40

MICROBIOLOGICAL PROBLEMS INVOLVED IN PACKAGED

JOHN C. AYRES

DEPARThqENT OF DAIRY AND FOOD INDUSTRY 1 OWA STATE UNl VERSl TY, AMES ......

It has been known for many years that fresh red meats develop maxi- mum scarlet red color (oxymyoglobin) when packaged in films having high oxy- gen transmission rates such as are provided by cellulose acetate, polystyrene, and cellophane, but that these meats have better keeping qualities when stored in films that are less permeable to oxygen (Ramsbottom et al, 1951; Kraft and Ayres, 1952; Ramsbottom, 1954; Brissey, 1963). Unfortunately, consumers have reacted unfavorably to the color of incompletely oxygenated red meats. For this reason, plastics permitting some oxygen diffusion in packaged fresh meats have been advocated, even though it is recognized that the desired color development is at the expense of longer storage life. On the other hand, cured meats most satisfactorily retain the desired pink-red color of nitric oxide myoglobin and myochromogen when the film in which they are packaged is impermeable to oxygen. In the presence of light, which be- haves as a catalyst, these last two pigments are rapidly converted to metmyo- globin and other oxidized pigments. It is for this purpose that much effort is being made to package cured products in films im-permeable to oxygen.

Several techniques have been developed for providing satisfactory gas and light relationships for packaged cured meats. Among those most commonly used are:

I. Wax paperboard trays with transparent plastic windows. The fillinix Company developed this type of container in 1945 (viz Modern Packaging, 1945, 1951) to block the entrance of light except when the meat was to be viewed.

11. Entirely transparent packaging materials except for backing board or metal disk support.

a. Compression and use of a stretchable film such as polyethylene, rubber hydrochloride, etc.

b. Gas flushing and pouch packaging. LSuggested in the early 1950's by Ogilvy and Apes (1951) and &aft and Ayres (1953) and in general use since 1962.1

c. Film opaque to W or with a W absorber. As yet not used comercially.

d. Evacuation and use of:

1. Heat shrinkable film.

2. Form fitting plastics. In the last five years, packages having the contour of the products being packaged have gained in popularity. These form-fitting materials are quite popular for products such as wieners, sausages, etc. The most successful of all packaging films are those that fit around the product and then can be evacuated and shrunk as in the Cryovac process so that very little, if any, free air remains.

In addition to furnishing better color protection, these packaging innovations provide greater sanitation in the handling of meat products and thus improve the keeping quality of packaged meats. Neverthe- less, there are always a few that are present and ultimately grow. The nature and extent of such proliferation is a matter of importance. In the presence of proper moisture conditions, microorganisms initially present on the meat grow rapidly even at low temperatures. Unfortunately, there are many consumers who believe that cured meats packaged in the newer types of transparent, close fitting materials have an unduly extended storage life. To illustrate: one of our assistants wa8 searching in the market for a package of wieners that had not been in the refrigerated display case longer than two hours and asked one of the clerks arranging meats in a large supermarket. This employee indicated complete unconcern regarding the ages of the packages and expressed the opinion that age was unimpor- tant because the meat was packaged in these new types of packages and should keep.

It long has been recognized (Glage, 1901; Hichardson and Scherubel, 1909; Scott, 1936) that bacterial slime is produced only at high humidity and that desiccation serves as a preventative. Bacterial slime develops at 99% RH and above. At 98qb RH, growth becomes visible but colonies do not coalesce. At 96 1/2-97$ RII, the number of bacteria/cm2 reaches lo9 without growth be- coming visible to the naked eye.

I. Fresh Meats

Fresh raw meats contain fairly low salt concentrations so that they are in equilibrium with a surrounding atmosphere of 99% relative humidity (Scott, 1936). In other words, their equilibrium relative humidity is 99 or their average water content approximates a water activity (aw) value of .99 where the aw of pure water is 1.00. If the atmosphere surrounding the meat is relatively dry, moisture dissipates from the surface and the outside layer becomes crustlike or horny. While such surface dehydration or "case harden- ing" provides unfavorable conditions for bacterial multiplication, the meat becomes excessively tough, undesirable in appearance, and dark in color and so the use of several of the more highly permeable plastics such as cellulose acetate, polystyrene, or plain cellophane is not recommended even though these films provide good oxygenation of the myoglobin to oxymyoglobin.

With relatively impermeable films, the a, value remains constant throughout the expected storage life of the meat. Grganisms such as Pseudomonas, Achromobacter, and Shigella, as well as many other bacteria, require very high water activities for multiplication. Scott (1957) has shown that Pseudomonas will not produce slime at a, less than .98 and that most of the bacteria associated with the spoilage of meats will not grow at a, less that .96. Thus, it is unlikely that fresh meat products could be stored at a water activity low enough to eliminate growth of such microorganisms. 42.

Organisms of possible public health significance in fresh meats are exotic or, at most, play a very minor role. One report (Ayres and Adams, 1953) indicated that the usual load of putrefactive anaerobic spores present in packaged raw beef ranged between 0.7-6.0 per 100 g. while in another investigation (Steinkraus and Ayres, 1964) a mean spore count of 6.5 per gram of fresh beef trimmings was reported. According to McKillop (1959), the most probable numbers of in uncooked meats is approximately 0.03 per gram while -C. botulinum has not been isolated from fresh meats.

Various genera of the Enterobacteriaciae, including Salmonella, Paracolobactrum and Escherichia, have been repeatedly recovered from ground meats. Occasionally, coagulase positive Staphylococci also have been found. In addition, the recent incrimination of packaged smoked fish as the source of C. botulinum E has served as a warning to the entire food packaging indGstry of the potential danger from pathogens when adequate safeguards in sanitation are not practiced.

Since the early work of Glage in 1901, there have been many studies confirming that almost all of the microorganisms found on raw meats are at the surface and that the internal flesh is relatively free of microorganisms. Thirty-six years ago, Koran and Smith (1929) reported a negligible increase in numbers of organisms in the deep flesh of beef stored for two weeks at 5OC. This led Noran (1935) to conclude that spoilage by bacteria in the deeper parts of the flesh is unimportant compared with that taking place at the surface. Depending upon the tempera- ture of storage and the age of cut-up meats, bacterial proliferation serves as an indicator for the freshness of the meat. For example (Fig. l), sliced beef initially containing less than 100 bacteria per square centimeter of surface area keeps for nine to ten days when stored at 5OC, while steaks having an initial load of 20,000 crganism develop off-odor within six days. Soon after the detection of off-odor, these meats become slimy. All too often, incipient slime has been observed in packaged meats, particularly in ground beef. Initial loads of freshly ground beef, even when carefully handled, were in the range of 75,000-150,000 per gram. As meat is cut and passed through the grinder, the load becomes redistributed. When the grinder is not cleaned immediately before re-use, levels of a million or more per gram may be expected. =aft and Ayres (1952) and Ayres (1960) refer to con- centrations ranging from 60-100 million organisms per cm2 by the time that slime becomes manifest. There is a time lapse of only a few hours between recognition of off-odor and production of slime. The resident flora at this time consists almost entirely of pseudomonads although, to a lesser degree, Achromobacter, Micrococcus, and Flavobacterium may also be present. These organisms are psychrotrophs and persist even though the meat is stored at low temperatures.

11. Cured meats As indicated earlier, pigments in these meats rapidly undergo deterioration in the presence of oxygen and light and so the industry has developed films and packaging equipment that prevent the combined effects of these deleterious agents. 43, Fig. 1. Effect of initial bacterial populations on storage 1 i fe of sl iced and ground beef.

STORAGE TEMPERATURE 4.4OC

II I I I I I 0 2 4 6 8 IO I; TIME IN DAYS 44.

