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Fate of Listeria monocytogenes During the Souring of Ergo, a Traditional Ethiopian Fermented Milk
Fate of Listeria monocytogenes During the Souring of Ergo, a Traditional Ethiopian Fermented Milk M. ASHENAFI Department of Basic Sciences Awassa College of Agriculture Addis Ababa University PO Box 5 Awassa, Sidamo, Ethiopia ABSTRACT
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The growth potential of three strains of Listeria monocytogenes during the natural souring of raw milk into Ergo was studied to detennine the effect of souring and container smoking on their inactivation. All three strains of L. monocytogenes grew well in sterile milk in unsmoked containers, reaching counts > 107 cfulml within 24 h at ambient temperature. Smoking of containers decreased the growth rate of the strains for 24 h but had varying effects on the strains. For all test strains, the count increased slightly (1.0 to 1.7 log units) until 12 h during the souring of Ergo in unsmoked containers. After 12 h of souring, the counts decreased steadily to undetectable levels at 48 or 60 h. Smoking of containers increased the rate of inactivation of the test organisms, and inactivation was complete at 36 h. Because Ergo is made by the natural souring of raw milk, consumers must be made aware of the potential dangers of acquiring listeriosis from fresh Ergo from raw milk. (Key words: Listeria monocytogenes, Ergo, sour milk, fennented milk) Abbreviation key: LAB = lactic acid bacteria LSD = Lactobacillus-Streptococcus differenti~ ation medium. INTRODUCTION
In Ethiopia, a significant proportion of milk is consumed in its fennented state as Ergo. The fermentation, usually natural, is effected
Received May 3. 1993. Accepted August 24. 1993. 1994 J Dairy Sci 77:696-702
through the spontaneous proliferation of the initial raw milk flora. In most urban homes raw milk is left at ambient temperatures kept in warmer places to fennent. In rural areas, particularly among the pastoralists, raw milk is usually kept in a fennenting vessel, which is smoked with Acacia nilotica wood (17) and, in certain cases, is even inoculated with a small amount of old fennented milk. Smoking of containers with olive wood is common in the highlands. Contamination of raw milk with various substances and pathogens has been reported (9, 15, 18, 24, 26). The contamination of milk and Listeria other dairy products with monocytogenes is also well documented (19, 22). Listeria monocytogenes has long been recognized as a veterinary pathogen (13) that causes abortion in pregnant women and meningitis and encephalitis, particularly in neonates and immunocompromised hosts (20). Listeria monocytogenes is present in soil, decaying vegetation, and the intestinal tracts of various animals (10, 11). As a food pathogen, L. monocytogenes is nearly as important as Salmonella, Staphylococcus aureus, and Clostridium botulinum for the microbial safety of foods (14). Various food-related outbreaks of listeriosis have been due to the presence of L. monocytogenes in food products. Dairy products such as pasteurized milk and soft cheese have been associated with such outbreaks (6, 12). Inadequate sanitation, crosscontamination, and inadequate processing of raw milk can lead to contamination of products with Listeria. Therefore, raw milk is often suggested as a source of dairy product contamination (19). Reports on the microbiological quality and safety of milk and milk products from Ethiopia are rare (1, 2, 3). A previous study (5) on
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LISTERIA MONOCYTOGENES IN FERMENTING ERGO
growth potential of salmonellae in Ergo indicated that Salmonella strains were not completely inactivated at about 24 h of fermentation, the time at which Ergo is preferably consumed. Thus, the wholesomeness of Ergo has to be determined because it is prepared from raw milk without further processing. The aim of this study was to evaluate the growth potential and inactivation of L. monocytogenes during the making of Ergo. The impact of the smoking of the container, a traditional processing method, on the behavior of this microorganism was also assessed. MATERIALS AND METHODS Collection of Samples
Milk (2 L) was collected from a lactating cow from a dairy farm for every experiment. The milk was boiled in steam (lOO°C for 1 h) and distributed in 250-ml amounts in eight sterile screw-capped bottles, four of which were previously smoked by inversion over smoking olive wood splinters for 2 min. Milk samples from five cows were used separately in this study as replicates, and results are means.
697
final inoculum of about 106 cfufml. Milk in the other containers served as a LAB-free control. All samples were incubated at ambient temperature (around 25 ± 2°C). Analysis of Samples
Fresh inoculated milk in the different bottles was sampled (10 rnI) aseptically, and appropriate dilutions were spread on modified McBride agar (Oxoid) to determine the inoculum level of L. monocytogenes. The inoculum level of LAB was determined on LSD plates. Modified McBride agar plates were incubated at 32°C for 48 h, and LSD plates were incubated in an anaerobic jar (Oxoid) at 32°C for 48 h. Souring milk and controls were sampled (10 ml) at l2-h intervals until 60 h and similarly processed for the enumeration of the L. monocytogenes strains. The pH of milk samples was measured using a digital pH meter (model 10; Corning Glass Works, Medfield, MA). A volume of .9 ml of milk samples was titrated against .IN NaOH to determine the percentage of titratable acidity as lactic acid. RESULTS
Cultures
The following bacterial cultures were used in this study: L. monocytogenes strains WS 2300, WS 2301, and WS 2302. The cultures were isolated from dairy products and obtained from the culture collection of Bakteriologisches Institute, (Sudd. Versuchs-u. Forschungsanstalt fur Milchwirtschaft, Weihenstephan, Germany). For the initiation of lactic fermentation in milk, lactic acid bacteria (LAB) were isolated from a naturally soured Ergo on LactobacillusStreptococcus differentiation medium (LSD; Oxoid, Basingstoke, England). The isolates were suspended and kept in sterilized milk. Inoculation of Milk with Test Organisms
Listeria monocytogenes strains were separately inoculated into milk in two unsmoked and two smoked containers for a final inoculum of around 1()3 cfulml. Milk in one smoked and one unsmoked container was further inoculated with LAB suspension for a
Listeria monocytogenes WS 2300 grew rapidly in milk in unsmoked containers and reached counts > 107 cfufml within 24 h (Figure lA). Variations in counts of L. monocytogenes WS 2300 were marked; the coefficients of variation ranged between 11.1 and 19.9%. Counts declined after 48 h. Growth pattern and variation were similar for L. monocytogenes WS 2301 (Figure lB). Coefficients of variation ranged between 11.2 and 17%. Listeria monocytogenes WS 2302 showed minimum variation (CV <10%) at all times and tended to maintain high numbers throughout incubation (Figure lC). Listeria monocytogenes strains WS 2300 and WS 2302 proliferated faster, increasing by 3 log units in the first 12 h. A slight fall in pH was observed during the growth of L. monocytogenes WS 2300 in milk in unsmoked containers (Figure 2A). The pattern was similar for the other L. monocytogenes test strains (Figure 2, B and C). Mean decreases in pH of .8, 1.0, and .6 units occurred at 36 h during the growth of L. Journal of Dairy Science Vol. 77, No.3, 1994
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ASHENAFI
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Time (h) Figure I. Growth of Listeria monocytogenes strains WS 2300 (A), WS 2301 (B), and WS 2302 (C) in milk in unsmoked (0) and smoked (t.) containers and their inactivation in souring Ergo in unsmoked (e) and smoked (A) containers. Journal of Dairy Science Vol. 77, No.3, 1994
699
LISTERIA MONOCITOGENES IN FERMENTING ERGO
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Time (h) Figure 2. Changes in pH during the growth of Listeria monocyrogenes strains WS 2300 (A), WS 2301 (B), and WS 2302 (C) in milk in unsmoked (0) and smoked (~) containers and in souring Ergo in unsmoked (e) and smoked (A) containers. Journal of Dairy Science Vol. 77, No.3. 1994
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ASHENAFI
