Behavior of Listeria monocytogenes in the Presence of Gluconic Acid and During Preparation of Cottage Cheese Curd Using Gluconic Acid MOUSTAFA A. EL·SHENAWY and ELMER H. MARTH Department of Food Science
and the Food Research Institute University of Wisconsin-Madison Madison 53706 ABSTRACT
Unrestricted or minimally restricted growth of Listeria monocytogenes strain V7 occurred 1) at 13"C in tryptose broth with .125 or .25% gluconic acid or .1 to .3% glucono-delta-lactone, 2) at n"C in milk with .125 to 1.0% gluconic acid or .5 or 1.0% gluconoOIt""IV'lV"l
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Figure 6. Behavior of Listeria monocytogenes at 13'C
9
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--
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Figure 7. Behavior of Listeria monocytogenes at 13'C
in milk containing gluconic acid.
in broth containing glucono-delta-Iactone.
or .5%, GA did not affect the lag phase, but GT were increased to 110 h as concentration of acid was increased to .5%. Addition of .75% GA prolonged the lag phase to 2 d followed by growth with a GT of 14.5 b. Presence of 1.5% GA inhibited growth of the pathogen, but after 4 wk of incubation (the end of the experiment) the population increased slightly.
complete, inactivation of the pathogen. Complete inactivation after 3 wk of incubation was accomplished by 3.0% GDL (Figure 8). Survival of Listeria monocytogenes During Preparation of Cottage Cheese Curd
Cottage cheese curd was prepared from pasteurized skim milk inoculated to contain ca. 2.5
Behavior of Listeria monocytogenes In the Presence of Glucono-Delta-Lactone at 13'C
L. monocytogenes grew in unacidified broth (control) after less than 1 d with a GT of 3.6 h. Presence of .1 % GDL did not affect bacterial growth, whereas .2% delayed onset of growth to 1 d followed by a GT of 6.5 h. At .3%, GDL delayed onset of growth to 2 d, followed by a GT of 14 h. Concentrations of .4 or .5% GDL inhibited growth for up to 12 d followed by some increase in numbers. Presence of .75 or 1.0% of the lactone completely inhibited growth of the pathogen with some inactivation as the incubation progressed (Figure 7). Incubation of milk containing .5 or 1.0% GDL at 13°C allowed growth of L. monocytogenes after a lag phase of 2 and 3 d; GT were 7.2 and 10.3 h, respectively. The pathogen was inhibited for 2 wk followed by a small increase in numbers when milk contained 1.5% GDL. Presence of 2.0 or 2.5% GDL completely inhibited growth with some, but not Iournal of Dairy Science Vol. 73. No.6. 1990
10 8
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6 9 12 15 3 4 Days Weeks Control ---..-.5% ----a-- 1.0% 1.5% ~ 2.0% - - 0 - - 2.5% --....-
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Figure 8. Behavior of Listeria monocytogenes at 13'C in milk containing glucono-delta-Iactone.
1435
GLUCONIC ACID CONTROLS USTERIA
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_1)
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_____ 2
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Figure 9. Survival of Listeria monocytogenes during preparation of cottage cheese curd made with rennet, HeI, or gluconic acid. 0) Immediately after cutting, I) after temperature was increased to 48.9·C, 2) after temperature was increased to 54.4"C, 3) after temperature was increased to S7.2·C, 4) after 15 min of cooking, and 5) after 30 min of cooking. TA Tryptose agar, MLA McBride Listeria agar, and TSA = tryptose-salt agar.
=
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Points in manUfacturing process
Figure 10. Survival of Listeria monocytogenes in the whey during preparation of cooked cottage cheese curd made with rennet, HCI, or gluconic acid. Symbols and lines
are the same as in Figure 9.
=
x 1()6 cfulml L. monocytogenes/ml. Bovine rennet, HCI, or GA were used separately to coagulate the milk. The skim milk was free of L. monocytogenes before inoculation. After curd foonation, the pathogen was more concentrated in curd than in whey (Figures 9 and 10). Immediately after cutting rennet curd, the pathogen was recovered from curd or whey at a level similar to that initially in milk. With acid treatments, recovery of the organism was .5 and 1.5 orders of magnitude less in curd or whey, respectively, than the initial count of milk. After the temperature was raised to 48.9°C, numbers of the pathogen in rennet curd or rennet whey had not changed, whereas a further reduction in numbers (from that observed initially) occurred in curd and whey prepared with HCI or GA. After the temperature was raised to 54.4°C, the pathogen started to grow in the rennet curd, but numbers in rennet whey decreased somewhat. There was a decrease of 1 or 2 orders of magnitude, respectively, in curd made with HCI or GA, but there were no appreciable differences in numbers of the pathogen in accompanying wheys when tested by plating on TA. After the temperature was raised to 57.2°C,
numbers of the pathogen in rennet curd increased further but continued to decrease in rennet whey. Numbers in both types of acid curd decreased further; reduction in numbers in acid whey was greater than in acid curd. After 15 min of cooking, numbers of the bacterium in rennet curd decreased to the initial population; whereas the decrease in w~ey was about 1 order of magnitude. After the same treatment, there was a decrease of 3 to 4 orders of magnitude in population of acid curd with the reduction being greater in GA than in HCI curd. The pathogen was still detected (10 cfu/ml) in whey of HCI curd, whereas it was not detectable in GA whey with our method. At the end of cooking (30 min at 57.2°C) numbers decreased by about 1.5 and 2.5 orders of magnitude, respectively, in curd or whey from the rennet treatment. After completion of the cooking process, the pathogen was not completely eliminated from curd made with HCI, but it could not be detected in whey. No viable cells of L. monocytogenes could be detected in GA curd or GA whey when the cooking process was complete. Listeria monocytogenes was not recovered from GA curd after cold enrichment in TB for up to 8 wk. However, the pathogen was recovered through cold enrichment (after 6 wk) of Journal of Dairy Science Vol. 73,
No.6, 1990
1436
EL-SHENAWY AND MARnI
HCI whey taken at the end of cooking and (after 4 wk) from GA whey taken after 15 min of cooking. Numbers of L. monocytogenes in curd or whey plated on TA, MLA, or TSA were a~ proximately the same until immediately after cutting the curd After the temperature was increased to over 48.9°C, differences in counts obtained with the different media began to increase. Differences in numbers of the bacterium when plating was on TA and MIA were always small, but this was not true between TA or MLA and TSA. DISCUSSION
Gluconic acid and GDL are the most common acid and acidogen products suggested for use in making certain kinds of cheese. Our results indicate that GA and GDL at various concentrations can affect behavior of L. monocytogenes. The effect depended, in part, on incubation temperature. The pathogen was more susceptible to effect of GA or GDL at the higher (35°C) rather than lower (13°C) temperature. For example, .75% GA in TB completely inactivated the pathogen after 5 d at 35°C, whereas the same concentration or more (1.0%) of the acid only inhIbited growth of the pathogen at 13°C. When the medium was milk, 1.5% GA completely inhIbited growth of the bacterium and caused some decrease in population at 35°C, whereas the same concentration of GA permitted a small increase in numbers at 13°C. The same was true with GDL in TB or milk. As expected, GA became more effective as the concentration was increased; the higher the concentration, the lower the pH and the greater the inhibition. For example, .125% GA in TB at 35°C gave a pH of 5.9 and did not affect growth of the pathogen (Figure 1). However, when the concentration of GA was raised to .25%, the pH was reduced to 5.3, the lag phase was prolonged from 3 to 6 h, and the GT was increased from 54 to 90 min. Listeria monocytogenes was more susceptible to effects of GA or GDL in TB than in milk. For example, .75% GA in TB completely inactivated the bacterium after 5 d at 35°C; however, in milk, 1.5% GA failed to cause complete inactivation at 35°C. This can be explained by differences in the pH achieved in the two media. In the first instance, .75% GA in Journal of Daily Science Vol. 73,
No.6, 1990
TB reduced the pH from 7.0 to 4.13, whereas the same concentration of GA in milk reduced the pH from 6.4 to 4.98. The same was true when GDL was used Hydrolysis of lactone was more rapid in TB than in milk (fable I), and the buffering action of milk was greater than of TB. The lactone hydrolyzed rapidly in broth during the first 2 to 3 h, and then only small additional decreases in pH were observed unti16 h (fable 1). Hydrolysis was less rapid in milk; the lactone continued to hydrolyze during the first 5 to 6 h and then only a small further decrease in pH was observed by 24 h. The concentration of GDL required to reduce the pH of TB was less than half that needed to reduce the pH of milk to the same value. This explains why the pathogen was more susceptible to GDL in TB than in milk and why higher concentrations were needed to inhibit or inactivate L. monocytogenes in milk than in TB. Shahamat et al. (23) concluded that inhibition of L. monocytogenes by sodium nitrite depended on the nature of the substrate, pH, and storage temperature. E1-Shenawy and Marth (lO, 11, 12, 13) found that susceptibility of L. monocytogenes to sorbic, benzoic, or propionic acids depended on incubation temperature and pH of the medium either singly or in combination as well as on concentration of preservative. Our data support conclusions made from the earlier observations. To study the effect of GA on survival of L. monocytogenes during preparation of cooked cottage cheese curd, milk was inoculated with the pathogen and then rennet curd was prepared; it served as the unacidified control. We did not prepare the second control, cottage cheese made with a lactic starter culture, because such work was done earlier by Ryser et al. (22). Hydrochloric acid served to prepare curd by lowering th pH via hydrogen ion concentration, and GA served in a similar way, but with undissociated acid molecules present. Mter cutting the curd, lower levels of the pathogen were recovered from curd or whey, resulting from action of the acids rather than rennet. This can be attributed to the low pH needed to coagulate the milk with the acids. Raising the temperature for cooking (S7.2°C) did not appreciably reduce numbers of the pathogen in rennet curd but did reduce them in whey. Evidently cells in curd were afforded greater protection from heat than those in whey.
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GLUCONIC ACID CONTROLS USTERIA
In this study, numbers of L. monocytogenes were determined with TA and MLA to assess populations of injured and uninjured cells. Differences between C01Dlts obtained with TA and MLA were small for curd or whey receiving any of the treatments (Figures 9 and 10). This is in accord with work of Golden et at (17), who found that MLA is one of the best selective media to recover injured cells of L. monocytogenes. We also used TSA to assess the population of uninjured cells. In earlier studies, this medium was used to assess injury of L. monocytogenes caused by heat (8), acids (1), gamma radiation (14), and extended incubation in a minimal medium at different temperatures and in the presence of benzoic acid (26). In the present study, numbers of L. monocytogenes in curd or whey, immediately after cutting the curd, were approximately the same (Figures 9 and 10), regardless of the medium that was used to enumerate the bacterium. Raising the temperature of curd during cooking to 54.4·C or above was accompanied by differences in C01Dlts of the pathogen when plating was on TA and TSA, indicating injury of L. monocytogenes. The degree of injury was related to the coagulant used and temperature during cooking of the curd (Figures 9 and 10). This can be seen from differences in C01Dlts when rennet (less injury) or acids (HCl or GA) (more injury) served as coagulants. Injury of the pathogen in rennet curd resulted largely from heat alone, whereas injury in acid curd resulted from the combined effects of heat and acid. Injury in whey (Figure 10) was greater than in curd (Figure 9), probably because curd afforded some protection to the bacterial cells. This may explain why the bacterium at one point was recovered by cold enrichment in TB from GA curd but not GA whey. Ryser et al. (22) reported that small numbers of L. monocytogenes survived the cooking process when cottage cheese was manufactured using milk inoculated with 1()4 to lOS L. monocytogenes and coagulated with acid from lactic acid bacteria. The viable survivors initially were f01Dld by cold enrichment and not by direct plating of freshly made cottage cheese curd. Listeria monocytogenes also has been reported to survive during manufacture of Kachkaval cheese made from artificially contaminated ewe's milk (18). In this instance, the bacterium survived a heat treatment of 75 to
76·C. Our results indicate that GA more effectively aided in inactivation of L. monocytogenes during cooking of cottage cheese curd than did HCl. This may be explained by the antimicrobial action of GA, which is related both to pH and to degree of dissociation. Undissociated organic acids are more readily soluble in the bacterial cell membrane and thus are more bactericidal than are dissociated acids (21). Although the bacterium was not completely eliminated from cooked HCI curd, as noted earlier when a lactic starter culture was used (22), there were fewer survivors in it than in the rennet curd. Undoubtedly this resulted from the lower pH (4.7) in the former than in the latter, where enzymatic clotting of curd occurred without an appreciable change in pH. In conclusion, these data demonstrate that inhibition or inactivation of L. monocytogenes by GA was affected by 1) temperature: inactivation was more rapid at 35 than at 13"C; 2) concentration of acid or acidogen: the greater the concentration, the lower the pH and the greater the efficacy of the acid or acidogen; 3) form of acid: GA was more effective than GDL; and 4) the medium: the pathogen was more susceptible to GA or GDL in TB than in milk. Our data suggest that using GA and probably GDL at concentrations high enough to coagulate milk for cottage cheese production, when combined with the heat of cooking as done in these experiments, should result in Listeria-free cottage cheese curd. Appropriate hygienic practices then must be used to keep it Listeria-free during the remainder of the processing procedures.
ACKNOWLEDGMENTS Research supported by the College of Agricultural and Life Sciences and by the Center for Dairy Research, University of Wisconsin-Madison.
REFERENCES 1 Ahamad, N., and E. H. Marth. 1989. Behavior ofListeriJl morwcytogeMS at 7, 13,21, and 3S'C in tryptosc broth acidifIed with acetic, citric or lactic acid. I. Food Prot. S2:688.
2 Anis, S.M.K., and B.G Ladkani. 1988. Effect of kind of acidulaDt and pH level on some characteristics of Iownal of Dairy Science Vol. 73,
No.6, 1990
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EL-SHENAWY AND MARTH
Mozzarella cheese curd made by direct acidification. Egypt. J. Dairy Sci. 16:267. 3 Anonymous. 1985. Listeriosis ontbreak associated with Mexican-style cheese - California. Morbid. Mortal. Weeldy Rep. 34:357. 4 Anonymous. 1985. Recalls of soft ripened and Camembert cheese suspected to be contaminated with Listeria. FDA Enforcement Rep. Sept. 4. FDA, Washington, DC. 5 Anonymous. 1986. Finds Listeria in Brie from Frenchcertified plant. Food Chern. News 27(50):2. 6 Bayoumi, S., and S. Madkor. 1988. The user of GDL in the manufacture of yoghurt. Egypt. J. Dairy Sci. 16:233. 7 Bayoumi, S., and H. Reuter. 1986. The user of GDL in Domiati cheese made from UFmilk concentrate. Minia J. Agric. Res. Dev. 8:271. 8 Dallmier, A. W., and S. E. Martin. 1988. Catalase and superoxide dismutase activities after beat injury of L. monocytogenes. Appl. Environ. Microbiol. 54:581. 9 Deane, D. D., and E. G. Hammond. 1960. Coagulation of milk for cheese making by ester hydrolysis. J. Dairy Sci. 43:1421. 10 EI-Shenawy, M A., and E. H. Marth. 1988. Sodium benzoate inhibits growth of or inactivates Listeria monocytogenes. J. Food Prot. 51:525. 11 EI-Shenawy, M. A., and E. H. Marth. 1988. Inhibition and inactivation of Listeria monocytogenes by sorbic acid. J. Food Prot. 51:842. 12 EI-Shenawy, M. A., and E. H. Marth. 1989. Behavior of Listeria monocytogenes in the presence of sodium propionate. Intern. J. Food Microbiol. 8:85. 13 EI-Shenawy, M A., and E. H. Marth. 1989. Inhibition or inactivation of Listeria mollocytogelles by sodium benzoate together with some organic acids. J. Food Prot 52: 771. 14 El-Shenawy, M. A., A. E. Yousef, and E. H. Marth. 1989. Radiation sensitivity of Listeria mollOcytogenes in broth or in raw ground beef. Lebensm. Wiss. Technol. 22:387. 15 Emstrom, C. A. 1965. Mechanized "pizza cheese" making by means of continnous direct acidification method. Manuf. Milk Prod. J. 56:7. 16 Fleming, D. W., S. L. Cacm, K. R. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audrier, 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.
Joumal of Dairy Science Vol. 73,
No.6, 1990
17 Golden, D. A., L. R Beuchal, and R. E. Bracket. 1988. Evaluation of selective direct plating media for their suitability to recover uninjured, heat injured, and freezeinjured LiSteria mollOcytogenes from foods. Appl. Environ. Microbiol. 54:1451. 18 Ikonomov, L., and D. Todorov. 1964. Studies of the viability of Listeria mollocytogenes in ewe's milk and dairy products. Vet Med. Nauki, Sofiya 1:23 (Dairy Sci. Abstr. 27:2). 19 Mabbill, L. A., H. R. Chapman, and N.J. Berridge. 1955. Experiments in cheesemaking without starter. J. Dairy Res. 22:365. 20 Rosenow, E. M., and E. H. Marth. 1987. Listeria, listeriosis and dairy foods: A review. Cult Dairy Prod. J. 22(4):13. 21 Ryser, E. T., and E. H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food Prot 51:615. 22 Ryser, E. T., E. H. Marth, and M P. Doyle. 1985. Survival of Listeria mollOcytogenes during manufacture and storage of cottage cheese. J. Food Prot. 48:746. 23 Shahamat, M., A. Seaman, and M. Woodbine. 1980. Influence of sodium chloride, pH and temperature on the inhibitory activity of sodium nitrite on Listeria monocytogelles. Page 227 ill Microbial growth and survival in extremes of environment. J .EL. Corry and G. W. Gould, ed. Academic Press, London, Engl. 24 Shehata, A. E., M Iyer, N. F. Olson, and T. