7 days. This indicates either that proteins formed during maturation prevent the appearance of SEC, or that the amount of SEC1 required for serological detection is decreased by the influence of proteolysis or denaturation. This disappearance of toxin was not observed in the case of positive SEA toxin samples. Earlier research has shown that enterotoxins in an active state are not deactivated by proteolytic enzymes (3). The formation of enterotoxin B was not observed in any of the experiments, although quite large inocula of SEB-producing strains were introduced into the sausage meat. The
18
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
TABLE 3. Enterotoxin A, B, and C1 production during ripening in dry sausages with and without starter culture Bacterial inoculum Sausage anaDuplolyzed (lot numferS. aureus strain ber)
ETa and THNb detection after mixing for:
mente
ET
THN
-
+-
++++-
ET
THN
-
++-
30 days
14 days
7 days
3 days
THN
ET
THN
++++-
NAd
-
NA NA NA
+++-
+++ +-
NA NA NA NA
+++ +-
ET
1.1 1.2 1.3 1.4
510 510 530 530
+ +
2.1 2.2 2.3 2.4
510 510 530 530
+ +
-
++-
+
--
+-
-
+-
-
3.1 3.2
510 + 530 510 + 530
+
+ -
+
+ -
+ +-
NA NA
+ +-
+
+-
-
-
4.1 4.2 4.3 4.4 4.5 4.6
510 510 510 510 510 510
-
+
+
+
-
+ -
++ ++ + +
+ + -
++
+ -
NA NA NA NA NA NA
++
+
5.1 5.2 5.3 5.4 5.5 5.6
511 511 511 511 511 511
6.1 6.2 6.3 6.4 6.5 6.6
510 510 754 754 754 754
-
-
-
-
-
++ ++-
+
+
-
++ + ++ ++ + ++
-
-
++-
-
++-
-
+++
-
+++ +
+ ++ +
+ -
+
+
+
+
+
+
+
+-
+
+-
+ -
-
+++ +-
NA NA NA NA NA NA
+ +++ ++-
+ -
+++ ++ + +
+
-
+-
-
++ ++ + +-
NA NA NA NA
NA NA NA NA NA NA
NA NA NA NA
+ +++ ++-
NA NA NA NA NA NA
NA NA NA NA NA NA
++-
++-
enterotoxin detected; + -, weak positive reaction for toxin; +, toxin detected. see Table 4. c Symbols for Duploferment (starter culture): -, not added; +, added. d NA, Not analyzed. a ET, Enterotoxin. Symbols: -, no b THN, Thermonuclease. Symbols:
dry sausage at least at the beginning of maturation is not a barrier to the formation of enterotoxins A and C, (29). The fall in pH value to 5.5 Thermonuclease Marking in difduring the first 3 days of maturation is not Diam of zones of (,ug/ml of exferent groups sufficient to prevent continued production of DNAa hydrolytract) correof thermonuenterotoxins (7, 9, 27). On the other hand, the sis (mm/5-jul exclease activisponding to zones of DNA hytract) combined effect of different factors inhibiting ties drolysis the production of enterotoxins, such as a , salts pH, smoking, and NO2, may concentration, '0.001 s3.2 have a significant effect in combination with a +3.3-5.1 0.001-0.01 starter culture (12). + 0.01-0.1 5.2-6.9 The use of thermonuclease determination as ++ 7.0-8.7 0.1-1.0 a screening method for toxicity has been sugDNA, Deoxyribonucleic acid. gested (25). The lowest level of staphylococci at which thermonuclease may be detected was 105 reason for this may lie in the physicochemical cells/g, indicating that nuclease assay is more conditions prevailing in dry sausage. The a, is sensitive than enterotoxin assay. On the other too low for the synthesis of SEB toxin, as was hand, the fact that some very high levels of shown by Troller (28), whereas the aw value of staphylococci resulted in only a very weak therTABLE 4. Grouping and marking thermonuclease activities detected in dry sausages
a
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
monuclease reaction indicates either that the method is not reliable or that the production of thermonuclease was significantly depressed in these samples. As Erickson et al. (6) have shown, the production of thermonuclease is inhibited by falling oxygen tension and pH. The high thermonuclease activities observed in lot 4 are presumably a result of this pH effect. In the other lots, larger numbers of lactobacilli may have inhibited the formation of staphylococcal nuclease as a result of reduction of pH or some other reason. In earlier reports reduction of staphylococcal nuclease activity has only been shown for growing cultures of Bacillus subtilis and S. faecalis var. liquefaciens (18). Changes in the actual nuclease activity as a function of maturation time were not observed in this research, which may indicate that the influence of lactobacilli is purely a function of pH reduction. Because the correlation between thermonuclease and the production of enterotoxin in dry sausage does seem not to be unambiguous, it cannot be recommended that assays for enterotoxins be replaced solely by thermonuclease determinations in cases ofsuspected contamination by staphylococcal enterotoxins. The thermonuclease test would, however, be suitable as a screening test. All samples giving a positive reaction could then be examined as soon as possible for the presence of enterotoxins. ACKNOWLEDGMENTS We would like to express our gratitude to Helsingin Kauppiaat Oy for the opportunity to use the necessary maturation and smoking facilities. Excellent technical assistance in the preparation of sausages was provided by Pauli Hill and in the assays of enterotoxin and thermonuclease by Marja Hukkanen, Aulikki Koskimiiki, Lea Kallio, and Aila Tuomolin. The manuscript was translated by Michael Bailey. LITERATURE CITED 1. Barber, L. E., and R. H. Deibe. 1972. Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage. Appl. Microbiol. 24:891-898. 2. Casman, E. P., R. W. Bennett, A. E. Dorsey, and B. S. Stone. 1969. The micro-slide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6:185-198. 3. Chu, F. S., K. Thadhani, E. J. Schantz, and M. S. Bergdoll. 1966. Purification and characterization of staphylococcal enterotoxin A. Biochemistry 5:32813289. 4. Donnelly, C. B., J. E. Leslie, and L. A. Black. 1968. Production of enterotoxin A in milk. Appl. Microbiol. 16:917-924. 5. Donnelly, C. B., J. E. Leslie, L. A. Black, and K. H. Lewis. 1967. Serological identification of enterotoxigenic staphylococci from cheese. Appl. Microbiol. 15:1382-1387. 6. Erickson, A., and R. H. Deibel. 1973. Production and heat stability of staphylococcal nuclease. Appl. Microbiol. 25:332-336.
19
7. Genigeorgis, C., M. S. Foda, A. Mantis, and W. W. Sadler. 1971. Effect of sodium chloride and pH on enterotoxin C production. Appl. Microbiol. 21:862866. 8. Genigeorgis, C., H. Riemann, and W. W. Sadler. 1969. Production of enterotoxin-B in cured meats. J. Food Sci. 34:62-67. 9. Genigeorgis, C., M. Savoukidis, and S. Martin. 1971. Initiation of staphylococcal growth in processed meat environments. Appl. Microbiol. 21:940-942. 10. Gilliland, S. E., and M. L. Speck. 1974. Antagonism of
11.
12. 13. 14.
15.
16.
17.
18. 19.
20.
21. 22. 23. 24.
25.
26.
27.
lactic streptococci toward Staphylococcus aureus in associative milk cultures. Appl. Microbiol. 28:10901093. Haines, W. C., and L. G. Harmon. 1973. Effect of selected lactic acid bacteria on growth on Staphylococcus aureus and production of enterotoxin. Appl. Microbiol. 25:436-441. Heidelbauer, R. J., and P. R. Middaugh. 1973. Inhibition of staphylococcal enterotoxin production in convenience foods. J. Food Sci. 38:885-888. Hirsch, A., and L. M. Wheater. 1951. The production of antibiotics by staphylococci. J. Dairy Res. 18:193197. Jarvis, A. W., R. C. Lawrence, and G. G. Pritchard. 1973. Production of staphylococcal enterotoxins A, B, and C under conditions of controlled pH and aeration. Infect. Immun. 7:847-854. Lachica, R. V., C. Genigeorgis, and P. D. Hoeprich. 1971. Metachromatic agar-diffusion methods for detecting staphylococcal nuclease activity. Appl. Microbiol. 21:585-587. Lachica, R. V., K. F. Weiss, and R. H. Deibel. 1969. Relationships among coagulase, enterotoxin, and heat-stable deoxyribonuclease production by Staphylococcus aureus. Appl. Microbiol. 18:126-127. Lachica, R. V. F., P. D. Hoeprick, and C. E. Frantti. 1972. Convenient assay for staphylococcal nuclease by the metachromatic well-agar diffusion technique. Appl. Microbiol. 24:920-923. Lachica, R. V. F., P. D. Hoeprick, and H. P. Riemann. 1972. Tolerance of staphylococcal thermonuclease to stress. Appl. Microbiol. 23:994-997. McCoy, D. W., and J. E. Faber. 1966. Influence of food microorganisms on staphylococcal growth and enterotoxin production in meat. Appl. Microbiol. 14:372377. Nurmi, E. 1966. Effect of bacterial inoculations on characteristics and microbial flora of dry sausage. Acta Agric. Fenn. 108:1-77. Oxford, A. C. 1944. Diplococcin, an antibacterial protein elaborated by certain milk streptococci. Biochem. J. 38:178-182. Plitman, M., G. Park, R. Gomez, and A. J. Sinskey. 1973. Viability of Staphylococcus aureus in intermediate moisture meats. J. Food Sci. 38:1004-1008. Reiser, R., D. Conaway, and M. S. Bergdoll. 1974. Detection of staphylococcal enterotoxin in foods. Appl. Microbiol. 27:83-85. Scott, W. J. 1953. Water relations of Staphylococcus aureus at 30°C. Aust. J. Biol. Sci. 6:549-564. Tatini, S. K. 1975. Application of heat-stable nuclease for screening foods for the likely presence of enterotoxins. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Ref. 242:137-138. Thompson, R., and A. Johnson. 1951. The inhibitory action of saliva on the diphtheria bacillus. Hydrogen peroxide, the inhibitory agent produced by salivary streptococci. J. Infect. Dis. 88:81-85. Tompkin, R. B., M. M. Ambrosino, and S. K. Stozek. 1973. Effect of pH, sodium chloride and sodium nitrite on enterotoxin A production. Appl. Microbiol.
20
NISKANEN AND NURMI
26:883-837. 28. Troller, J. A. 1971. Effect of water activity on enterotoxin B production and growth of Staphylococcus aureus. Appl. Microbiol. 21:435-439. 29. Troller, J. A. 1972. Effect of water activity on enterotoxin A production and growth of Staphylococcus aureus. Appl. Microbiol. 24:440-443.
APPL. ENVIRON. MICROBIOL. 30. Troller, J. A., and W. C. Frazier. 1963. Repression of Staphylococcus aureus by food bacteria. Appl. Microbiol. 11:163-165. 31. Woodburn, M., T. N. Morita, and S. Z. Venn. 1973. Production of staphylococcal enterotoxins A, B, and C in collodial dispersions. Appl. Microbiol. 25:825-833.
p. 11-20
Vol. 31, No. 1 Printed in U.SA.
Effect of Starter Culture on Staphylococcal Enterotoxin and Thermonuclease Production in Dry Sausage AIMO NISKANEN*
AND
ESKO NURMI
Technical Research Centre of Finland, Food Research Laboratory, Biologink. 1, SF-02150 Espoo 15,* and State Veterinary Medical Institute, Hameetie 57, SF-00550 Helsinki 55, Finland Received for publication 12 August 1975
Different amounts of enterotoxin A-, B-, and C,-producing staphylococci were added to dry sausage prepared by normal processes, either alone or in conjunction with a starter culture (micrococci and lactobacilli). The sausage was examined after 0, 3, 7, 14, and 30 days for staphylococci, micrococci, and lactobacilli, and measurements were made of water activity, pH, enterotoxin, and thermostable nuclease. The results showed that in the absence of starter culture measurable amounts of entertoxin A were formed in a 200-g sample of dry sausage in 3 days, the level of Staphylococcus aureus infection being over 106 cells/g. Enterotoxin B was not found, although the total number of staphylococci was over 108 cells/g. Enterotoxin C1 was observed when the Staphylococcus count was about 8 x
107 cells/g, but
was no
longer detectable after 7 days. The starter culture
prevented the production of enterotoxin A in all cases investigated. By contrast, a very high-level inoculation of an enterotoxin Cl-producing strain gave a positive result after 3 days of incubation even in the presence of a starter culture. Heat-stable nuclease was observed in all sausages to which enterotoxinproducing staphylococci were added. The cell count determined in a sample of sausage had no definite correlation with the thermonuclease activity of the sample. Several earlier workers have shown that many microbes, when used as starter cultures, have an inhibitory effect on the growth of staph-
ylococci in experimental liquid cultures (10, 11, 19, 30). This effect has been assumed to be a result of the production of substances with antibiotic properties (13, 21, 30) and, by some Streptococcus lactis and Pediococcus cerevisiae strains, a result of the production of substances other than antibiotics (11). Competition among microorganismus for essential nutrients such as biotin and niacin has also been regarded as having an antagonistic effect (11). It has been shown that lactic acid and hydrogen peroxide produced by competing microbes can inhibit the growth of Staphylococcus aureus (11, 26). On the other hand, it has been demonstrated that certain microbes, although not significantly affecting the growth of staphylococci, do, howprevent the formation of enterotoxins (19). In addition, certain microorganisms that stimulated the production of enterotoxins without affected cell growth have been observed (19). Different lactic acid bacteria have been shown to have an inhibitory effect on growth and enterotoxin production by S. aureus in liquid cultures (31), in the order streptococci, P.
ever,
cerevisiae, and lactobacilli. Lactobacilli do not, in fact, appear to affect growth but do have a slight inhibitory effect on the production of enterotoxins (11). However, observations from experimental liquid cultures cannot be directly applied to foodstuffs, as staphylococci have been shown to behave differently in colloidal dispersions than in solutions (31). Most foods are complex colloidal systems, the physical properties of which are influenced by aeration, buffer capacity, water activity, (a,), adsorption of metabolites, availability of nutrients, etc. The properties of dry sausage vary continuously during maturation. Apart from changes in microbial populations, some varying factors are NO2, a,,, and pH. The latter has also been shown to have a significant effect on growth and enterotoxin production by S. aureus (1, 7, 14). It has been shown that by adding a suitable starter culture to sausage it is possible to influence maturation time, keeping qualities, aroma, and consistency, as well as to prevent taste and color defects (20). There are still some manufacturers of dry sausage who do not employ any addition of microorganisms and some who stabilize the process by addition of glucono11
12
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
8-lactone, either alone
or
combined with
a
starter culture.
The technological advantages of the use of a starter culture are already recognized, but in addition the effects of the starter culture on the microflora of the sausage have also been studied (20). Aerobic sporulating bacteria, gramnegative bacteria, and lipolytic and proteolytic bacteria have been controlled by adding starter cultures (20). Barber et al. (1) have shown the growth and enterotoxin production by staphylococci are dependent on the available oxygen in the sausage. The biological production of an acid environment by inoculation of the sausage with large amounts of P. cerevisiae prevents anaerobic growth of staphylococci, but does not completely prevent aerobic growth (1). After addition of 1.5% glucono-&lactone and a large inoculum of P. cerevisiae, over 107 S. aureus cells per g of sausage were necessary before measurable amounts of enterotoxins were observed after 24 h of incubation at 37 C (1). In normally processed dry sausage, such quantities of staphylococci are hardly possible other than in the case of an extremely highlevel initial contamination. In practice this level of contamination is possible only if an S. aureus abcess inside the frozen meat escapes observation at the stage of meat inspection. The correlation between thermoresistent nuclease and both growth and enterotoxin production by S. aureus is considered to be very high (16). For this reason a screening test for thermonuclease has been suggested for high-risk foodstuffs as an indictor of enterotoxin (25). The main aims of the work were to clarify: (i) the level of staphylococcal infection of the sausage meat necessary for the subsequent development in the normally processed dry sausage of staphylococcal populations sufficient to produce measurable amounts of enterotoxin, (ii) the effect of starter cultures containing lactobacilli and micrococci on the growth of staphylococci and the production of enterotoxins, and (iii) the use of measurement of staphylococcal thermonuclease as an indicator of the toxicity of a sample without the necessity of the difficult and comparatively laborious determination of enterotoxin. MATERIALS AND METHODS Dry sausage. The dry sausage used was prepared according to a dry sausage recipe by the normal commercial production method. The average composition of sausage was: beef, 44%; pork, 24%; pork fat, 28%; and 3.75% of a seasoning-salt mixture (final concentrations; sodium chloride, 3.0%; glucose, 0. 6%; potassium nitrate, 0.05%; white pepper, 0.1%).
TABLE 1. Ability of staphylococcal strains to produce enterotoxins and nucleasea
.au s.strain
Source
510 530
ATCC 196 Food poisoning outbreak in Finland ATCC 14458 Food poisoning outbreak in Finland
511 754
Enterotoxin Thermonuclease Type ,g/ml (.±g/ml) A A
4 10
18 190
B
30 128
21 6
C,
a Conditions of production: sac culture technique of Donnelly et al. (5), using BHI broth as nutrient; incubation, +37 C for 24 h.
Each production lot consists of six to eight different dry sausages, each type having three parallel sausages: (i) sausage with no added microorganisms; (ii) sausage containing starter culture according to normal production procedures (Duploferment, Rudolf Muller and Co., Giessen, Germany) (the starter culture contained lactobacilli and micrococci in 1:1 proportion; lyophilized or frozen micrococci and lactobacilli were added to the sausage meat according to the manufacturer's recommendations); (iii) sausage to which was added, in addition to the starter culture, two to three different doses of an enterotoxin-producing S. aureus strain per lot (properties and inoculum strengths of the enterotoxigenic strains can be seen in Tables 1 and 2); and (iv) sausage inoculated with two to three different doses of the S. aureus strain per lot without the starter culture. The S. aureus inoculum was grown in nutrient broth (Difco) at 37 C for 18 h. The cells were washed twice with saline before inoculation of the sausage meat lots were carefully mixed and packed into artificial skins (Cutisin R, Prago Export, Czechoslovakia), 65 mm in diameter and 300 mm in length. The sausages were then incubated at 23 to 24 C and (RH) 96% relative humidity for 2 days, after which they were smoked at 21 to 22 C, RH 80 to 85%, for 5 days. Final ripening and storage was at 17 C and RH 75 to 80%. Samples were taken from each sausage of every lot 0, 3, 7, 14, and/or 30 days after mixing. The microbiological examinations and measurements of pH and a ,, were performed immediately, after which the remainder of the sample was frozen to -30 C before assay of the enterotoxin and thermonuclease. The first assays were carried out within 1 month of freezing, and the necessary duplicate assays were carried out after 6 to 12 months. Microbiological assays. The numbers of micrococci and S. aureus present at each sampling were determined from two 10-g duplicate samples, after using suitable dilution series in phosphate-buffered saline. Colony-forming units (CFU) of S. aureus were counted from both the calf blood agar and Staphylococcus Medium no. 110 (Difco). All the inoculated S. aureus strains produced hemotoxins and yellow or orange pigment on Staphylococcus medium no. 110.
VOL. 31, 1976
13
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE co
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14
NISKANEN AND NURMI
APPL. ENVIRON. MICROBIOL.
Counting of CFU from calf blood agar. All colo- was dissolved in 0.2 ml of distilled water. The connies, surrounded after 24 h of incubation at +37 C centrated sample obtained by this method was asby hemotoxic reaction and having typical shape, sayed for enterotoxin by the microslide method. size, and color, were considered as S. aureus. Some When it was required to assay enterotoxins from a typical colonies were investigated for coagulase pro- 200-g sample, this was done by carrying out the duction. This investigation was made more often procedure outlined above on two 100-g samples. Bewith samples from control sausages because they fore the final lyophilization, the toxin-containing fractions were concentrated to 1 to 1.5 ml, conbined, were without S. aureus inoculum. Counting of CFU from Staphylococcus medium and washed in chloroform when necessary. In addino. 110. From separate colonies, representative of tion to the method described above, some of the different types of colonies occurring on the plate, samples (Table 2, samples 6.1 through 6.6) were color, size, shape, Gram staining, catalase produc- examined by the method described by Reiser et al. tion, aerobic acid production from mannitol, gela- (23). Detection of enterotoxins by the microslide tine hydrolyzation, and anaerobic fermentation of glucose were estimated. Some colonies were exam- method. Determinations of enterotoxins from concentrated extracts were carried out by the gel diffuined for coagulase production. All mannitol-positive, gelatine-hydrolyzing, sion test of Casman et al. (2). The concentration of gram-positive, catalase-positive, yellow or orange the control toxin was 0.5 ,ug/ml. Before dying of colonies, which had typical size and shape and also microslides, these were immersed in 0.2% Haemoproduced coagulase, were considered as S. aureus. Sol solution (Merz and Dade AG, 3018 Bern) at room White or gray colonies, with size and shape typical temperature for 1 to 1.5 h. The nonspecific precipiof micrococci and caused by gram-positive, catalase- tate caused by proteins originating from the sausage positive cocci, incapable offermenting glucose anaer- was thus removed, and the line of precipitation was clarified (1). obically, were considered as micrococci. Extraction of thermonuclease. Thermonuclease CFU of lactobacilli were estimated using Rogosa was assayed from 30-g samples taken to 1 cm below SL agar (Difco). pH measurement. The pH of all samples was the surface and homogenized in 60 ml of distilled water. Otherwise the assay was carried out accordmeasured with a Beckman Zeromatic pH meter. a ,. The a,, of five different sausages from each lot ing to Tatini (26), except that the final volume of the was measured using a Sina water activity meter sample before heat treatment was 3 ml, which sim(type SMT-C). Measurement was made at 20 C in a plified pH control using a glass electrode. Assay of thermonuclease. Thermonuclease was thermostat. In addition, the a, of 80-g samples was measured at 20 C using an electric hygrometer-indi- assayed by a modification of the metachromatic cator water activity meter (Hygrodynamics Inc.) for well-agar-diffusion technique (17). Deoxyribonucleic acid agar was prepared by the method described by purposes of comparison. Enterotoxins and antisera. Staphylococcal enter- Lachica et al. (15). Amounts of 12.1 ml were poured otoxin A (SEA) and enterotoxin A antiserum were into petri dishes (diameter, 89 mm). After cooling, obtained from Serva Feinbiochemica GmbH and 18 wells (diameter, 2 mm) were made in each dish. Co., Heidelberg, Germany. The other purified enter- ITo four dishes were pipetted 5-gl aliquots of nine otoxins (Staphylococcal enterotoxin B [SEBI and different levels (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, Staphylococcal enterotoxin Cl [SECJ]) and their spe- and 10.0 Ag/ml) of micrococcal nuclease (Worthingcific antisera were obtained from M. S. Bergdoll, ton Biochemical Corp. Freehold, N.J.) for use in the Food Research Institute, University of Wisconsin, standard line. The enzyme was dissolved in 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 8.5. Madison. Extraction of enterotoxins from sausage. En- Each sample under investigation (5-,ul aliquots) was terotoxins were extracted from samples of sausage pipetted to two dishes in a random order. Incubation by a method which will be fully described in a later was for 4 h at 37 C, and the results were read simulpublication. The main features of the method were taneously by two people with a stereomicroscope as follows. The analysis unit (100 g) was taken to (American Optical Co., model 570) at x 10 magnificadepth of about 10 mm and ground in 200 ml of 0.2 N tion, using the internal scale of measurement in NaCl until a uniform broth was obtained. This sus- each dish and bottom illumination. A semilogarithpension was cold centrifuged at 25,000 x g for 15 mic plot of concentration of nuclease in standard min. The precipitate was washed twice in 0.2 N solutions versus the diameters of zones of hydrolysis saline, and the insoluble fraction was discarded. The of deoxyribonucleic acid were drawn. With the aid of soluble fraction was washed in chloroform at pH 7.5, this standard line the thermonuclease activity of after which proteins were acid precipitated at pH each sausage was determined. The activities thus 4.5. After precipitation the pH of the solution was obtained were assigned to four groups (Table 4). Enterotoxigenicity and production of thermorestored to 7.0, and a 20x concentration was affected by dialysis against Carbowax CM20. The concen- nuclease by the S. aureus strains mixed in dry trate was subjected to gel filtration on Sephadex G- sausage mass. These investigations were carried 25 gel. Fractions containing toxin were collected and out on the buffer solution of dialysis sac cultures further concentrated, after which they were treated incubated for 24 h at 37 C in brain heart infusion on Bio-Gel P-60. Toxin-containing fractions were (BHI; Difco), as described by Donnelly et al. (5). washed in chloroform and concentrated to 2 ml. The Enterotoxin and thermonuclease determinations concentrate was freeze dried, and the dry material were carried out as described above.
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
15
RESULTS Growth of S. aureus in different types of dry sausage. Details of the bacteriological 7 changes taking place in the sausages from each lot, which were inoculated either with different amounts of S. aureus (sausage type A) or with 6 S. aureus and starter culture (type B), are given in Table 2. In Fig. 1 the average bacteriological changes as a function of maturation 5 time are given for types A and B and also for controls C and D (see footnote to Table 2). From these results it can be seen that the starter 3 culture has inhibited the growth of S. aureus throughout the maturation. Nearly as great an effect can be seen both in sausages into which 4 S. aureus has been inoculated (type B) and in respective control sausages (type C). However, even in sausages to which starter culture has been added, some increase in S. aureus is found 7 during the first 3 days, after which the level 4 decreases continuously throughout the maturation. The initial level in type A sausage is LL 6 attained after 10 days (Fig. 1) and in type B 0 after 7 days. The number of viable staphylococci remains high, however, even after 30 days 5 of maturation, in type A over 105 cells/g and in type B 104 cells/g. In controls C and D the numbers of staphylococci are lower than the z 4 values shown in Fig. 1, because the method used is not quantitative in all cases, where CFU, in fact, have been below 102, 103, or 104/g. 0-I Therefore, in calculating averages the corresponding logarithms of the limits 102, 103, or 104 have been used. 7 Changes in the starter culture microbes in dry sausage during ripening. The relative rates of additions of micrococci and lactobacilli 6 are approximately in the ratio 1:1, except in the case of lot 4 in which the relation is about 103: 1 (Table 2). The number of micrococci in type B 5 and C sausages increases only slightly after 3 days of maturation, after which a steady decrease is observable throughout storage. Simi4 lar changes occur also in types A and D. It appears that the presence of S. aureus does not affect the starter culture micrococci (type B), but by contrast the growth of micrococci entering sausage type A by natural contamination is 14 30 0 3 7 reduced by addition of S. aureus. In the sausages with starter culture (B and DAYS AFTER MIXING C) the maximum number of lactobacilli is found after 3 days of incubation, whereas in sausage 1. Changes in bacterial count (mean values) types A and D the maximum number, after in FIG. different types of dry sausage during ripening. growth from natural contamination, occurs Symbols: 0, With S. aureus inoculum (sausage type after 7 days (Fig. 1). A); *, with S. aureus and starter culture (type B); Ol, The level of lactobacilli falls to approxi- without S. aureus inoculum and with starter culture mately its initial level after about 14 days in inoculum (type C); *, without S. aureus inoculum the sausages with starter culture. Thereafter and without starter culture inoculum (type D).
16
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
there is no significant decrease in the numbers of lactobacilli. In the sausages without starter culture the behavior of lactobacilli follows a similar pattern, but the final level of lactobacilli is in fact higher than that in the sausages with starter culture. This is probably because the micrococci of the starter culture have an
inhibitory effect on lactobacilli. A similar inhibition of natural lactobacilli flora by S. aureus can be observed in sausage type A. Changes in pH and aw in different types of dry sausage during ripening. In dry sausages with starter culture the pH changes during maturation are dominated by the lactic acid produced by lactobacilli. The sausage meat used in these experiments had a pH of 5.95 + 0.40 immediately after mixing (Fig. 2). The initial pH of lot 4 was 6.4, indicating that the meat used was unsatisfactory. In sausage types B and C the pH fell after 7 days of maturation to an average of 5.3, which is considered desirable in normal production (20). The lowest average pH recorded was 5.1, after 14 days. In sausages without starter culture the pH did not fall below 5.5 in general at any time during maturation, and in lot 4 the pH remained above 5.75 in all cases, probably because the starter culture inoculum was too small and the quality of the meat too
poor.
The average a w changes occurring in the control sausage (type D) as a function of maturation time are shown in Fig. 2. The other types of sausage showed no significant deviation from this curve. No significant change in aw was observed during the first 3 days. This is reasonable since the first stage (2 days) took place under conditions of 96% RH. During the next stage (smoking), under conditions of 80 to 85% RH, the sausages began to desorb water so that
the final a, reached an average value of 0.948. During storage (75 to 80% RH) the sausages lost water continuously by evaporation, the a, falling below 0.880 by day 30 after mixing. This value is quite near the lower limit for multiplication of S. aureus (22, 24). Capability of the inoculated S. aureus strains to secrete enterotoxins and thermonuclease in BHI broth. The different S. aureus strains added to different lots are shown in Table 2. Strain 510 was added to four different lots, strain 530 to three, and both 511 and 754 to one lot only. The ability of these strains to produce enterotoxins and thermonuclease in 24 h at 37 C in BHI broth is shown in Table 1. From these results it can be seen that strains 510 and 530 produced comparable levels of enterotoxin and thermonuclease. In the case of strains 511 and 754, no correlation exists. S. aureus and detectable SEA in different types of dry sausage. In this investigation two SEA-producing S. aureus strains were used (Table 1). These strains were added to the sausage meat during production with different inoculum sizes (Table 2), either separately or in combination. After 3 days of maturation, a weak positive reaction in two duplicates was observed in sausage 1. CFU of S. aureus was determined as 2.2 x 106/g. In the control sample 1.2 the level of CFU was 6.9 x 105/g, and no enterotoxin was detected. At lower inoculation levels strain 510 did not produce measurable levels of SEA. Large inocula of strain 510 were added to the sausage meat of lot 4. SEA was detected in all sausages without starter culture after 3 days of maturation. In the sausages with starter culture enterotoxin was not detected, although the number of S. aureus cells exceeded that in sausage 1.1. Another interesting WATE R
_1-6
0,96 ,
ACT I V ITYY 5,7
0,94
5,5
0.92
5,3
0.90 0.88
0
3
DAYS
7
14
AFTER
30 MIXING
.
t
.
0
3
7
DAYS
14
AFTER
30 MIXING
FIG. 2. Changes in pH and a,, (mean values) in different types of dry sausage during ripening. Symbols: 0, With S. aureus inoculum; 0, with S. aureus and starter culture inoculum; O, without S. aureus inoculum and with starter culture inoculum; *, without S. aureus inoculum and without starter culture inoculum.
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
result from the experiments with lot 4 is that the formation of SEA is apparently prevented by the presence of the micrococci, although the numbers of staphylococci and micrococci are almost the same. On the other hand, the number oflactobacilli present after 3 days ofmaturation are relatively small, with the result that the fall in pH did not take place normally. Strain 530 did not produce appreciably more enterotoxin than strain 510 in dry sausage, although in BHI broth it was a more efficient producer. SEB and SEC, production. Three levels of SEB-producing strain 511 were inoculated into sausages of lot 5. The samples taken were negative for enterotoxin B, in all cases, although high levels of staphylococci were present (Table 2). Strain 754, producing SEC,, was added to lot 6. After 3 days, SEC, was found in sausages 6.3, 6.5, and 6.6. The presence of enterotoxin in sausage 6.6 is of interest because in other cases the presence of a starter culture prevented the formation of enterotoxins, even though the numbers of S. aureus in samples 4.6 and 5.6 were in excess of those in sample 6.6. After 7 days, on the other hand, no SEC, was detectable in any of the sausages, whereas SEA was detectable after 7, 14, and 30 days in all sausages in which it was detectable after 3 days, with the exception of sausage 1.1. In some sausages containing SEA, toxin could still be detected after 1 year at -30 C, despite organoleptic changes taking place during this time. Relation of staphylococcal thermonuclease to numbers of S. aureus and enterotoxin production. In this research it was shown that, with a few exceptions, thermonuclease was formed more in sausage of type A than in the corresponding type B sausage prepared with starter culture. The results are presented in Table 3, using the groupings in Table 4. The greatest amounts of thermonuclease, over 0.45 ,ug/ml of extract, were found in lot 4, in which pH values remained exceptionally high throughout the maturation time. Despite high cell counts in sausages 6.3 and 6.5, the levels of thermonuclease were low. On the other hand, as can be seen from the results in lot 4, the nuclease was detectable in the absence ofdetectable enterotoxin. Although strains 510 and 511 produced nearly identical amounts of nuclease in BHI, strain 510 seems to be a better producer in normally fermented sausage (compare samples 5.5 and 5.6 and 6.1 and 6.2 after 3 days) and much better in sausages of low lactobacilli level (compare samples 4.5 and 4.6 and 5.5 and 5.6). From these results it can be seen that the numbers of staphylococcal cells present in the
17
sausage did not correlate with the thermonuclease level detected in the 30-g sample. No significant changes were observed in the thermonuclease content of one sausage as a function of maturation time.
DISCUSSION The results obtained showed that contaminations of over 105 cells/g are required in the production stage, and over 2 x 106 cells/g in the final product, before enterotoxin is produced in quantities detectable from a 200-g sample of dry sausage. These results are in agreement with those obtained by Barber et al. (1) for fermented sausage, by Donnelly et al. (4) for milk, and by Genigeorgis et al. (8) for salted meat. In examining the results, however, it should be noted that dry sausage is an extremely difficult material for the isolation of enterotoxin, making precise estimation impossible for any strain. An interesting observation was that, although the starter culture had a definite inhibitory effect on the growth ofStaphylococcus only when the Staphylococcus inoculum was smaller than the starter culture inoculum, the starter culture without exception prevented the formation of detectable levels of enterotoxin. This result is apparently dependent on the presence of micrococci in the starter culture used. A similar effect has been determined by McCoy et al. (19) for Serratia marcescens and two Escherichia coli strains. On the other hand the lactobacilli of starter cultures have, at comparable levels of micrococci, an inhibitory effect on staphylococcal growth, as can be seen in samples 4.2 and 6.2. In this way the lactobacilli apparently decrease enterotoxin formation. Thus the number of staphylococcal cells present cannot be used as a measure of toxicity. The inability of the starter culture to prevent the formation of SEC, indicates a difference either in production mechanism or quantity compared to SEA. SEC, was observed after 3 days but not after 7 days. This indicates either that proteins formed during maturation prevent the appearance of SEC, or that the amount of SEC1 required for serological detection is decreased by the influence of proteolysis or denaturation. This disappearance of toxin was not observed in the case of positive SEA toxin samples. Earlier research has shown that enterotoxins in an active state are not deactivated by proteolytic enzymes (3). The formation of enterotoxin B was not observed in any of the experiments, although quite large inocula of SEB-producing strains were introduced into the sausage meat. The
18
APPL. ENVIRON. MICROBIOL.
NISKANEN AND NURMI
TABLE 3. Enterotoxin A, B, and C1 production during ripening in dry sausages with and without starter culture Bacterial inoculum Sausage anaDuplolyzed (lot numferS. aureus strain ber)
ETa and THNb detection after mixing for:
mente
ET
THN
-
+-
++++-
ET
THN
-
++-
30 days
14 days
7 days
3 days
THN
ET
THN
++++-
NAd
-
NA NA NA
+++-
+++ +-
NA NA NA NA
+++ +-
ET
1.1 1.2 1.3 1.4
510 510 530 530
+ +
2.1 2.2 2.3 2.4
510 510 530 530
+ +
-
++-
+
--
+-
-
+-
-
3.1 3.2
510 + 530 510 + 530
+
+ -
+
+ -
+ +-
NA NA
+ +-
+
+-
-
-
4.1 4.2 4.3 4.4 4.5 4.6
510 510 510 510 510 510
-
+
+
+
-
+ -
++ ++ + +
+ + -
++
+ -
NA NA NA NA NA NA
++
+
5.1 5.2 5.3 5.4 5.5 5.6
511 511 511 511 511 511
6.1 6.2 6.3 6.4 6.5 6.6
510 510 754 754 754 754
-
-
-
-
-
++ ++-
+
+
-
++ + ++ ++ + ++
-
-
++-
-
++-
-
+++
-
+++ +
+ ++ +
+ -
+
+
+
+
+
+
+
+-
+
+-
+ -
-
+++ +-
NA NA NA NA NA NA
+ +++ ++-
+ -
+++ ++ + +
+
-
+-
-
++ ++ + +-
NA NA NA NA
NA NA NA NA NA NA
NA NA NA NA
+ +++ ++-
NA NA NA NA NA NA
NA NA NA NA NA NA
++-
++-
enterotoxin detected; + -, weak positive reaction for toxin; +, toxin detected. see Table 4. c Symbols for Duploferment (starter culture): -, not added; +, added. d NA, Not analyzed. a ET, Enterotoxin. Symbols: -, no b THN, Thermonuclease. Symbols:
dry sausage at least at the beginning of maturation is not a barrier to the formation of enterotoxins A and C, (29). The fall in pH value to 5.5 Thermonuclease Marking in difduring the first 3 days of maturation is not Diam of zones of (,ug/ml of exferent groups sufficient to prevent continued production of DNAa hydrolytract) correof thermonuenterotoxins (7, 9, 27). On the other hand, the sis (mm/5-jul exclease activisponding to zones of DNA hytract) combined effect of different factors inhibiting ties drolysis the production of enterotoxins, such as a , salts pH, smoking, and NO2, may concentration, '0.001 s3.2 have a significant effect in combination with a +3.3-5.1 0.001-0.01 starter culture (12). + 0.01-0.1 5.2-6.9 The use of thermonuclease determination as ++ 7.0-8.7 0.1-1.0 a screening method for toxicity has been sugDNA, Deoxyribonucleic acid. gested (25). The lowest level of staphylococci at which thermonuclease may be detected was 105 reason for this may lie in the physicochemical cells/g, indicating that nuclease assay is more conditions prevailing in dry sausage. The a, is sensitive than enterotoxin assay. On the other too low for the synthesis of SEB toxin, as was hand, the fact that some very high levels of shown by Troller (28), whereas the aw value of staphylococci resulted in only a very weak therTABLE 4. Grouping and marking thermonuclease activities detected in dry sausages
a
VOL. 31, 1976
STAPHYLOCOCCAL ENTEROTOXIN IN DRY SAUSAGE
monuclease reaction indicates either that the method is not reliable or that the production of thermonuclease was significantly depressed in these samples. As Erickson et al. (6) have shown, the production of thermonuclease is inhibited by falling oxygen tension and pH. The high thermonuclease activities observed in lot 4 are presumably a result of this pH effect. In the other lots, larger numbers of lactobacilli may have inhibited the formation of staphylococcal nuclease as a result of reduction of pH or some other reason. In earlier reports reduction of staphylococcal nuclease activity has only been shown for growing cultures of Bacillus subtilis and S. faecalis var. liquefaciens (18). Changes in the actual nuclease activity as a function of maturation time were not observed in this research, which may indicate that the influence of lactobacilli is purely a function of pH reduction. Because the correlation between thermonuclease and the production of enterotoxin in dry sausage does seem not to be unambiguous, it cannot be recommended that assays for enterotoxins be replaced solely by thermonuclease determinations in cases ofsuspected contamination by staphylococcal enterotoxins. The thermonuclease test would, however, be suitable as a screening test. All samples giving a positive reaction could then be examined as soon as possible for the presence of enterotoxins. ACKNOWLEDGMENTS We would like to express our gratitude to Helsingin Kauppiaat Oy for the opportunity to use the necessary maturation and smoking facilities. Excellent technical assistance in the preparation of sausages was provided by Pauli Hill and in the assays of enterotoxin and thermonuclease by Marja Hukkanen, Aulikki Koskimiiki, Lea Kallio, and Aila Tuomolin. The manuscript was translated by Michael Bailey. LITERATURE CITED 1. Barber, L. E., and R. H. Deibe. 1972. Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage. Appl. Microbiol. 24:891-898. 2. Casman, E. P., R. W. Bennett, A. E. Dorsey, and B. S. Stone. 1969. The micro-slide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6:185-198. 3. Chu, F. S., K. Thadhani, E. J. Schantz, and M. S. Bergdoll. 1966. Purification and characterization of staphylococcal enterotoxin A. Biochemistry 5:32813289. 4. Donnelly, C. B., J. E. Leslie, and L. A. Black. 1968. Production of enterotoxin A in milk. Appl. Microbiol. 16:917-924. 5. Donnelly, C. B., J. E. Leslie, L. A. Black, and K. H. Lewis. 1967. Serological identification of enterotoxigenic staphylococci from cheese. Appl. Microbiol. 15:1382-1387. 6. Erickson, A., and R. H. Deibel. 1973. Production and heat stability of staphylococcal nuclease. Appl. Microbiol. 25:332-336.