monocytogenes strains WS 2300, WS 2301, and WS 2302, respectively. Final pH and titratable acidity at 60 h remained >5.2 and <.45%, respectively, in all cases. In milk in smoked containers, pH and titratable acidity remained at about 6.0 and .20%, respectively, for the test strains throughout incubation. For L. monocytogenes WS 2300, however, pH fell to <6.0 at 60 h. The pH varied little among samples (CV <10%). Smoking of containers decreased the growth rate of the three test strains until 24 h but had varying effects on the strains. Strain WS 2301, which appeared to be the least sensitive to container smoking, had a bacterial count less than that of the control at 24 h of only .4 log units; the counts of strain WS 2302 decreased by .9 and .8 log units and strain WS 2302 by .8 and 1.3 log units at 12 and 24 h, respectively. However, variations in counts among samples were markedly high in strains WS 2301 and 2302 (CV >20%). The pH and titratable acidity remained almost unchanged during growth of strains WS 2301 and WS 2302. For strain WS 2300, however, pH was <6, and titratable acidity was >.2%, at 60 h. The count of all test strains increased slightly (1.0 to 1.7 log units) until 12 h during the souring of Ergo in unsmoked containers (Figure 1). After 12 h, the count became undetectable at 48 h for strains WS 2300 and WS 2302 and 60 h for strain WS 2301. The variation in counts among samples was, however, higher in all strains until 36 h (CV >30%). Smoking of containers increased rate of inactivation of the test organisms, and counts were reduced to undetectable levels at 36 h. Strain WS 2302 was inactivated at 48 h. Marked variations were observed among samples until 36 h in strains WS 2301 and 2302 (CV >30%). Rate of pH fall and increase in percentage of titratable acidity had quite similar patterns during the inactivation of the test strains in souring Ergo in unsmoked and smoked containers. Titratable acidity and pH reached >.55% and :5:4, respectively, at 12 h in all treatments. Differences in pH of souring milk in smoked and unsmoked containers ranged from .5 to .8 units at 12 h. Differences in percentage of titratable acidity were also higher at 12 h. Variations in pH and percentage of titratable acidity among samples in the various treatments were low (CV < 10%). Journal of Dairy Science Vol. 77, No.3. 1994
The lactic acid microflora during souring was dominated by coccobacillus-shaped lactobacilli (data not given). DISCUSSION
The three L. monocytogenes strains could reach > 107 cfu/ml in milk in unsmoked containers when the initial inoculum level was between 102 and 104 cfu/ml. Although smoking of containers appeared to decrease the rate of L. monocytogenes growth slightly in the first 24 h, the count was > 106 cfu/ml thereafter. The response of the strains to smoking was variable, but smoking did not seem to be effective in controlling L. monocytogenes growth in raw milk, although it appeared to interfere with acid production by the L. monocytogenes strains, as indicated by the higher pH at all times in milk in smoked containers. Inoculation of milk with Ergo starter cultures resulted in production of acid and subsequent drop in pH of milk. Although the strains used in this study proliferated markedly while the pH decreased to <5.0 in the first 24 h, count had declined slightly by 24 h, when pH fell to 4.0. However, a substantial number of microorganisms still survived. Earlier reports (16) indicated that L. monocytogenes could not grow at pH of slightly <5.5. Listeria monocytogenes was inactivated in fermentation of legumes at pH of 4.5 to 4.7 (4). Other workers (8) have reported the ability of L. monocytogenes to withstand a large variety of environmental conditions, including pH as low as 5.0. The strains in this study could, however, withstand pH as low as 3.9 for more than 24 h at 25 ± 2°C. However, these strains did not grow in souring milk at pH of <5.0, although other workers (21, 23) reported the ability of L. monocytogenes to grow at pH as low as 4.4 and 4.5. Smoking of containers resulted in the inactivation of the test strains in Ergo within 36 or 48 h. Because the amount of acid produced in fermenting Ergo in smoked containers was lower than that in unsmoked containers at least until 36 h and because the L. monocytogenes strains were not completely inactivated at 36 h at higher acidities in unsmoked containers, container smoking could be assumed to have a marked inhibitory effect on L. monocytogenes strains during the souring of Ergo.