Several studies have been made of the microbiology of vacuum- packed sliced processed meats (Leistner, 1956, 1957; Allen and Foster, 1960; Brown and Schmucker, 1960; Ingram, 1960; Linderholm, 1960; Alm --et al, 1961; Hansen and Riemann, 1962). While these packaging procedures were uniformly reported to improve appearance and acceptability, reports differed concerning the influence that evacuation had on microbial proliferation. Leistner (1957), Ingram (1960) and Linderholm (1960) reported insignificant differences in total counts between stored vacuum-packed and nonvacuum products. According to Ingram, reducing the oxygen tension in packaged Wiltshire bacon did not limit the rate of bacterial multiplication since most of the micrococci derive "oxygen from nitrate instead of air and the lactobacilli do not need it." Likewise, Niven (1961) reported that most cured meat spoilage organ- isms are capable of growing either aerobically or anaerobically but that anaerobic packaging provides some extension of shelf life due to the inhibi- tion of growth of and and other aerobic microorganisms. However, the other investigators attributed differences in dominant flora to vacuum- packaging. According to Alm --et a1 (1961), this shift was from a mixture of Bacillus sp., Mcrococcus sp. and Lactobacillus sp. to almost a pure cul+,ure of Lactobacillus sp. Similarly, Allen and Foster (1960) reported a change from heterofermentative organisms to homofermentative Lactobacillus sp. Hansen (1960) and, later, Hansen and Riemann (1962) indicated that the flora in vacuum-packed sliced bacon held at 2OoC shifted from almost equal propor- tions of Lactobacillus (30), (30), and Micrococcus (40%) at two days to predominantly lactic acid bacteria (70%) at the expense of the streptococci (20%) and micrococci (19%), but with bacon held at 50C for six days, the flora was constituted entirely of Lactobacilli (75%) and Strep- tococci (25%) and after 24 days was in this same proportion.

The use of vacuum or gas packaging for frankfurters has become increasingly popular during the past decade. Inasmuch as a study had been made in this laboratory prior to that time (Ogilvy, 1950; Ogilvy and Ayres, 1951, 1953), it was deemed important to find if there was a change in the predominant flora as a result of the use of evacuation or gassing. As may be seen in Fig. 2, differences in counts on frankfurters in evacuated packages (dotted lines) were insufficient to conclude that vacuum packing caused any reduction in population. (The points for each of the two curves represent median values for five separate determinations; the vertical lines represent ranges for each test.) Initial counts of the control wieners (containing added cereal), while somewhat higher than those recorded in a previous study for an all meat frankfurter (Ogilvy and Ayres, 1953; viz Fig. 3 were again quite consistent, the usual surface load being within the range 7,000-25,000 per frankfurter. During the first four days in the refrigerated display case, these numbers remained at about the same level but by the sixth day, some of the samples had begun to show evidence that the resident flora was increasing and counts were higher in each subsequent test. While there was a slight reduction in numbers during the first three days of storage, it is not known if any significance can be attached to this phenomenon. It is quite possible that during handling and packaging a number of microorganisms that were merely adventitious contaminants may have come into contact with the wiener surface and found the substrate unsuitable for growth. 45.

Fig. 2. Changes in bacterial loads on vacuum packaged VS. conventional ly packaged frankfurters.

W m 2

I-o am LL 0

3t - CONTROL PACKAGE

STORAGE TEMPERATURE 4.4"C

21 I I I I I J 0 2 4 6 8 IO 12 TIME IN DAYS 46.

Fig. 3. Effect of carbon dioxide on growth of micro- organi sms on frankfurters stored at II.Ic°C.

[FROM OGILVY a AYRES(l953)l

I I I I I I 1 5 IO 15 20 25 30 TIME IN DAYS 47.

As early as 1882, Kolbe recognized that C02 interfered with the normal development of micrcorganisms on meat. Since that time, many inves- tigators have tried to account for the inhibition of microorganisms by C02 (Frankel, 1889; Jacobs, 1920; Pruchas --et al, 1922; Brown, 1922; Valley and Rettger, 1927; Killeffer, 1930; Tomkins, 1950; Moran, --et al, 1932; Callow, 1932; Haines, 1933; Scott, 1938; Mallman --et al, 1940; Golding, 1940, 1945; Ruyle et al, 1946; Ogilvy and Ayres, 1951, 1953; Hays and Riester, 1958; Hays -et -9a1 1959). Carbon dioxide improves the storage life of frankfurters. Organisms originally found on this type of meat were predominantly Gram positive types such as micrococci, bacilli and sarcinae being most numerous with lactobacilli, Gram negative bacteria, and yeasts being less com- monly encountered. As the level of CO2 was raised, growth of molds, micrococci and yeasts was progressively retarded while that of the lactic acid bacteria was not (Fig. 3). In atmospheres containing 50 to 96% COzp the surface flora was composed almost entirely of Lactobacillaceae and Microbacterium. The product, while tart, contained no organisms of public health significance.

