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Effect of Sodium Nitrite on Clostridium botulinum in Canned Luncheon Meat: Evidence for a Perigo-type Factor in the Absence of Nitrite
Effect of Sodium Nitrite on Clostridium botulinum in Canned Luncheon Meat: Evidence for a Perigo-type Factor in the Absence of Nitrite Pin-Chuan Chang 1, Syed Mumtaz Akhtar 1, T. Burke2 and Hilliard Pivnick 1
Abstract Canned shelf-stable luncheon meat containing varying amounts of sodium nitrite, prepared according to a commercial formula, was processed at 110°C to Fo = 0.4 and held at 35°C until nitrite was no longer detectable. The meat was then inoculated with spores of Clostridium botulinum separately heated to Fo = 0.4 and the cams were incubated at 30°C. Swelling of cans with concomitant toxin production was related directly to the size of the inoculum and inversely to the concentration of nitrite at time of manufacture. Meat containing 200 ppm of nitrite at time of manufacture (less tham 2 ppm when inoculated) inhibited 1.43 !OglO more spores than meat that was manufactured without nitrite. It appears that an inhibitor, which we call Perigo-type Factor (PTF), is formed during commercial processing of shelf-stable luncheon meat that contains nitrite.
Resume De la viande it collation en conserves contenant diverses quantites de nitrite de sodium, preparee selon une formule commerciale, a ete chauffee a 110°C jusqu'a Fo = 0.4 et maintenue a 35°C jusqu'a destruction complete des nitrites. La viande a ete ensuite inoculee avec des spores de Clostridium botulinum chauffees separement jusqu'a Fo = 0.4 et les boites de conserves ont ete incuooes a 30°C. Le gonflement des boites avec la production concomitante de toxine a ete relie directement a la taille de !'inoculum et inversement it la concentration de nitrite au moment de la fabrication. La viande qui contenant 200 ppm de nitrite au moment de la fabrication (moins que ~ ppm it l'inoculation) a inhibe 1.43 10glO plus de spores que la viande fabriquee sans nitrite. II nous apparait qu'un inhibiteur, que nous appelons Facteur du type Perigo (FTP), est forme au cours de la preparation commerciale de viande a collation en conserves qui ne contient pas de nitrite.
Introduction In canned meat one spore of Clostridium botulinum may cause food poisoning if it survives the heatprocess, germinates, multiplies and produces toxin. Interruption of anyone of the above processes will prevent toxin formation. Thus, no toxin will be formed if sufficient heat is applied to kill all spores. Even if some spores survive the heat-treatment, the meat will still he safe provided that the spores are p.revented from: (a) germination; or (b) multiplicatIOn after germination; or (c) toxin production despite multiplication. Shelf·stable canned cured meat receives a heat process ·of Fo = 0.1 - 0.6 which may permit spores to survive, yet cured meats are stable and safe. Thus, there must be other factors, in addition to the heating process, which contribute to safety. These factors are generally thought to be salt (sodium chloride) and sodium nitrite. The concentrations of salt used in such products are not inhibitory to undamaged spores of 1 ~crobiology Division, Research Laboratories, Food Directorate, nealth Protection Branch, Health and Welfare Canada, Ottawa, Ontario. 2 Canada Packers Limited, Research and Development Laboratories, Toronto, Canada. Can. lnst. Food Sci. Technol. J. Vol. 7, No.3, 1974
a. botulinum, bnt are inhibitory to the heat-damaged spores that survive thermal processing (Pivnick and Thacker, 1970a.; 1970b). The exact mode of action of nitrite is not well known, but its contribution to the stability of shelf-stable meat is well documented (Silliker et al.) 1938; Pivnick et al., 1969). At sufficient concentratron and appropriate pH, nitrite is inhibitory to growth of a. botulinum. However, about 75-85% of the nitrite is lost during thermal processing of shelf-stable meat, and the loss continues during storage at ambient temperature (Nordin, 1969; Olsman and Krol, 1972). These losses mitigate against consideration of a major role for nitrite per se in inhibiting a. botulinum in shelf-stable products. A possible explanation for this paradox was presented by Perigo et al. (1967) who found that heating nitrite in a bacteriological medium resulted in the formation of an antimicrobial factor - the so·called Perigo factor or Perigo inhibitor. The phenomenon is called the Perigo effect. Because of apparent misconceptions in citing our reports, we have presented in the following paragraph the essence of our work and that ·of others regarding the Perigo effect in meat. Johnston et al. (1969), in experiments similar to those of Perigo et al. (1967), confirmed that a potent antibotulinal factor (Perigo inhibitor) could be formed when nitrite was heated with a bacteriological medium similar to that used by Perigo, but Johnston and Loynes (1971) showed that some other types of media heated with nitrite did not form an inhibitor. Moreover, Johnston et al. (1969) found that if meat was added to medium containing the Perigo inhibitor, the inhibitory activity was lost. For this reason we now call the inhibitor that is formed in meat, the "Perigo-type Factor (PTF)". In further studies, Johnston et al. (1969) canned shelf-stable meat with 0-200 ppm of nitrite and, after thermal processing, homogenized the meat with an equal weight of water and dispensed it in test tubes. These tubes were challenged with 6-8 spores of a. botulinum. All tubes permitted growth and toxinogenesis; there appeared to be no inhibitory activity. However, recognizing that there might be a slight Perigo-type effect not demonstrable by even this very small challenge, they supplemented similarly-prepared homogenates with sterile nitrite to obtain 80,100,120 and 150 ppm of nitrite and challenged each tube with 6-8 spores. They did not find inhibitory activity in homogenates with 80 ppm of supplemental nitrite regardless of the nitrite added to the meat at time of manufaeture. However, when supplemented with 100 and 120 ppm of nitrite, the homogenates obtained from meat manufactured with nitrite were more inhibitory than hom 0 g e nat e s 209
Table 1.
Residual sodium nitrite in canned pork luncheon meat held at 35°C. Lot 2.
