Microbiology of the Frankfurter Process: Salmonella and Natural Aerobic Flora

Salmonella senftenberg 775W added to frankfurter emulsion was killed during normal processing in the smoke house when internal product temperature was 71.1 C (160 F) or above. The thermal destruction point of S. senftenberg 775W in frankfurters (temperature at which no viable cells were detected) was a function of the length of time of the process rather than of the starting number of cells. Heating of frankfurters to 73.9 C (165 F) substantially reduced the total non-salmonella count. For total non-salmonella bacterial flora and salmonella, relatively little thermal destruction occurred below 43.3 C (110 F). The heating step can bring about a 7-log cycle decrease (10(8) to 10(1)/g) of bacteria present in the raw emulsion. The flora of this high-bacteriological-count raw emulsion was predominantly gram-negative rods. Variation in the number of bacteria (both total and salmonella) surviving at various temperatures during processing was attributed to slight variations in the temperature pattern of the smoke house during its operation. An integration process was devised which allowed calculation of exposure to temperatures above 110 F (43.3 C) on the basis of degree-minutes. Plots of degree-minutes versus log of surviving bacteria were linear. The salmonella plot had a greater slope than that of the total non-salmonella flora, indicating that salmonellae are more heat sensitive than the bacterial population as a whole. The predominant bacteria surviving the heating step were micrococci. These micrococci were able to increase in number in or on the frankfurters during storage at 5 C.     

Growth of Bacteria on Meat at Room Temperatures

The development of spoilage flora and the growth of individual psychrotrophs and pathogens on meat held at 20 or 30°C were studied. Under aerobic conditions psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C, but <20% at 30°C where they were displaced by species of Acinetobacter and Enterobacteriaceae which included both mesophilic and psychrotrophic strains. Mesophiles dominated the anaerobic spoilage flora at 30°C when clostridia were the major species, but at 20° the flora contained mesophiles and psychrotrophs in similar proportions and was dominated by Enterobacteriaceae. These results were largely predictable from the growth rate data for individual organisms.
Interactions between species occurred more frequently at 20°C than at 30°C. When pathogenic species were grown at 20 or 30°Cin competition with equal numbers of psychrotrophic spoilage organisms, no interactions were observed. When pathogens were grown in competition with high numbers of psychrotrophs, only Lactobacillus growing anaerobically inhibited Salmonella typhimurium and Escherichia coli , but other pathogens were inhibited to varying degrees depending on the competing species and the incubation conditions. In general, the degree of inhibition was greater at 20 than at 30°C and facultative organisms were more susceptible under anaerobic than aerobic conditions. It appears that the cumulative stresses of low pH, suboptimal temperatures and competition with large numbers of saprophytic organisms can inhibit many of the pathogens likely to be present on meat. The organisms least affected by the conditions on meat surfaces, Salmonella and Esch. coli , are likely to be the main hazards on meat of normal pH held at room temperatures.     

Destruction of Staphylococcus aureus during frankfurter processing.

We studied the thermal resistance of Staphylococcus aureus during frankfurter processing in respect to whether staphylococci are killed by the heating step of the process and whether heat injury interferes with the quantitative estimation of the survivors. With S. aureus 198E, heat injury could be demonstrated only when large numbers of cells (10(8)/g) were present and at a product temperature of 140 degrees F (60 degrees C). On tryptic soy agar and tryptic soy agar plus 7% NaCl media, at temperatures less than 140 degrees F, the counts were virtually identical; above 140 degrees F, the counts converged, with the organisms dying so rapidly that heat injury was not demonstrable. Heat injury was thus judged not to interfere with the quantitative estimation of staphylococci surviving the normal commercial heating given frankfurters. By using a combination of direct plating on tryptic soy agar and a most-probable-number technique, we detected no viable cells (less than 0.3/g) of several strains of S. aureus in frankfurters heated to 160 degrees F (71.1 degrees C). This temperature is compatible with the normal final temperature to which federally inspected processors heat their frankfurters and with the temperature needed to destroy salmonellae.     

Microbial Studies on Shelf Life of Cabbage and Coleslaw

The microbiology of a common commercial type of coleslaw was investigated with the objective of extending its shelf life at refrigerator temperature by delaying microbiological spoilage. Cabbage, its principal ingredient, had a total bacterial count of about 10⁵/g. Microbial growth in cabbage was prevented by storage at 1 C but not at 10 C or above. In coleslaw, the cabbage flora died and was replaced by the flora of the cultured sour cream contained in the dressing. At 14 C, the total count increased and the coleslaw deteriorated organoleptically. At 7 C, bacterial growth was suppressed but organoleptic deterioration occurred as rapidly as at 14 C. Thus, the deterioration was caused primarily by the physiological breakdown of plant tissue rather than by microorganisms, as was the original premise.     