Most cured meats contain sufficient sodium chloride, sugar and nitrate-nitrite to reduce the water-activity to a, = .90-.95. As may be seen in Fig. 4, Hansen and Riemann observed that requires an equilibrium relative humidity of 98 (aw = .98) for germination and does not grow below 94. Hence, for these meats - even though they may be vacuum or gas packaged - this organism does not pose a real threat. Cn the other hand, the Danish investigators reported that grew readily at a, values of .90-.95 and so was considered important as a possible pathogen. Cavett (1962) indicated that the incidence of coagulase positive S. aureus was low in vacuum packed sliced Wiltshire bacon but that these organisms were occasionally detected. Large numbers of these organisms are occasionally recovered from frankfurters (Plate 1). The majority of the S. aureus cells present in this particular petri plate were coagulase positive and conceivably constitute a real danger. It is recognized that recovery of such staphylococci - even from food implicated in a food poisoning outbreak - is only circumstantial evidence of the proper etiological agent (Hall --et al, 1963) and that proof requires dem- onstration of enterotoxin in the food. Still, coagulase positive staphylococci have often been associated with pyemic infections and, therefore, their pres- ence is viewed with grave suspicion. Since sufficient heat and salts are applied during processing of most cured meats to destroy almost all vegetative cells, the principal problem is one involving subsequent contamination. Ultimate microbial populations directly reflect sanitation during handling, packaging and storage. For example, Hansen and Riemann (1962) indicated the possibility of packing cooked ham with less than ten bacteria per slice, but in a study made by Miller (1960) of 113 retail packages of sliced cooked ham, counts ranging from fewer than 1000 to more than 52 million per inch2 of surface area were recovered. According to Casman et a1 (1963), meat products that are consumed shortly after heating or eaten without-- being cooked are rarely responsible for staphylococcal food poisoning; those generally involved have been heated before being subjected to careless handling and inadequate refrigeration. 48 Fig. 4. Comparison of water activity values for various meats with those limiting the germination or growth of microorganisms. Distilled water 1.0- (lowest aw permitting growth in artificial media)

Fresh meat Slime production Bacillus mycoides

Clostridium botulinum (germination) Pseudomonas (growth) (germination)

Achromobacter (growth)

- 0.95 Salmonella newport, Bacillus subtilis Bacillus cereus (growth) Clostridium botulinum (growth)

Frankfurter

Sausage Sarcina sp.

Sliced pressed ham Salami Micrococcus roseus Sliced bacon - 0.90 Staphylococcus aureus (anaerobic)

Cured pork Fermented sausage

Lower growth limit for several yeasts

Smoked pork Staphylococcus aureus (aerobic) Aspergillus niger

- 0.85

Aspergillus glaucus

Fermented sausage - - 0.80 Xeromyces with high salt content

(From Hansen & Riemann, 1962; also data from Scott, 1957) 49.

In conclusion, the length of storage life expected for fresh or cured meats will directly reflect the sanitation practiced during handling, packaging and storage. Adequate refrigeration will prolong the keeping quality of both products, but this safeguard alone is insufficient to prevent undesired bacteriological and chemical changes. Evacuation and the replace- ment of air by C02 may have salutary action for cured meats but the effect of water activity is probably of more importance. Since such meats are relatively free of microorganisms immediately after , the human factor is of con- siderable consequence. With proper sanitary precautions and the use of appropriate films, packaging techniques, and storage temperatures; it should be possible to extend the safe storage life of cured meats. It js advocated that fresh meats be kept at as low temperature and held for as short a time as possible.

Reference s

1. Allen, J. H. and Foster, E. M. Spoilage of vacuum-packed sliced processed meats during refrigerated storage. Food Research 25, 19 (1960).