Nitrite in formulation (ppm)
Nitrite after weeks at 35°C (ppm)
NDa 8.9 17.3 31.1 53.5
0 50 100 150 200
2
4
6
14
ND 3.0 8.7 12.0 22.1
ND 2.0 6.2 8.5 12.0
ND 1.6 3.4 4.7 7.5
ND 0.6 0.8
0
l.l
2.1
a Not done
obtained from meat manufactured without nitrite. Supplementation with 150 ppm prevented growth even in meat manufactured without nitrite. They concluded: "The nitrite that disappears during normal thermal processing of meat does not form a potent antimicrobial agent although under special conditions (small numbers of spores and high concentrations of supplemental nitrite) some inhibitory activity could be demonstrated." Ashworth and Spencer (1972) confirmed the results of Johnston et al. (1969); they too found that ment heated without nitrite required more free nitrite (added after heating) than meat heated with nitrite to inhibit identical inocula. Their conclusions were similar to those of Johnston et al. (1969) : "from the work reported here no evidence has been obtained which would indicate that a Perigo effect is likely to be involved, under practical conditions, with the safety and stability of canned cured meats." It appeal'ed to us that confirmation of PTF l'equired conditions prevailing in shelf-stable cured meat. These could be better approximated by inoculating, with spores that had survived normal thermal processing, into meat with a low residual concentraHan of nitrite. This would permit detection of a Perigo effect without a superimposed effect of residual free nitrite. Using this approach we now report the presence of PTF in meat made with nitrite and challenged with heat-damaged spores of C. botulinu1n. At the time of challenge the residual nitrite was less than 2 ppm. Table 2.
Cumulative number of swollen cans out of 20 cans inoculated. Lot 1.
Nitrite a in formulation (ppm) 0 100 200 300
Spores per can 22 220 2,200 22 220 2,200 290 2,200 22,000 220 2,200 22,000 220,000
Weeks at 30°C 1 0 0 4 0 0 0 0 0 0 0 0 0 0
2 2 9 13 0 4 13 2 8 19 1 3 8 9
4
5
9 18 20 2 20 18 18 19
_b
12 12 18 20
3 8 16 18 0 18 18 10 18 20 6 10 18 19
a Residual nitrite at time of inoculation was No further change.
b
210
L.
6
2
4
20 19 20
20
16 15 18
17 18 19
2 ppm.
7
18 19 20
Materials and Methods Can,ned Meat. Cans (300 x 308) of luncheon meat,
varying only in the level of sodium nitrite, were processed according to industrial practice. The formula for a 100 pound batch was: Meat Pork meat (25% fat) 61.0 lb. 10.0 lb. ingredients Pork head meat (15% fat) Pork back fat (91% fat) 8.5 lb. Water (cold tap) 16.5 lb. Corn starch 4.0 lb. 2.3 lb. !i~'i;.i:':~?:A"~" Sodium chloride Sufficient sodium nitrite was added to obtain concentrations shown in Tables 1 and 2. The meat ingredients were ground through a 3/16" holed plate using a Weiler grinder and then blended for 5 min. in a Ruffalo mixer (1000 pound capacity). The temperature of the mix was -1.7°C after blending. The mix was divided into equal sized portions and stored in plastic containers at -5.6°C until used, a maximum of three days. Two mixes were prepared and used for the two experiments described in Lot 1 and r~ot 2 under Results and Discussion. Each portion of the meat ingredients was reground throngh a 3/16" holed plate using a Biro grinder (7.5 hp) and then transferred to a Buffalo mixer (100 ponnd capacity) and the salt, starch and solution of sodium nitrite were added while mixing. A vacuum of 635 mm Hg was applied and the portion was mixed for 5 min. The temperature after mixing was -1DC. The emulsion was transferred to a Peerless stuffer, stuffed into 300 x 308 cans, and capped nnder 660 mm Hg vacuum. The cans were retorted in steam at 110°C for 85 min. (Fo = 0.4), cooled and stored at 35°C. Samples were withdrawn periodically for nitrite determination and, when the level in tbe meat made with tbe highest concentration of nitrite bad decreased to 2 ppm or less, all remaining cans were withdrawn and challenged. Inoculu1n, Challenge and Incubation. Spore suspensions of C. bof;ulinu1n 13983B were prepared according to Johnston et al. (1969). The spores were heatdamaged (Fo 0.4, 100°C for 50 min.) in a particlefree meat extract. This extract was prepared aseptically by homogenizing radiation-sterilized raw meat (Johnston and J,oynes, 1971) in sterile water, (1:4), then removing: meat particles by filtration through cheese doth and centrifuging. The pH of the extract was 6.2. Sodium nitrite (150 ppm), when required, and sodium chloride (4.5%) were added aseptically to the supernatant fluid. Viable spores were enumerated in Prickett tuhes using Wynne's agar (Difco) layered with Vaspar. Tbe heated spore suspension was diluted in sterile distilled water to obtain a known number of viable spores. Then 0.1 ml of suitable dilutions was introduced with a syringe and needle into each can of meat through a hole aseptically pierced in the lid. The inoculated cans were resealed (Chang and Akhtar, 1973) and incubated at 30°C. The cans were examined weekly, and swollen cans were removed and tested for toxin. Some cans of meat were inoculated with 0.1 ml
=
J.
Inst. Can. Sc,l. Technol. Aliment. Vol. 7, No.3, 1974
Table 3.
Effect of size of inoculum and nitrite in fonnulation on time for 50% of inoculated cans to swell (tso). Lot 1.
Nitrite a in fonnulation (ppm)
Spores per can 22 >l1 b >11 NDc ND
0 100 200 300
220
2,200
22,000
1.6 1.9 2.5 3.2
1.2 1.4 1.7 2.5
ND ND 1.3 1.6
220,000 ND ND ND 1.5
a At time of inoculation, meat contained L2 ppm NaN0 2• tso in weeks. c Not done.
b
sterile water and incubated as controls; none of the controls swelled. Toxin Test. Toxin was detected by methods described bv PivniCk and Bird (1965). Chemical Analyses. Moisture, fat and dextrose equivalent were determined by the official methods (A.O.A.C., 1970). Protein was determined by a rapid Kjeldahl procedure (Perrin, 1953). Salt was extracted according to D~Ter (1943) and determined electrometrically with a Model 41 Titralyzer (Fisher Sci. Co.). Nitrite at time of manufacture was determined by the official method (A.O.A.C., 1970) and in meat held at 35°C, by the method of Kamm et al. (1965). In our experience, the Kamm method is more suitable for determining nitrite at less than 10 ppm in meat.