Application of Replica Plating and Computer Analysis for Rapid Identification of Bacteria in Some Foods: II. Analysis of Microbial Flora in Irradiated Dover Sole (Microstomus pacificus)

This investigation was carried out to determine the nature of the microbial flora shifts in dover sole as a result of irradiation and storage at 6 C. The relationship was determined between the microorganisms which initially survive irradiation and those making up the final spoilage flora. A total of 2,723 isolates were examined by use of the replica-plating and computer analysis method. The spoilage of the unirradiated control samples during storage at 6 C was almost entirely due to the growth of Pseudomonas. This group, which occupied 25% of the fresh flora, grew to nearly 100% in 2 days of storage. In contrast, irradiation doses of 0.1, 0.2, 0.3, and 0.4 Mrad favored the growth of Achromobacter and yeasts. The Micrococcus, which survived radiation, did not grow at 6 C. At 0.5 Mrad, spoilage of fish samples was due entirely to the growth of yeasts.     

Nitrite loss and spoilage microflora development in chub-packed luncheon meat

Residual nitrite was lost from chub-packed luncheon meat during storage through both chemical breakdown and microbial consumption. The relative importance of these mechanisms in this pasteurized product was determined by the speed of development of the spoilage microflora, which is influenced by storage conditions. The nitrite half-life due to chemical loss was 13 d at 25°C and 36 d at 10°C. When microbial growth occurred these half-lives were reduced to 2.6 d and 21 d, respectively. Qualitative differences in the microflora that developed at these two temperatures (denitrifying Bacillus spp. at 25°C and non-denitrifying Streptococcus spp. at 10°C) account for the large temperature effect. Growth of Streptococcus spp. increased the rate of chemical nitrite loss in chubs by reducing the pH value. Nitrite did not inhibit the aerobic growth of either Bacillus or Streptococcus species associated with spoilage but did inhibit the anaerobic growth of Bacillus spp. This bacteriostatic effect of residual nitrite in anaerobic conditions will decrease during storage as nitrite level falls and oxygen penetrates the chub pack. Nitrite-mediated bacteriostasis does not obviate the need for refrigerated storage but does afford a real, if ephemeral, safeguard against spoilage occurring during short periods of temperature abuse.     

Conditions affecting growth and enterotoxin production by Staphylococcus aureus on temperature-abused chicken meat

Growth of Staphylococcus aureus at 15°C, with and without addition of representative spoilage bacteria, was studied in cooked, whole chicken meat and chicken broth. In the absence of competitors, the organism grew better in broth culture than on whole meat, but multiplied more slowly in broth when other organisms were present, even from twice the previous level of inoculum. The presence of competitors had no marked effect on the growth of Staph. aureus on whole meat. Enterotoxin A was not produced at 15°C on either whole meat or in broth, and occurred at 20°C only in pure culture. At 30° and 37°C, toxin was produced whether or not competitors were present. Toxin production by Staph. aureus appeared to be influenced more by growth temperature than by bacterial competition.     

Chemical and Microbiological Changes during Storage of Vacuum Packed Sliced Bacon

Summary: The micro-ecology of block cured, sliced, vacuum packed bacon has been studied during storage at 20 and 30°; high salt (8–12% NaCl, based on the aqueous phase) and low salt (5–7% NaCl) bacon were used. The dominant flora of all samples during the first 9 days' storage comprised catalase positive cocci. They persisted in the high salt bacon but, under lower salt conditions, group D streptococci and motile lactic acid bacteria became dominant.
Of the chemical changes studied, proteolysis, lipolysis and reduction of nitrate and nitrite were due largely to coagulase negative subgroup II staphylococci. Micrococci which predominated at 20° were capable of reducing nitrate to nitrite but of only slightly increasing free amino or fatty acids.
Inoculation of low count bacon with subgroup II staphylococci confirmed that this group was responsible for putrefaction in low salt bacon stored at temperatures above 20°. Similarly micrococci which tend to delay spoilage did not contribute much to the off odours and it was thought that the heterofermentative catalase negative organisms could contribute to typical sour spoilage.     

Bacteriology of Spoilage of Fish Muscle: I. Sterile Press Juice as a Suitable Experimental Medium

A sterile raw fish muscle press juice, diluted 1:4 with saline, has been prepared. This dilution greatly facilitated Seitz filtration and affected the spoilage properties of the medium only negligibly. At 5.5 C, the spoilage pattern of naturally contaminated diluted juice was almost identical to that of naturally contaminated fillets. This was shown by comparing the quantitative and qualitative aspects of the bacterial flora on the two substrates and by measuring the production of volatile reducing substances (VRS) and of trimethylamine (TMA). With the sterile raw muscle press juice, some preliminary data showed that individual members of the genera Achromobacter and Pseudomonas differ markedly in their spoilage capabilities: some grew but did not produce spoilage detectable either organoleptically or chemically; others gave rise to strong off odors and to high levels of VRS and TMA.     