2. Alm, F., Erichsen, I., and Molin, N. The effect of vacuum packaging on some sliced processed meat products as judged by organoleptic and bacteriologic analysis. Food Technol. -15, 199 (1961).

3. Ayres, J. C. Temperature relationships and some other characteristics of the microbial flora developing on refrigerated beef. Food Research -25, 1 (1960).

4. Ayres, J. C. and Adams, A. T. Cccurrence and nature of bacteria in canned beef. Food Technol. -7, 318 (1953). 5. Brown, W. On the germination and growth of fungi at various temperatures and in various concentrations of oxygen and of carbon dioxide. Ann. Botany 2, 257.

6. Brown, W. L. and Schmucker, M. L. The influence of low level gamma irradiation, antibiotic treatment, storage temperature and vacuum packaging on flavor and bacterial changes in cured bacon. Food Technol. -14, 92 (1960).

7. Callow, E. H. Gas storage of pork and bacon. I. Preliminary experiments. J. Soc. Chem. Ind. (London) -51, 116T. (1932).

8. Casman, E. P., McCoy, D. W. and Brandly, P. J. Staphylococcal Growth and Enterotoxin Froduction in Neat. Appl. Microbiol. -11, 1 (1963). 9. Cavett, J. J. The Microbiology of Vacuum Packed Sliced Bacon. J. Appl. Bact. 25, 2. (1962).

10. Frankel, C. Die Einwirkring der KohlensLure auf die Lebenstatigkirt der Microorganismen. Z. f. Hyg. -5, 332 (1889). 50.

11 * Glage, F. Ueber die Bedeutung der Aromabakterium fir die Fleischhygiene. Z. Fleisch-u, Milchhyg. -11, 131 (1901).

12. Gelding, N. S. The gas requirements of molds. 111. The effect of various concentrations of carbon dioxide on the growth of Penicillium rogueforti in air. J. Dairy Sei. -23, 891 (1940).

13. Golding, N. S. The gas requirements of molds. IV. A preliminary in- terpretation of growth rates of four common molds on the basis of absorbed gases. J. Dairy Sci. 737 (1945).

14. Haines, R. B. The influence of carbon dioxide on the rate of multiplica- tion of certain bacteria as judged by visible counts. J. SOC. Chem. Ind. (London) 52, 13T. (1933).

15. Hall, H. E., Angelotti, R. and Lewis, K. H. Quantitative detection of staphylococcal enterotoxin B in food by gel-diffusion methods. Public Health Reports (U.S. ) -78, 1089 (1963).

16. Hansen, N. H. Quality deterioration and bacterial growth in prepacked bacon. Publication No. 28, Danish Meat Research Inst. (Roskilde) 1960.

17. Hansen, N. H. and Riemann, H. Mikrobiologische Beschaffenheit von vorverpacktem Fleisch and vorvepackten Fleischwaren. Fleischwirtschaft -14, 861 (1962).

18. Hays, G. R., Burroughs, J. D. and Warner, R. C. Microbiological aspects of pressure packaged foods. 11. The effect of various gases. Food Technol. -13, 567 (1959).

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23. Kolbe, H. Antiseptische Eigenschaften der Kohlensaure. J. Prakt. Chem. -26, 249 (1882). 24. Kraft, A. A. and Apes, J. C. Post-mortem changes in stored meats. IV. Effect of packaging materials on keeping quality of self-service meats. Food Technol. -6, 8 (1952).

25. Leistner, L. Die Neuzeitliche Verpackung in der Fleischwirtschaft. I. Fleischwirtschaft -8, 422 (1956).

26. Leistner, L. Die Neuzeitliche Verpackung in der Fleischwirtschaft. 11. Fleischwirtschaft -9, 262 (1957). 51.

27. Linderholm, K. G. Bakteriologiska och hygieniska undersokningar av vakuumforpackade charkuterivaror. Nord. Hygienisk Tidskr. XLI (1-2), 17. (1960).

28. Mallmann, W. L., Zaikowski, L. and Ruster, M. The effect of carbon dioxide on bacteria with particular reference to food poisoning organisms. Mich. Agr. Exp. Sta. Bull -489 n.s. (1940). 29. McKillop, E. J. Bacterial contamination of hospital food with special reference to -C1. welchii. J. Hyg. 57, 31 (1959).