liSTERIA MONOCYTOGENES IN FERMENTING ERGO
In most cases, household preparation of Ergo requires a l-d incubation at ambient temperatures (about 25°C). The milk coagulates within 24 h, and Ergo is usually consumed preferably at this time because of its good flavor. Observations in this study indicated that the L. monocytogenes test strains were not inactivated at 24 h and that the count ranged between 103 and 105 cfulml. By the time that the test organisms were inactivated, the Ergo was usually considered to be too sour to consume. Because Ergo is traditionally made by the spontaneous souring of raw milk, L. monocytogenes, which is introduced into raw milk from an infected cow or milking utensils, milkers, or even consumers, could reach a high number at the early stages of Ergo souring and could survive in 24-h Ergo, thus resulting in listeriosis following consumption. Despite the general assumption that the low pH in Ergo controls the proliferation of undesirable microorganisms, the dangers of listeriosis from fresh Ergo must not be underestimated. The milk samples in this study were initially steam heated at l00'C for I h before inoculation with the test organisms and LAB. Thus, the lactoperoxidase system in raw milk, which can contribute to the inactivation of pathogens, may have been inactivated (7). However, in other studies (25), pathogens survived spontaneous souring of milk. Thus, Ergo should be made by boiling raw milk, cooling it to ambient temperatures, and inoculating it with a small amount of starter from a 3-d-old Ergo that is free from pathogens but contains enough LAB to initiate the required souring. Smoking of containers may help suppress the growth of undesirable microorganisms in the initial stages of souring and in hastening their disappearance from the product. ACKNOWLEDGMENTS
The technical assistance of Haile Alemayehu and Tsigereda Bekele is acknowledged. REFERENCES I Ashenafi, M. 1989. Proteolytic, lipolytic, and fermentative properties of yeasts isolated from ayib, a traditional Ethiopian cottage cheese. SINET (Addis Ababa) 12:131.
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2 Ashenafi, M. 1990. Microbiological quality of ayib, a traditional Ethiopian cottage cheese. Int. J. Food Microbiol. 10:263. 3 Ashenafi, M. 1990. Effect of curd-cooking temperatures on the microbiological quality of ayib. a tradilional Ethiopian cottage cheese. World J. Microbiol. Biotechnol. 6:159. 4 Ashenafi, M. 1991. Growth of Listeria monocytogenes in fennenting tempeh made of various beans and its inhibition by Lactobacillus plantarum. Food Microbiol. 8:303. 5 Ashenafi, M. 1993. Fate of Salmonella enteritidis and Salmonella typhimurium during the fermentation of Ergo, a traditional Ethiopian sour milk. Ethiop. Med. J. 31:91. 6 Beckers, H. J., H. J. &kers. P.S.S. Soentoro, and E.H.M. DeUgou-van Asch. 1987. The occurrence of Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4: 249. 7 Beumer, R. R., A. Noomen, and E. H. Kampelmacher. 1985. The effect of lactoperoxidase system on reduction of Campylobacter jejuni in raw milk. Antonie Leeuwenhoek. 51 :501. 8 Connor, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Effect of temperature, sodium chloride. and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59. 9 Ewald. S. 1987. Enterotoxin production by Staphylococcus au reus strains isolated from Danish foods. Int. J. Food Microbiol. 4:207. 10 Farber, J. M., M. A. Johnston, V. Purvis, and A. Loit. 1987. Surveillance of soft and semi-soft cheeses for the presence of Listeria spp. Int. J. Food Microbiol. 5: 157. II Fenlon, D. R. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environment. J. Appl. Bacteriol. 59:537. 12 Aeming. D. W., S. L. Cochi, K. L. Macdonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A.C.V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New England J. Med. 312:404. 13 Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309. 14 Hoover, D. G .. K. J. Dishart, and M. A. Hennes. 1989. Antagonistic effect of Pediococcus spp. against Listeria monocytogenes. Food Biotechnol. 3:183. 15 Huyneh, C. H., and S. Huyneh. 1985. Microbial contamination of some foods in Morocco: etiological and epidemiological studies. Sci. Aliments 5:51. 16 Irvin, A. D. 1968. The effect of pH on the multiplication of Listeria monocytogenes in grass silage media. Vet. Rec. 82:115. 17 Kassaye, T., B. K. Simpson, J. P. Smith, and C. B. O'Connor. 1991. Chemical and microbiological characteristics of Ititu. Milchwissenschaft 46:649. 18 Korlath, J. A., M. T. Osterholm, L. A. Judy, J. C. Forfang, and R. A. Robinson. 1985. A point-source outbreak of campylobacteriosis associated with consumption of raw milk. J. Infect. Dis. 152:592. 19 Lund, A. M., E. A. Zottola, and D. J. Pusch. 1991. Comparison of methods for isolation of Listeria from raw milk. J. Food Prot. 54:602. Journal of Dairy Science Vol. 77, No.3, 1994
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20 Mclauchlin, J. 1987. Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacterial. 63:1. 21 Parish, M. E., and D. P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52:144. 22 Rodriguez, L. D., 1.F.F. Garayzabal, JAV. Boland, E. R. Ferri, and G. S. Fernandez. 1985. Isolation de micro-organismes du genre Listeria partir de lait crn destine 11 la consomrnation humaine. Can. 1. Microbial. 31 :938. 23 Sorrells, K. M., D. C. Enigle, and 1. R. Hatfield. 1989. Effect of pH, acidulant, time, and temperature on the
Journal of Dairy Science Vol. 77, No.3, 1994
growth and survival of Listeria monocytogenes. J. Food. Prot. 52:571. 24 Tibana, A., M. B. Warnken, M. P. Nunes, I. D. Ricciardi, and A.L.S. Noleta. 1987. Occurrence of Yersinia species in raw and pasteurized milk in Rio de Janiero, Brazil. 1. Food Prot. 50:580. 25 Uradzinski, J., S. Kafel, and J. Szteyn. 1985. Effect of heat treatment on survival of salmonellae in naturally souring milk. Moo. Weter. 41:658. 26 White, C. H. 1986. Occurrence of foodbome pathogens in dairy products J. Dairy Sci. 69(Suppl. 1): 224.(Abstr.)
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The growth potential of three strains of Listeria monocytogenes during the natural souring of raw milk into Ergo was studied to detennine the effect of souring and container smoking on their inactivation. All three strains of L. monocytogenes grew well in sterile milk in unsmoked containers, reaching counts > 107 cfulml within 24 h at ambient temperature. Smoking of containers decreased the growth rate of the strains for 24 h but had varying effects on the strains. For all test strains, the count increased slightly (1.0 to 1.7 log units) until 12 h during the souring of Ergo in unsmoked containers. After 12 h of souring, the counts decreased steadily to undetectable levels at 48 or 60 h. Smoking of containers increased the rate of inactivation of the test organisms, and inactivation was complete at 36 h. Because Ergo is made by the natural souring of raw milk, consumers must be made aware of the potential dangers of acquiring listeriosis from fresh Ergo from raw milk. (Key words: Listeria monocytogenes, Ergo, sour milk, fennented milk) Abbreviation key: LAB = lactic acid bacteria LSD = Lactobacillus-Streptococcus differenti~ ation medium. INTRODUCTION
In Ethiopia, a significant proportion of milk is consumed in its fennented state as Ergo. The fermentation, usually natural, is effected
Received May 3. 1993. Accepted August 24. 1993. 1994 J Dairy Sci 77:696-702
through the spontaneous proliferation of the initial raw milk flora. In most urban homes raw milk is left at ambient temperatures kept in warmer places to fennent. In rural areas, particularly among the pastoralists, raw milk is usually kept in a fennenting vessel, which is smoked with Acacia nilotica wood (17) and, in certain cases, is even inoculated with a small amount of old fennented milk. Smoking of containers with olive wood is common in the highlands. Contamination of raw milk with various substances and pathogens has been reported (9, 15, 18, 24, 26). The contamination of milk and Listeria other dairy products with monocytogenes is also well documented (19, 22). Listeria monocytogenes has long been recognized as a veterinary pathogen (13) that causes abortion in pregnant women and meningitis and encephalitis, particularly in neonates and immunocompromised hosts (20). Listeria monocytogenes is present in soil, decaying vegetation, and the intestinal tracts of various animals (10, 11). As a food pathogen, L. monocytogenes is nearly as important as Salmonella, Staphylococcus aureus, and Clostridium botulinum for the microbial safety of foods (14). Various food-related outbreaks of listeriosis have been due to the presence of L. monocytogenes in food products. Dairy products such as pasteurized milk and soft cheese have been associated with such outbreaks (6, 12). Inadequate sanitation, crosscontamination, and inadequate processing of raw milk can lead to contamination of products with Listeria. Therefore, raw milk is often suggested as a source of dairy product contamination (19). Reports on the microbiological quality and safety of milk and milk products from Ethiopia are rare (1, 2, 3). A previous study (5) on
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LISTERIA MONOCYTOGENES IN FERMENTING ERGO
growth potential of salmonellae in Ergo indicated that Salmonella strains were not completely inactivated at about 24 h of fermentation, the time at which Ergo is preferably consumed. Thus, the wholesomeness of Ergo has to be determined because it is prepared from raw milk without further processing. The aim of this study was to evaluate the growth potential and inactivation of L. monocytogenes during the making of Ergo. The impact of the smoking of the container, a traditional processing method, on the behavior of this microorganism was also assessed. MATERIALS AND METHODS Collection of Samples
Milk (2 L) was collected from a lactating cow from a dairy farm for every experiment. The milk was boiled in steam (lOO°C for 1 h) and distributed in 250-ml amounts in eight sterile screw-capped bottles, four of which were previously smoked by inversion over smoking olive wood splinters for 2 min. Milk samples from five cows were used separately in this study as replicates, and results are means.
697
final inoculum of about 106 cfufml. Milk in the other containers served as a LAB-free control. All samples were incubated at ambient temperature (around 25 ± 2°C). Analysis of Samples
Fresh inoculated milk in the different bottles was sampled (10 rnI) aseptically, and appropriate dilutions were spread on modified McBride agar (Oxoid) to determine the inoculum level of L. monocytogenes. The inoculum level of LAB was determined on LSD plates. Modified McBride agar plates were incubated at 32°C for 48 h, and LSD plates were incubated in an anaerobic jar (Oxoid) at 32°C for 48 h. Souring milk and controls were sampled (10 ml) at l2-h intervals until 60 h and similarly processed for the enumeration of the L. monocytogenes strains. The pH of milk samples was measured using a digital pH meter (model 10; Corning Glass Works, Medfield, MA). A volume of .9 ml of milk samples was titrated against .IN NaOH to determine the percentage of titratable acidity as lactic acid. RESULTS
Cultures
The following bacterial cultures were used in this study: L. monocytogenes strains WS 2300, WS 2301, and WS 2302. The cultures were isolated from dairy products and obtained from the culture collection of Bakteriologisches Institute, (Sudd. Versuchs-u. Forschungsanstalt fur Milchwirtschaft, Weihenstephan, Germany). For the initiation of lactic fermentation in milk, lactic acid bacteria (LAB) were isolated from a naturally soured Ergo on LactobacillusStreptococcus differentiation medium (LSD; Oxoid, Basingstoke, England). The isolates were suspended and kept in sterilized milk. Inoculation of Milk with Test Organisms
Listeria monocytogenes strains were separately inoculated into milk in two unsmoked and two smoked containers for a final inoculum of around 1()3 cfulml. Milk in one smoked and one unsmoked container was further inoculated with LAB suspension for a