Results and Discussion Two lots of canned meat were produced and challenged. Lot 1 c'Ontained 0-300 ppm of nitrite and Lot 2 contained 0-200 ppm. In other respects the two lots were very similar) 57.3 - 60% moisture; 23 - 25% fat; 10.8 - 12.3% protein; 3.7 - 4.2% dextrose equivalent, 2.2 - 2.4% Halt and 3.7 - 3.9% brine (gm salt/gm salt gm water) X 100. Depletion of Nitrite. Meat stored at 35°C was tested at intervals for residual nitrite. In Lot 1, meat made with 300 ppm of nitrite contained 2.2 ppm after 15 weeks. In IJot 2, the highest concentration of nitrite used was 200 ppm; it declined to 2.1 ppm in 14 weeks (Table 1). The decay of nitrite in Lot 2 during the first six weeks followed a first order reaction; for this period an equation, log ColC = 0.126t, was obtained by averaging the slopes of the regression lines for the 4 levels of nitrite (S.E. < 10%). In the equation, Co = the concentration of nitrite at the beginning of incubation and C = the concentration at time t in weeks.
+
Table 4.
Effect of inoculum on toxinogenesis in luncheon meat made with varying amounts of nitrite. Lot 2.
Nitrite a in fonnulation (ppm)
o
50 100 150 200
Spores per can
Total toxic
8
16
32
64
128
Inoculated
Ib 1
6
.'3 6 2 4
9 7 5
12 9 10 7
33/80 25/80 18/80 21/80 2/80
o o 1
2 1
3
o
o
7 1
o
a ~2 ppm at time of inoculation. b [lumber of cans, out of 16 inoculated, that became toxic during 11 weeks at 30°C. Can. lnst. Food ScI. Technol. J. Vol. 7, No.3, 1974
Table 5.
Inhibition (In) due to salt and Perigo-type factor (PTF) in canned luncheon meat. Lot 2. NaN0 2 in Sporesa Ratio spores: Totalb Units fonn-
that
Added
Grew in salt
units
of In
ulation (ppm)
grew
Grew
Grew in salt plus nitrite
of In
due to PTF
54.3 35.3 22.6 27.6 2.0
83.1 112.3 175.3 143.8 1974.1
1.9 2.1 2.2 2.2 3.3
0.2 0.3 0.3 1.4
o
50 100 150 200
1.5 2.4 2.0 27.0
a Spores added at each nitrite level = 16(8+16+32+64+128) 3968. These spores had survived heating to Fo = 004. b One unit of In = inhibition of 1 loglO or 90% of spores.
=
The half-life was 2.4 weeks. The data for Lot 1 were less extensive and are not presented. Lnoculation amd Incu,bation of Meat. Lot 1 was inoculated with spores that survived heating to Fa = 0.4 in the meat extract (see lfaterials and Methods) with and without 150 ppm of nitrite. No appreciable difference waH f.ound between cans inoculated with the two preparations and the results were, therefore, combined. Most of the cans swelled within 4 weeks of inoculation and none swelled after 7 weeks; the experiment was terminated after 11 weeks. Table 2 shows the cumulative number of swollen -cans, all of which contained toxic meat. There is no evidence that the nitrite concentration added at the time of manufacture prevented the inoculum from growing and causing swelling; one possible exception is found by comparing an inoculum of 22 spores in meat manufactured with 0 and 100 ppm of nitrite. There was, however, an indication that nitrite did have an effect on the time for swelling to occur. Table 3 shows that as the nitrite at the time of manufacture increased, the time for swelling of 50% of cans (t so ) at each inoculum level increased. As expected, an increase in size of inoculum decreased the time for swelling. As it was obvious that we had inoculated Lot 1 with too many spores to obtain quantitative information on the number of spores required to initiate growth, Lot 2 was inoculated with fewer spores than Lot 1. For Lot 2, spores were heated in the meat extract containing 150 ppm of nitrite, but not in extract without nitrite (cf. Lot 1). Table 4 shows the number of spores added and the number of cans that swelled. The results are similar to thm:e obtained with Lot 1, with the major exception that, at the end of the experiment, each group contained unswollen cans: this was probably due to the smaller number of spores inoculated per can in Lot 2. Quantitation of Inhibition of Outgrowth. Assuming that inhibition of the inoculum was due only to salt and PTF we have calculated the inhibition due to salt for Lot l' and for both factors for I.. ot 2 (Table 5). The extent of 'inhibition was calculated (Pivnick and Petrasovits, 1973) from the ratio: number of spores added/ number ·of spores that grew. The number of spores that grew was estimated by a Most Probably Number Method based on the ratio: cans inoculated/cans that 211
did not swell (Stumbo et al., 1950). Inhibition is expressed in units of In; each unit represents inhibition of outgrowth of 90% of spores (1 loglO) (Pivnick, 1970) . Salt in canned meat may inhibit outgrowth 'Of heat-damaged spores. Meat made with salt, but lacking nitrite, was inoculated in both experiments. In Lot 1, each of 20 cans was inoculated with 22 spores (total 440) and 9 of the 20 cans swelled. The estimated number of spores that grew was 11.86, and 328.14 (97.3%) failed to grow. The ratio of spores added/spores that grew (440/11.86) was 37.08 (loglO 1.57 1.6 units 'of In). In Lot 2 (see Table 5), salt was responsible for inhibiting 98.63% of the added spores (1.9 units of In). For spores treated at Fo 0.4, this degree of inhibition is consif.1tent with that found by Pivnick and Thacker (19703) in 3.7 - 3.9% brine. Inhibition due to PTF was calculated by subtracting units of In due to salt alone from units of In due to salt PTF (Table 5). Nitrite added at concentrations 'Of 50-] 50 ppm to the product prior to thermal processing created a relatively small amount of PTF whereas 200 ppm of added nitrite produced sufficient PTF to cause 1.4 units of In. Two salient points emerge from these experiments: (1) spores that survive Fo = 0.4 are strongly inhibited by salt (cf. Pivnick and Thacker, 1970a; Roberts and lng-ram, 1966) ; and (2) there appears to be a threshold level of nitrite, somewhere between 150 and 200 ppm added before heat processing, which is necef.1sary to produce an appreciable amount of PTF. There is substantial evidence for the inhibitory activity of salt towards heat-damaged spores, but there is no supportive literature concerning inhibition of spores by PTF in meat in the absence of free nitrite. Cel·tainly, 150 ppm of nitrite added to raw meat inoculated with a. botulinum prior to processing to Fo = 0.6 cauf.1pd substantial inhibition of toxigenesis (1.64 units of In) (Pivnick et al., 1969; Pivnick and Petrasovits, 1973) and prevented spoilage in uninoculated packs processed to an Fo of about 0.1 (Silliker et al., 1958). However, when canned meat is processed, ther~ is always residual nitrite immediately after processmg, and for a period during storage (Nordin 1969; Christiansen et al., 1973). Assuming that most of the spores germinate within a day of processing (Pivnick et aT., 1970), it is quite likely that PTF and residual nitrite interact to prevent outgrowth. In model systems, Johnston et al. (1969) had to supplement meat heated with nitrite, with additional nitrite after heating, in order to demonstrate a PTF. Similarly, Ashworth and Spencer (1972) and Ashworth et al. (1973) demonf.1trated a PTF in meat heated with sufficient nitrite to have appreciable residual nitrite after heating. The recent finding by Ashworth et al. (1973) that PTF can be formed in pork during a simulated pasteurhdng process (20°C increased to 70°0 in 4 hr.) m.ay explain the inhibition of toxinogenesis by a. botul~num In inoculated, pasteurized, canned pork
=
=
+
212
=
incubated at 27°C (Christiansen et al., 1973). Indeed, "it seems not unreasonable to suggest that the presence of a thermally-derived inhibitor based on nitrite may be a major contributory factor to the safety and stability of pasteurized cured pork products" (Ashworth et al., 1973). Our results indicate that a similar statement may be made for cured, shelf-stable canned meats that are usually processed to obtain centre temperatures above 100°C. However, in such products, the inhibitory effect of salt against heat-damaged spores may be even more important than PTF.