30. Miller, W. A. The microbiology of self-service, packaged square slices of cooked ham. J. Milk and Food Technol. -23, 311 (1960).

31. Moran, T. Post-mortem and refrigeration changes in meat. J. SOC. Chem. Ind. 54, Pt. 2 trans. 149T (1935).

32. Moran, T. and Smith, E. C. Post-mortem changes in animal tissues. The conditioning or ripening of beef. Gr. Brit. Dept. of Sci. and Ind. Res. H. M. Stationery Office. Food Invest. Bd. Special Rept. -36, (1929).

33. Moran, T., Smith, E. C. and Tomkins, R. G. The inhibition of mould growth on meat by carbon dioxide. J. SOC. Chem. Ind. (London) -51, 114T. (1932).

34. Ogilvy, W. S. Storage of meat in carbon dioxide atmospheres at tenperatures above freezing. (Unpublished Ph. D. thesis, Ames, Iowa, Iowa State Univ. Library) (19 50 ) .

35. Ogilvy, W. S. and Apes, J. C. Post-mortem changes in stored meats. 111. The effect of atmxpk-eres containing carbon dioxide in prolonging the storage life of frankfurters. Focd Technol. 5, ZOO (1951).

36. Ogilvy, W. S. and Apes, J. C. Post-mortem changes in stored meats. V. The effect of carbon dioxide on microbial growth on stored frankfurters and characteristics of some microorganisms isolated from them. Food Research -18, 121 (1953).

37. Prucha, M. J., Brannon, J. M. and hbrose, A. S. Does C02 in carbonated milk and milk products destroy bacteria? Univ. of Ill. Agr. Coll. and Exp. Sta. Bull. -256 (1922).

38. Ramsbottom, J. M. Meat-packaging criteria. Modern Packaging (Feb.) (1954).

39. Richardson, W. D. and Scherubel, E. F. The deterioration and commercial preservation of flesh foods. Second paper - The storage of beef at temperatures above the freezing point. J. Ind. Eng. Chem. -1, 95 (1901).

40. Ruyle, E. H., Pearce, W. E. and Hayes, G. L. Prevention of mold in kettled blueberries in No. 10 cans. Food Research -11, 274 (1946).

41. Scott, W. The grGwth of microorganisms on ox nuscle. 1. The influence of water content of substrate on rate of growth at -1OC. Australia J. Coun. Sci. Ind. Research -9, 177 (1936).

42. Scott, W. J. The growth of microorganisms on ox muscle. 111. The influence of 10 per cent carbon dioxide on rates of growth at -1OC. Australia J. Coun. Sci. Ind. Research -11, 266 (1938). 52.

43. Scott, W. J. Water relations of food spoilage microorganisms. Advances _.-in Food Research -7, 83. Academic Press Inc., New York, N. Y. 404 pp. (1957). 44. Steinkraus, K. H. and Apes, J. C. Incidence of putrefactive anaerobic spores in meat. J. Food Sci. -29, 87 (1964).

45. Tomkins, R. G. Studies of the growth of moulds. Proc. Roy. SOC. (London) ser. B -105, 375 (1930).

46. Valley, G. and Rettger, L. F. The influence of carbon dioxide on bacteria. J. Bact. 714, 101 (1927).

47. Williams, 0. B. and Furnell, H. G. Spore germination, growth and spore formation by Clostridium botulinum in relation to the water content of the substrate. Food Research -18, 35 (1953).

NEIL WEBB: ...... in introduction of Dr. H. E. Hall, who has prepared a paper for the group. -- A brief background on Dr. Hall, he spent 5 years in the U. S. Army in Clinical bacteriology and from there he moved to Boston City Hospital, a division of Hartford University, working with Dr. Nax Finland. He came South to Kentucky where he finished his academic work Ph D in 1955 and worked in bacterial allergies and as a research associate at the University of Kentucky. He later worked as a private laboratory technician in Akron, Ohio working on clinical mycology and bacteriology. After joining the Taft Center for sometime, he is Acting Chief of the Food Microbiological Section at the Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio. It is a privilege to present Dr. Hall.

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