19
7. Genigeorgis, C., M. S. Foda, A. Mantis, and W. W. Sadler. 1971. Effect of sodium chloride and pH on enterotoxin C production. Appl. Microbiol. 21:862866. 8. Genigeorgis, C., H. Riemann, and W. W. Sadler. 1969. Production of enterotoxin-B in cured meats. J. Food Sci. 34:62-67. 9. Genigeorgis, C., M. Savoukidis, and S. Martin. 1971. Initiation of staphylococcal growth in processed meat environments. Appl. Microbiol. 21:940-942. 10. Gilliland, S. E., and M. L. Speck. 1974. Antagonism of
11.
12. 13. 14.
15.
16.
17.
18. 19.
20.
21. 22. 23. 24.
25.
26.
27.
lactic streptococci toward Staphylococcus aureus in associative milk cultures. Appl. Microbiol. 28:10901093. Haines, W. C., and L. G. Harmon. 1973. Effect of selected lactic acid bacteria on growth on Staphylococcus aureus and production of enterotoxin. Appl. Microbiol. 25:436-441. Heidelbauer, R. J., and P. R. Middaugh. 1973. Inhibition of staphylococcal enterotoxin production in convenience foods. J. Food Sci. 38:885-888. Hirsch, A., and L. M. Wheater. 1951. The production of antibiotics by staphylococci. J. Dairy Res. 18:193197. Jarvis, A. W., R. C. Lawrence, and G. G. Pritchard. 1973. Production of staphylococcal enterotoxins A, B, and C under conditions of controlled pH and aeration. Infect. Immun. 7:847-854. Lachica, R. V., C. Genigeorgis, and P. D. Hoeprich. 1971. Metachromatic agar-diffusion methods for detecting staphylococcal nuclease activity. Appl. Microbiol. 21:585-587. Lachica, R. V., K. F. Weiss, and R. H. Deibel. 1969. Relationships among coagulase, enterotoxin, and heat-stable deoxyribonuclease production by Staphylococcus aureus. Appl. Microbiol. 18:126-127. Lachica, R. V. F., P. D. Hoeprick, and C. E. Frantti. 1972. Convenient assay for staphylococcal nuclease by the metachromatic well-agar diffusion technique. Appl. Microbiol. 24:920-923. Lachica, R. V. F., P. D. Hoeprick, and H. P. Riemann. 1972. Tolerance of staphylococcal thermonuclease to stress. Appl. Microbiol. 23:994-997. McCoy, D. W., and J. E. Faber. 1966. Influence of food microorganisms on staphylococcal growth and enterotoxin production in meat. Appl. Microbiol. 14:372377. Nurmi, E. 1966. Effect of bacterial inoculations on characteristics and microbial flora of dry sausage. Acta Agric. Fenn. 108:1-77. Oxford, A. C. 1944. Diplococcin, an antibacterial protein elaborated by certain milk streptococci. Biochem. J. 38:178-182. Plitman, M., G. Park, R. Gomez, and A. J. Sinskey. 1973. Viability of Staphylococcus aureus in intermediate moisture meats. J. Food Sci. 38:1004-1008. Reiser, R., D. Conaway, and M. S. Bergdoll. 1974. Detection of staphylococcal enterotoxin in foods. Appl. Microbiol. 27:83-85. Scott, W. J. 1953. Water relations of Staphylococcus aureus at 30°C. Aust. J. Biol. Sci. 6:549-564. Tatini, S. K. 1975. Application of heat-stable nuclease for screening foods for the likely presence of enterotoxins. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Ref. 242:137-138. Thompson, R., and A. Johnson. 1951. The inhibitory action of saliva on the diphtheria bacillus. Hydrogen peroxide, the inhibitory agent produced by salivary streptococci. J. Infect. Dis. 88:81-85. Tompkin, R. B., M. M. Ambrosino, and S. K. Stozek. 1973. Effect of pH, sodium chloride and sodium nitrite on enterotoxin A production. Appl. Microbiol.
20
NISKANEN AND NURMI
26:883-837. 28. Troller, J. A. 1971. Effect of water activity on enterotoxin B production and growth of Staphylococcus aureus. Appl. Microbiol. 21:435-439. 29. Troller, J. A. 1972. Effect of water activity on enterotoxin A production and growth of Staphylococcus aureus. Appl. Microbiol. 24:440-443.
APPL. ENVIRON. MICROBIOL. 30. Troller, J. A., and W. C. Frazier. 1963. Repression of Staphylococcus aureus by food bacteria. Appl. Microbiol. 11:163-165. 31. Woodburn, M., T. N. Morita, and S. Z. Venn. 1973. Production of staphylococcal enterotoxins A, B, and C in collodial dispersions. Appl. Microbiol. 25:825-833.