References A.O.A.C. 1970. Official methods of analysis of the Association of Official Analytical Chemists. 11th Edition, Washington, D.C. Ashworth, J., and Spencer, R. 1972. The Perigo effect in pork. J. Food Techno!. 7 :111. Ashworth, J., Hargreaves, L. L., and Jarvis, B. 1974. The production of an antimicrobial effect in pork heated with sodium nitrite under simulated commercial pasteurization conditions. J. Food Technol. In Press. Chang, P-C., and Akhtar, S. M. 1974. Simplified method of resealing inoculated cans of food. Can. Inst. Food ScI. Techno!. J. 6:294. Christiansen, L. N., Johnston, R. W., Kautter, D. A., Howard, J. W., and Aunan, W. J. 1973. Effect of nitrite and nitrate on toxin production by Clostridium botulinum and on nitrosamine formation In perishable canned comminuted cured meat. Appl. Mlcrobio!. 25 :357. Dyer, W. J. 1943. Rapid determination of sodium chloride in the presence of protein. Ind. Eng. Chem. Ana!. edn. 15 :439. Johnston, M. A., and Loynes, R. 1971. Inhibition of Clostridium botulinum by sodium nitrite as affected by bacteriological media and meat suspensions. Can. Inst. Food Techno!. J. 4:179. Johnston, M. A., Pivnlck, H., and Samson, J. M. 1969. Inhibition of Ciostridium botulinum by sodium nitrite in a bacteriological medium in meat. Can. Inst. Food Techno!. J. 2:52. Kamm, L., McKeown, G. G., and Smith, D. M. 1965. New colorimetric method for the determination of the nitrate and nitrite content of baby foods. J.A.O.A.C. 48:892. Nordin, H. R. 1969. The depletion of added sodium nitrite in ham. Can. Inst. Food Techno!. J. 2:79. Olsman, W. J., and Krol, B. 1972. Depletion of nitrite in heated meat products during storage. Proc. 18th Meeting, Meat Research Workers, Guelph, Can., pp. 409-415. PerillO, J. A.. Whitinll', E., and Bashford. T. E. 1967. Observations of the inhibition of vegetative cells of Clostridium sporogenes by nitrite which has been autoclaved in a laboratory medium, discussed in the context of sub-lethally processed cured meats. J. Food Techno!. 2:377. Perrin, C. H. 1953. Rapid modified procedures for determination of Kjeldahl nitrogen. Anal. Chern. 25 :968. Pivnlck, H. 1970. The inhibition of heat-damaged spores of C!. botulinum by sodium chloride and sodium nitrite Symp. MlcrobioI. Semi-preserved Foods. Prague. Pivnick, H., and Bird, H. 1965. Toxinogenesis by Clostridium botulinum types A and E in perishable cooked meats vacuum-packed In plastic pouches. Food Techno!. 19:132. Pivnick, H., and Petrasovlts, A. 1973. A rationale for the safety cf canned shelf-stable cured meat: Protection = Destruction + Inhibition. Proc. 19th Meeting, European Meat Research Workers, Paris. Plvnick, H., and Thacker, C. 197090. Effect of sodium chloride and pH on initiation of growth by heat-damaged spores of Clostridium botulinum. Can. Inst. Food Techno!. J. 3:70. Plvnick, H., and Thacker, C. 1970b. Time-saving system for enumeration of anaerobes by the MPN method. Can. Inst. Food Techno!. J.3:9. Pivnick, H., Barnett, H. W., Nordin, H. R., and Rubin, L. J. 1969. Factors affecting the safety of canned, cured, shelf-stable luncheon meat inoculated with Clostridium botulinum. Can. lost. Food Techno!. J. 2:141. Pivnlck, H., Johnston, M. A., Thacker, C., and Loynes, R. 1970. Effect of nitrite on destruction and germination of Clostridium botulinum and putrefactive anaerobes 3679 and 3679 h in meat and in buffer. Can. Inst. Food Techno!. J. 3:103. Roberts, T. A.. and Ingram, M. 1966. The effect of sodium chloride. potassium nitrite and sodium nitrite on the recovery of heated bacterial spores. J. Food Technol. 1:147. Sllliker, J. H., Greenberg, R. A., and Schack, W. R. 1958. Effect of indiVidual curing ingredients on the shelf stablllty of canned comminuted meats. Food Techno!. 12:551. Stumbo. C. R., Murphy, J. R., and Cochran, J. 1950. Nature of thermal death time r,llrves for P.A. 3679 and Clostridium botulinum. Food Techno!. 4 :321.