Listeria monocytogenes WS 2300 grew rapidly in milk in unsmoked containers and reached counts > 107 cfufml within 24 h (Figure lA). Variations in counts of L. monocytogenes WS 2300 were marked; the coefficients of variation ranged between 11.1 and 19.9%. Counts declined after 48 h. Growth pattern and variation were similar for L. monocytogenes WS 2301 (Figure lB). Coefficients of variation ranged between 11.2 and 17%. Listeria monocytogenes WS 2302 showed minimum variation (CV <10%) at all times and tended to maintain high numbers throughout incubation (Figure lC). Listeria monocytogenes strains WS 2300 and WS 2302 proliferated faster, increasing by 3 log units in the first 12 h. A slight fall in pH was observed during the growth of L. monocytogenes WS 2300 in milk in unsmoked containers (Figure 2A). The pattern was similar for the other L. monocytogenes test strains (Figure 2, B and C). Mean decreases in pH of .8, 1.0, and .6 units occurred at 36 h during the growth of L. Journal of Dairy Science Vol. 77, No.3, 1994
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ASHENAFI
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Time (h) Figure I. Growth of Listeria monocytogenes strains WS 2300 (A), WS 2301 (B), and WS 2302 (C) in milk in unsmoked (0) and smoked (t.) containers and their inactivation in souring Ergo in unsmoked (e) and smoked (A) containers. Journal of Dairy Science Vol. 77, No.3, 1994
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LISTERIA MONOCITOGENES IN FERMENTING ERGO
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Time (h) Figure 2. Changes in pH during the growth of Listeria monocyrogenes strains WS 2300 (A), WS 2301 (B), and WS 2302 (C) in milk in unsmoked (0) and smoked (~) containers and in souring Ergo in unsmoked (e) and smoked (A) containers. Journal of Dairy Science Vol. 77, No.3. 1994
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monocytogenes strains WS 2300, WS 2301, and WS 2302, respectively. Final pH and titratable acidity at 60 h remained >5.2 and <.45%, respectively, in all cases. In milk in smoked containers, pH and titratable acidity remained at about 6.0 and .20%, respectively, for the test strains throughout incubation. For L. monocytogenes WS 2300, however, pH fell to <6.0 at 60 h. The pH varied little among samples (CV <10%). Smoking of containers decreased the growth rate of the three test strains until 24 h but had varying effects on the strains. Strain WS 2301, which appeared to be the least sensitive to container smoking, had a bacterial count less than that of the control at 24 h of only .4 log units; the counts of strain WS 2302 decreased by .9 and .8 log units and strain WS 2302 by .8 and 1.3 log units at 12 and 24 h, respectively. However, variations in counts among samples were markedly high in strains WS 2301 and 2302 (CV >20%). The pH and titratable acidity remained almost unchanged during growth of strains WS 2301 and WS 2302. For strain WS 2300, however, pH was <6, and titratable acidity was >.2%, at 60 h. The count of all test strains increased slightly (1.0 to 1.7 log units) until 12 h during the souring of Ergo in unsmoked containers (Figure 1). After 12 h, the count became undetectable at 48 h for strains WS 2300 and WS 2302 and 60 h for strain WS 2301. The variation in counts among samples was, however, higher in all strains until 36 h (CV >30%). Smoking of containers increased rate of inactivation of the test organisms, and counts were reduced to undetectable levels at 36 h. Strain WS 2302 was inactivated at 48 h. Marked variations were observed among samples until 36 h in strains WS 2301 and 2302 (CV >30%). Rate of pH fall and increase in percentage of titratable acidity had quite similar patterns during the inactivation of the test strains in souring Ergo in unsmoked and smoked containers. Titratable acidity and pH reached >.55% and :5:4, respectively, at 12 h in all treatments. Differences in pH of souring milk in smoked and unsmoked containers ranged from .5 to .8 units at 12 h. Differences in percentage of titratable acidity were also higher at 12 h. Variations in pH and percentage of titratable acidity among samples in the various treatments were low (CV < 10%). Journal of Dairy Science Vol. 77, No.3. 1994