Inhibition of Spores of Clostridium spp. by Sodium Nitrite

S ummary . Sodium nitrite heated in a laboratory medium was more inhibitory to spores of Clostridium spp. than nitrite added as a filter-sterilized solution to the same medium. Most spores remained refractile after inhibition for >3 months and some proved viable when inoculated into fresh nitrite-free medium. The inhibitory activity of heated nitrite medium was not stable indefinitely, growth sometimes occurred on re-inoculation with vegetative cells.     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

Effects of nisin on growth of bacteria attached to meat.

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.     

Thermal susceptibility of Streptococcus faecium strains isolated from frankfurters

The heat resistance of nine strains of Streptococcus faecium isolated from frankfurters was determined at 63 and 68 degrees C in brain heart infusion broth. Exponential-phase cultures (approximately 10(7) colonies/mL) were used as inoculants. The heat resistance of S. faecium DP2181, a moderately resistant isolate, was further examined in broth (55, 63, and 68 degrees C) and frankfurter emulsion (63 and 68 degrees C). The decimal reduction times (D values) were determined by regression. In broth, both time-temperature combinations resulted in a 3-4 log decline in bacterial numbers for the nine S. faecium strains tested. For S. faecium DP2181, the survivor curves deviated from the logarithmic order of death at all three heating temperatures. An initial slow period of death was evident at 55 degrees C and a resistant tail of organisms was observed at 55, 63, and 68 degrees C. The D55D63, and, D68 values for the logarithmic portion of the corresponding survivor curves were 105.6, 9.36, and 3.34 min, respectively. The survival of DP2181 was enhanced by the frankfurter emulsion. The results indicate that populations of S. faecium existed that were very heat resistant and could survive normal frankfurter processing if initially present in high numbers.     

Effects of nisin on growth of bacteria attached to meat

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.     

A note on microbial spoilage of sheep meat at ambient temperature

Shelf-life and microbial spoilage of sheep carcass meat at ambient temperature under commercial conditions were studied. The initial bacterial count of carcasses ranged 5.6-5.8 log/cm2. Staphylococcus spp. (48%) predominated in the initial microflora of carcasses followed by Micrococcus spp. (19%) and Escherichia spp. (12%). Microbial spoilage of carcasses occurred around 20 h when the bacterial count reached 8.0-9.0 log/cm2. Thus the shelf life of carcasses at ambient temperature was 19 h. The predominant micro-organisms at the time of spoilage were Escherichia and 'Acinetobacter-like' organisms. It was also observed that Enterobacter, Pseudomonas and Staphylococcus spp. could form a major part of the final flora. The presence of Escherichia and Staphylococcus spp. in higher percentages on the surface of carcasses at the time of spoilage presents the scope for health hazards.     

Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate.     

Formation of Antibacterial Factor by Heating System Containing Nitrite and Tryptone

A Perigo-type antibacterial factor (PTF) was produced when tryptone (a pancreatic digest of casein) medium was heated with nitrite at 121°C for 20 min. This PTF was inhibitory against Staphylococcus aureus, Bacillus subtilis and Clostridium botulinum, but was not against Escherichia coli and Salmonella typhimurium. The inhibitory activity varied with the concentration of nitrite (5 ~ 100 ppm) and tryptone (1, 2, 4%), and with pH (4, 5, 6, 7). The maximum inhibitory activity was observed when the medium containing 4% tryptone and 0.2% thioglycolate was heated with more than 50 ppm nitrite at pH 6. The tryptone was separated into three fractions by gel filtration and PTF was produced in every fraction, although the inhibitory activity was different in each. Our PTF might be unstable towards oxygen because its activity was lost completely by shaking for more than 16 hr.     

Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.     

Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.     

Effect of temperature on the microbial flora of herring fillets stored in air or carbon dioxide

The microbial development on fillets of herring from the Baltic Sea was studied at temperatures from 0-15 C in air or 100% carbon dioxide (96-100% CO2). The shelf-life of the fillets , defined as the time for the 'total aerobic count' to reach 1 X 10(7) c.f.u./g increased with decreased temperature from 1 d at 15 degrees C to 7 d at 0 degrees C (air). The corresponding values in CO2 were 3 d and 33 d, respectively. The initial flora of the herring fillets was dominated by Alteromonas putrefaciens and Pseudomonas spp. and so was the spoilage flora after storage in air (together 62-95% of the flora: all temperatures). Alteromonas putrefaciens predominated slightly at 2 degrees C to 15 degrees C, while Pseudomonas spp. dominated at 0 degrees C. The Pseudomonas flora was mainly split between Ps. fragi , Ps. fluorescens and a heterogenous group of unidentified Pseudomonas spp. The proportions were not influenced by temperature. In 100% CO2 at the time of spoilage the flora consisted of a significant number of Lactobacillus spp. Below 4 C the domination was almost complete while at 10 degrees C and 15 degrees C. Enterobacteriaceae, Vibrionaceae and Alt. putrefaciens was also found. It was concluded that the microbiological shelf-life of herring fillets is improved by refrigeration storage in 100% CO2 but for good results the temperature should not exceed 2 degrees C.