--------Received October 25, 1973
J. Inst. Can. SCI. Techno!. Aliment. Vol. 7, No.3, 1974
Abstract Canned shelf-stable luncheon meat containing varying amounts of sodium nitrite, prepared according to a commercial formula, was processed at 110°C to Fo = 0.4 and held at 35°C until nitrite was no longer detectable. The meat was then inoculated with spores of Clostridium botulinum separately heated to Fo = 0.4 and the cams were incubated at 30°C. Swelling of cans with concomitant toxin production was related directly to the size of the inoculum and inversely to the concentration of nitrite at time of manufacture. Meat containing 200 ppm of nitrite at time of manufacture (less tham 2 ppm when inoculated) inhibited 1.43 !OglO more spores than meat that was manufactured without nitrite. It appears that an inhibitor, which we call Perigo-type Factor (PTF), is formed during commercial processing of shelf-stable luncheon meat that contains nitrite.
Resume De la viande it collation en conserves contenant diverses quantites de nitrite de sodium, preparee selon une formule commerciale, a ete chauffee a 110°C jusqu'a Fo = 0.4 et maintenue a 35°C jusqu'a destruction complete des nitrites. La viande a ete ensuite inoculee avec des spores de Clostridium botulinum chauffees separement jusqu'a Fo = 0.4 et les boites de conserves ont ete incuooes a 30°C. Le gonflement des boites avec la production concomitante de toxine a ete relie directement a la taille de !'inoculum et inversement it la concentration de nitrite au moment de la fabrication. La viande qui contenant 200 ppm de nitrite au moment de la fabrication (moins que ~ ppm it l'inoculation) a inhibe 1.43 10glO plus de spores que la viande fabriquee sans nitrite. II nous apparait qu'un inhibiteur, que nous appelons Facteur du type Perigo (FTP), est forme au cours de la preparation commerciale de viande a collation en conserves qui ne contient pas de nitrite.
Introduction In canned meat one spore of Clostridium botulinum may cause food poisoning if it survives the heatprocess, germinates, multiplies and produces toxin. Interruption of anyone of the above processes will prevent toxin formation. Thus, no toxin will be formed if sufficient heat is applied to kill all spores. Even if some spores survive the heat-treatment, the meat will still he safe provided that the spores are p.revented from: (a) germination; or (b) multiplicatIOn after germination; or (c) toxin production despite multiplication. Shelf·stable canned cured meat receives a heat process ·of Fo = 0.1 - 0.6 which may permit spores to survive, yet cured meats are stable and safe. Thus, there must be other factors, in addition to the heating process, which contribute to safety. These factors are generally thought to be salt (sodium chloride) and sodium nitrite. The concentrations of salt used in such products are not inhibitory to undamaged spores of 1 ~crobiology Division, Research Laboratories, Food Directorate, nealth Protection Branch, Health and Welfare Canada, Ottawa, Ontario. 2 Canada Packers Limited, Research and Development Laboratories, Toronto, Canada. Can. lnst. Food Sci. Technol. J. Vol. 7, No.3, 1974
a. botulinum, bnt are inhibitory to the heat-damaged spores that survive thermal processing (Pivnick and Thacker, 1970a.; 1970b). The exact mode of action of nitrite is not well known, but its contribution to the stability of shelf-stable meat is well documented (Silliker et al.) 1938; Pivnick et al., 1969). At sufficient concentratron and appropriate pH, nitrite is inhibitory to growth of a. botulinum. However, about 75-85% of the nitrite is lost during thermal processing of shelf-stable meat, and the loss continues during storage at ambient temperature (Nordin, 1969; Olsman and Krol, 1972). These losses mitigate against consideration of a major role for nitrite per se in inhibiting a. botulinum in shelf-stable products. A possible explanation for this paradox was presented by Perigo et al. (1967) who found that heating nitrite in a bacteriological medium resulted in the formation of an antimicrobial factor - the so·called Perigo factor or Perigo inhibitor. The phenomenon is called the Perigo effect. Because of apparent misconceptions in citing our reports, we have presented in the following paragraph the essence of our work and that ·of others regarding the Perigo effect in meat. Johnston et al. (1969), in experiments similar to those of Perigo et al. (1967), confirmed that a potent antibotulinal factor (Perigo inhibitor) could be formed when nitrite was heated with a bacteriological medium similar to that used by Perigo, but Johnston and Loynes (1971) showed that some other types of media heated with nitrite did not form an inhibitor. Moreover, Johnston et al. (1969) found that if meat was added to medium containing the Perigo inhibitor, the inhibitory activity was lost. For this reason we now call the inhibitor that is formed in meat, the "Perigo-type Factor (PTF)". In further studies, Johnston et al. (1969) canned shelf-stable meat with 0-200 ppm of nitrite and, after thermal processing, homogenized the meat with an equal weight of water and dispensed it in test tubes. These tubes were challenged with 6-8 spores of a. botulinum. All tubes permitted growth and toxinogenesis; there appeared to be no inhibitory activity. However, recognizing that there might be a slight Perigo-type effect not demonstrable by even this very small challenge, they supplemented similarly-prepared homogenates with sterile nitrite to obtain 80,100,120 and 150 ppm of nitrite and challenged each tube with 6-8 spores. They did not find inhibitory activity in homogenates with 80 ppm of supplemental nitrite regardless of the nitrite added to the meat at time of manufaeture. However, when supplemented with 100 and 120 ppm of nitrite, the homogenates obtained from meat manufactured with nitrite were more inhibitory than hom 0 g e nat e s 209
Table 1.
Residual sodium nitrite in canned pork luncheon meat held at 35°C. Lot 2.
Nitrite in formulation (ppm)
Nitrite after weeks at 35°C (ppm)
NDa 8.9 17.3 31.1 53.5
0 50 100 150 200
2
4
6
14
ND 3.0 8.7 12.0 22.1
ND 2.0 6.2 8.5 12.0
ND 1.6 3.4 4.7 7.5
ND 0.6 0.8
0
l.l
2.1
a Not done
obtained from meat manufactured without nitrite. Supplementation with 150 ppm prevented growth even in meat manufactured without nitrite. They concluded: "The nitrite that disappears during normal thermal processing of meat does not form a potent antimicrobial agent although under special conditions (small numbers of spores and high concentrations of supplemental nitrite) some inhibitory activity could be demonstrated." Ashworth and Spencer (1972) confirmed the results of Johnston et al. (1969); they too found that ment heated without nitrite required more free nitrite (added after heating) than meat heated with nitrite to inhibit identical inocula. Their conclusions were similar to those of Johnston et al. (1969) : "from the work reported here no evidence has been obtained which would indicate that a Perigo effect is likely to be involved, under practical conditions, with the safety and stability of canned cured meats." It appeal'ed to us that confirmation of PTF l'equired conditions prevailing in shelf-stable cured meat. These could be better approximated by inoculating, with spores that had survived normal thermal processing, into meat with a low residual concentraHan of nitrite. This would permit detection of a Perigo effect without a superimposed effect of residual free nitrite. Using this approach we now report the presence of PTF in meat made with nitrite and challenged with heat-damaged spores of C. botulinu1n. At the time of challenge the residual nitrite was less than 2 ppm. Table 2.
Cumulative number of swollen cans out of 20 cans inoculated. Lot 1.
Nitrite a in formulation (ppm) 0 100 200 300
Spores per can 22 220 2,200 22 220 2,200 290 2,200 22,000 220 2,200 22,000 220,000
Weeks at 30°C 1 0 0 4 0 0 0 0 0 0 0 0 0 0
2 2 9 13 0 4 13 2 8 19 1 3 8 9
4
5
9 18 20 2 20 18 18 19
_b
12 12 18 20
3 8 16 18 0 18 18 10 18 20 6 10 18 19
a Residual nitrite at time of inoculation was No further change.
b
210
L.
6
2
4
20 19 20
20
16 15 18
17 18 19
2 ppm.
7
18 19 20
Materials and Methods Can,ned Meat. Cans (300 x 308) of luncheon meat,
varying only in the level of sodium nitrite, were processed according to industrial practice. The formula for a 100 pound batch was: Meat Pork meat (25% fat) 61.0 lb. 10.0 lb. ingredients Pork head meat (15% fat) Pork back fat (91% fat) 8.5 lb. Water (cold tap) 16.5 lb. Corn starch 4.0 lb. 2.3 lb. !i~'i;.i:':~?:A"~" Sodium chloride Sufficient sodium nitrite was added to obtain concentrations shown in Tables 1 and 2. The meat ingredients were ground through a 3/16" holed plate using a Weiler grinder and then blended for 5 min. in a Ruffalo mixer (1000 pound capacity). The temperature of the mix was -1.7°C after blending. The mix was divided into equal sized portions and stored in plastic containers at -5.6°C until used, a maximum of three days. Two mixes were prepared and used for the two experiments described in Lot 1 and r~ot 2 under Results and Discussion. Each portion of the meat ingredients was reground throngh a 3/16" holed plate using a Biro grinder (7.5 hp) and then transferred to a Buffalo mixer (100 ponnd capacity) and the salt, starch and solution of sodium nitrite were added while mixing. A vacuum of 635 mm Hg was applied and the portion was mixed for 5 min. The temperature after mixing was -1DC. The emulsion was transferred to a Peerless stuffer, stuffed into 300 x 308 cans, and capped nnder 660 mm Hg vacuum. The cans were retorted in steam at 110°C for 85 min. (Fo = 0.4), cooled and stored at 35°C. Samples were withdrawn periodically for nitrite determination and, when the level in tbe meat made with tbe highest concentration of nitrite bad decreased to 2 ppm or less, all remaining cans were withdrawn and challenged. Inoculu1n, Challenge and Incubation. Spore suspensions of C. bof;ulinu1n 13983B were prepared according to Johnston et al. (1969). The spores were heatdamaged (Fo 0.4, 100°C for 50 min.) in a particlefree meat extract. This extract was prepared aseptically by homogenizing radiation-sterilized raw meat (Johnston and J,oynes, 1971) in sterile water, (1:4), then removing: meat particles by filtration through cheese doth and centrifuging. The pH of the extract was 6.2. Sodium nitrite (150 ppm), when required, and sodium chloride (4.5%) were added aseptically to the supernatant fluid. Viable spores were enumerated in Prickett tuhes using Wynne's agar (Difco) layered with Vaspar. Tbe heated spore suspension was diluted in sterile distilled water to obtain a known number of viable spores. Then 0.1 ml of suitable dilutions was introduced with a syringe and needle into each can of meat through a hole aseptically pierced in the lid. The inoculated cans were resealed (Chang and Akhtar, 1973) and incubated at 30°C. The cans were examined weekly, and swollen cans were removed and tested for toxin. Some cans of meat were inoculated with 0.1 ml
=
J.
Inst. Can. Sc,l. Technol. Aliment. Vol. 7, No.3, 1974
Table 3.
Effect of size of inoculum and nitrite in fonnulation on time for 50% of inoculated cans to swell (tso). Lot 1.
Nitrite a in fonnulation (ppm)
Spores per can 22 >l1 b >11 NDc ND
0 100 200 300
220
2,200
22,000
1.6 1.9 2.5 3.2
1.2 1.4 1.7 2.5
ND ND 1.3 1.6
220,000 ND ND ND 1.5
a At time of inoculation, meat contained L2 ppm NaN0 2• tso in weeks. c Not done.
b
sterile water and incubated as controls; none of the controls swelled. Toxin Test. Toxin was detected by methods described bv PivniCk and Bird (1965). Chemical Analyses. Moisture, fat and dextrose equivalent were determined by the official methods (A.O.A.C., 1970). Protein was determined by a rapid Kjeldahl procedure (Perrin, 1953). Salt was extracted according to D~Ter (1943) and determined electrometrically with a Model 41 Titralyzer (Fisher Sci. Co.). Nitrite at time of manufacture was determined by the official method (A.O.A.C., 1970) and in meat held at 35°C, by the method of Kamm et al. (1965). In our experience, the Kamm method is more suitable for determining nitrite at less than 10 ppm in meat.
Results and Discussion Two lots of canned meat were produced and challenged. Lot 1 c'Ontained 0-300 ppm of nitrite and Lot 2 contained 0-200 ppm. In other respects the two lots were very similar) 57.3 - 60% moisture; 23 - 25% fat; 10.8 - 12.3% protein; 3.7 - 4.2% dextrose equivalent, 2.2 - 2.4% Halt and 3.7 - 3.9% brine (gm salt/gm salt gm water) X 100. Depletion of Nitrite. Meat stored at 35°C was tested at intervals for residual nitrite. In Lot 1, meat made with 300 ppm of nitrite contained 2.2 ppm after 15 weeks. In IJot 2, the highest concentration of nitrite used was 200 ppm; it declined to 2.1 ppm in 14 weeks (Table 1). The decay of nitrite in Lot 2 during the first six weeks followed a first order reaction; for this period an equation, log ColC = 0.126t, was obtained by averaging the slopes of the regression lines for the 4 levels of nitrite (S.E. < 10%). In the equation, Co = the concentration of nitrite at the beginning of incubation and C = the concentration at time t in weeks.
+
Table 4.
Effect of inoculum on toxinogenesis in luncheon meat made with varying amounts of nitrite. Lot 2.
Nitrite a in fonnulation (ppm)
o
50 100 150 200
Spores per can
Total toxic
8
16
32
64
128
Inoculated
Ib 1
6
.'3 6 2 4
9 7 5
12 9 10 7
33/80 25/80 18/80 21/80 2/80
o o 1
2 1
3
o
o
7 1
o
a ~2 ppm at time of inoculation. b [lumber of cans, out of 16 inoculated, that became toxic during 11 weeks at 30°C. Can. lnst. Food ScI. Technol. J. Vol. 7, No.3, 1974
Table 5.
Inhibition (In) due to salt and Perigo-type factor (PTF) in canned luncheon meat. Lot 2. NaN0 2 in Sporesa Ratio spores: Totalb Units fonn-
that
Added
Grew in salt
units
of In
ulation (ppm)
grew
Grew
Grew in salt plus nitrite
of In
due to PTF
54.3 35.3 22.6 27.6 2.0
83.1 112.3 175.3 143.8 1974.1
1.9 2.1 2.2 2.2 3.3
0.2 0.3 0.3 1.4
o
50 100 150 200
1.5 2.4 2.0 27.0
a Spores added at each nitrite level = 16(8+16+32+64+128) 3968. These spores had survived heating to Fo = 004. b One unit of In = inhibition of 1 loglO or 90% of spores.
=
The half-life was 2.4 weeks. The data for Lot 1 were less extensive and are not presented. Lnoculation amd Incu,bation of Meat. Lot 1 was inoculated with spores that survived heating to Fa = 0.4 in the meat extract (see lfaterials and Methods) with and without 150 ppm of nitrite. No appreciable difference waH f.ound between cans inoculated with the two preparations and the results were, therefore, combined. Most of the cans swelled within 4 weeks of inoculation and none swelled after 7 weeks; the experiment was terminated after 11 weeks. Table 2 shows the cumulative number of swollen -cans, all of which contained toxic meat. There is no evidence that the nitrite concentration added at the time of manufacture prevented the inoculum from growing and causing swelling; one possible exception is found by comparing an inoculum of 22 spores in meat manufactured with 0 and 100 ppm of nitrite. There was, however, an indication that nitrite did have an effect on the time for swelling to occur. Table 3 shows that as the nitrite at the time of manufacture increased, the time for swelling of 50% of cans (t so ) at each inoculum level increased. As expected, an increase in size of inoculum decreased the time for swelling. As it was obvious that we had inoculated Lot 1 with too many spores to obtain quantitative information on the number of spores required to initiate growth, Lot 2 was inoculated with fewer spores than Lot 1. For Lot 2, spores were heated in the meat extract containing 150 ppm of nitrite, but not in extract without nitrite (cf. Lot 1). Table 4 shows the number of spores added and the number of cans that swelled. The results are similar to thm:e obtained with Lot 1, with the major exception that, at the end of the experiment, each group contained unswollen cans: this was probably due to the smaller number of spores inoculated per can in Lot 2. Quantitation of Inhibition of Outgrowth. Assuming that inhibition of the inoculum was due only to salt and PTF we have calculated the inhibition due to salt for Lot l' and for both factors for I.. ot 2 (Table 5). The extent of 'inhibition was calculated (Pivnick and Petrasovits, 1973) from the ratio: number of spores added/ number ·of spores that grew. The number of spores that grew was estimated by a Most Probably Number Method based on the ratio: cans inoculated/cans that 211
did not swell (Stumbo et al., 1950). Inhibition is expressed in units of In; each unit represents inhibition of outgrowth of 90% of spores (1 loglO) (Pivnick, 1970) . Salt in canned meat may inhibit outgrowth 'Of heat-damaged spores. Meat made with salt, but lacking nitrite, was inoculated in both experiments. In Lot 1, each of 20 cans was inoculated with 22 spores (total 440) and 9 of the 20 cans swelled. The estimated number of spores that grew was 11.86, and 328.14 (97.3%) failed to grow. The ratio of spores added/spores that grew (440/11.86) was 37.08 (loglO 1.57 1.6 units 'of In). In Lot 2 (see Table 5), salt was responsible for inhibiting 98.63% of the added spores (1.9 units of In). For spores treated at Fo 0.4, this degree of inhibition is consif.1tent with that found by Pivnick and Thacker (19703) in 3.7 - 3.9% brine. Inhibition due to PTF was calculated by subtracting units of In due to salt alone from units of In due to salt PTF (Table 5). Nitrite added at concentrations 'Of 50-] 50 ppm to the product prior to thermal processing created a relatively small amount of PTF whereas 200 ppm of added nitrite produced sufficient PTF to cause 1.4 units of In. Two salient points emerge from these experiments: (1) spores that survive Fo = 0.4 are strongly inhibited by salt (cf. Pivnick and Thacker, 1970a; Roberts and lng-ram, 1966) ; and (2) there appears to be a threshold level of nitrite, somewhere between 150 and 200 ppm added before heat processing, which is necef.1sary to produce an appreciable amount of PTF. There is substantial evidence for the inhibitory activity of salt towards heat-damaged spores, but there is no supportive literature concerning inhibition of spores by PTF in meat in the absence of free nitrite. Cel·tainly, 150 ppm of nitrite added to raw meat inoculated with a. botulinum prior to processing to Fo = 0.6 cauf.1pd substantial inhibition of toxigenesis (1.64 units of In) (Pivnick et al., 1969; Pivnick and Petrasovits, 1973) and prevented spoilage in uninoculated packs processed to an Fo of about 0.1 (Silliker et al., 1958). However, when canned meat is processed, ther~ is always residual nitrite immediately after processmg, and for a period during storage (Nordin 1969; Christiansen et al., 1973). Assuming that most of the spores germinate within a day of processing (Pivnick et aT., 1970), it is quite likely that PTF and residual nitrite interact to prevent outgrowth. In model systems, Johnston et al. (1969) had to supplement meat heated with nitrite, with additional nitrite after heating, in order to demonstrate a PTF. Similarly, Ashworth and Spencer (1972) and Ashworth et al. (1973) demonf.1trated a PTF in meat heated with sufficient nitrite to have appreciable residual nitrite after heating. The recent finding by Ashworth et al. (1973) that PTF can be formed in pork during a simulated pasteurhdng process (20°C increased to 70°0 in 4 hr.) m.ay explain the inhibition of toxinogenesis by a. botul~num In inoculated, pasteurized, canned pork
=
=
+
212
=
incubated at 27°C (Christiansen et al., 1973). Indeed, "it seems not unreasonable to suggest that the presence of a thermally-derived inhibitor based on nitrite may be a major contributory factor to the safety and stability of pasteurized cured pork products" (Ashworth et al., 1973). Our results indicate that a similar statement may be made for cured, shelf-stable canned meats that are usually processed to obtain centre temperatures above 100°C. However, in such products, the inhibitory effect of salt against heat-damaged spores may be even more important than PTF.