The lactic acid microflora during souring was dominated by coccobacillus-shaped lactobacilli (data not given). DISCUSSION
The three L. monocytogenes strains could reach > 107 cfu/ml in milk in unsmoked containers when the initial inoculum level was between 102 and 104 cfu/ml. Although smoking of containers appeared to decrease the rate of L. monocytogenes growth slightly in the first 24 h, the count was > 106 cfu/ml thereafter. The response of the strains to smoking was variable, but smoking did not seem to be effective in controlling L. monocytogenes growth in raw milk, although it appeared to interfere with acid production by the L. monocytogenes strains, as indicated by the higher pH at all times in milk in smoked containers. Inoculation of milk with Ergo starter cultures resulted in production of acid and subsequent drop in pH of milk. Although the strains used in this study proliferated markedly while the pH decreased to <5.0 in the first 24 h, count had declined slightly by 24 h, when pH fell to 4.0. However, a substantial number of microorganisms still survived. Earlier reports (16) indicated that L. monocytogenes could not grow at pH of slightly <5.5. Listeria monocytogenes was inactivated in fermentation of legumes at pH of 4.5 to 4.7 (4). Other workers (8) have reported the ability of L. monocytogenes to withstand a large variety of environmental conditions, including pH as low as 5.0. The strains in this study could, however, withstand pH as low as 3.9 for more than 24 h at 25 ± 2°C. However, these strains did not grow in souring milk at pH of <5.0, although other workers (21, 23) reported the ability of L. monocytogenes to grow at pH as low as 4.4 and 4.5. Smoking of containers resulted in the inactivation of the test strains in Ergo within 36 or 48 h. Because the amount of acid produced in fermenting Ergo in smoked containers was lower than that in unsmoked containers at least until 36 h and because the L. monocytogenes strains were not completely inactivated at 36 h at higher acidities in unsmoked containers, container smoking could be assumed to have a marked inhibitory effect on L. monocytogenes strains during the souring of Ergo.
liSTERIA MONOCYTOGENES IN FERMENTING ERGO
In most cases, household preparation of Ergo requires a l-d incubation at ambient temperatures (about 25°C). The milk coagulates within 24 h, and Ergo is usually consumed preferably at this time because of its good flavor. Observations in this study indicated that the L. monocytogenes test strains were not inactivated at 24 h and that the count ranged between 103 and 105 cfulml. By the time that the test organisms were inactivated, the Ergo was usually considered to be too sour to consume. Because Ergo is traditionally made by the spontaneous souring of raw milk, L. monocytogenes, which is introduced into raw milk from an infected cow or milking utensils, milkers, or even consumers, could reach a high number at the early stages of Ergo souring and could survive in 24-h Ergo, thus resulting in listeriosis following consumption. Despite the general assumption that the low pH in Ergo controls the proliferation of undesirable microorganisms, the dangers of listeriosis from fresh Ergo must not be underestimated. The milk samples in this study were initially steam heated at l00'C for I h before inoculation with the test organisms and LAB. Thus, the lactoperoxidase system in raw milk, which can contribute to the inactivation of pathogens, may have been inactivated (7). However, in other studies (25), pathogens survived spontaneous souring of milk. Thus, Ergo should be made by boiling raw milk, cooling it to ambient temperatures, and inoculating it with a small amount of starter from a 3-d-old Ergo that is free from pathogens but contains enough LAB to initiate the required souring. Smoking of containers may help suppress the growth of undesirable microorganisms in the initial stages of souring and in hastening their disappearance from the product. ACKNOWLEDGMENTS
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