References A.O.A.C. 1970. Official methods of analysis of the Association of Official Analytical Chemists. 11th Edition, Washington, D.C. Ashworth, J., and Spencer, R. 1972. The Perigo effect in pork. J. Food Techno!. 7 :111. Ashworth, J., Hargreaves, L. L., and Jarvis, B. 1974. The production of an antimicrobial effect in pork heated with sodium nitrite under simulated commercial pasteurization conditions. J. Food Technol. In Press. Chang, P-C., and Akhtar, S. M. 1974. Simplified method of resealing inoculated cans of food. Can. Inst. Food ScI. Techno!. J. 6:294. Christiansen, L. N., Johnston, R. W., Kautter, D. A., Howard, J. W., and Aunan, W. J. 1973. Effect of nitrite and nitrate on toxin production by Clostridium botulinum and on nitrosamine formation In perishable canned comminuted cured meat. Appl. Mlcrobio!. 25 :357. Dyer, W. J. 1943. Rapid determination of sodium chloride in the presence of protein. Ind. Eng. Chem. Ana!. edn. 15 :439. Johnston, M. A., and Loynes, R. 1971. Inhibition of Clostridium botulinum by sodium nitrite as affected by bacteriological media and meat suspensions. Can. Inst. Food Techno!. J. 4:179. Johnston, M. A., Pivnlck, H., and Samson, J. M. 1969. Inhibition of Ciostridium botulinum by sodium nitrite in a bacteriological medium in meat. Can. Inst. Food Techno!. J. 2:52. Kamm, L., McKeown, G. G., and Smith, D. M. 1965. New colorimetric method for the determination of the nitrate and nitrite content of baby foods. J.A.O.A.C. 48:892. Nordin, H. R. 1969. The depletion of added sodium nitrite in ham. Can. Inst. Food Techno!. J. 2:79. Olsman, W. J., and Krol, B. 1972. Depletion of nitrite in heated meat products during storage. Proc. 18th Meeting, Meat Research Workers, Guelph, Can., pp. 409-415. PerillO, J. A.. Whitinll', E., and Bashford. T. E. 1967. Observations of the inhibition of vegetative cells of Clostridium sporogenes by nitrite which has been autoclaved in a laboratory medium, discussed in the context of sub-lethally processed cured meats. J. Food Techno!. 2:377. Perrin, C. H. 1953. Rapid modified procedures for determination of Kjeldahl nitrogen. Anal. Chern. 25 :968. Pivnlck, H. 1970. The inhibition of heat-damaged spores of C!. botulinum by sodium chloride and sodium nitrite Symp. MlcrobioI. Semi-preserved Foods. Prague. Pivnick, H., and Bird, H. 1965. Toxinogenesis by Clostridium botulinum types A and E in perishable cooked meats vacuum-packed In plastic pouches. Food Techno!. 19:132. Pivnick, H., and Petrasovlts, A. 1973. A rationale for the safety cf canned shelf-stable cured meat: Protection = Destruction + Inhibition. Proc. 19th Meeting, European Meat Research Workers, Paris. Plvnick, H., and Thacker, C. 197090. Effect of sodium chloride and pH on initiation of growth by heat-damaged spores of Clostridium botulinum. Can. Inst. Food Techno!. J. 3:70. Plvnick, H., and Thacker, C. 1970b. Time-saving system for enumeration of anaerobes by the MPN method. Can. Inst. Food Techno!. J.3:9. Pivnick, H., Barnett, H. W., Nordin, H. R., and Rubin, L. J. 1969. Factors affecting the safety of canned, cured, shelf-stable luncheon meat inoculated with Clostridium botulinum. Can. lost. Food Techno!. J. 2:141. Pivnlck, H., Johnston, M. A., Thacker, C., and Loynes, R. 1970. Effect of nitrite on destruction and germination of Clostridium botulinum and putrefactive anaerobes 3679 and 3679 h in meat and in buffer. Can. Inst. Food Techno!. J. 3:103. Roberts, T. A.. and Ingram, M. 1966. The effect of sodium chloride. potassium nitrite and sodium nitrite on the recovery of heated bacterial spores. J. Food Technol. 1:147. Sllliker, J. H., Greenberg, R. A., and Schack, W. R. 1958. Effect of indiVidual curing ingredients on the shelf stablllty of canned comminuted meats. Food Techno!. 12:551. Stumbo. C. R., Murphy, J. R., and Cochran, J. 1950. Nature of thermal death time r,llrves for P.A. 3679 and Clostridium botulinum. Food Techno!. 4 :321.
--------Received October 25, 1973
J. Inst. Can. SCI. Techno!. Aliment. Vol. 7, No.3, 1974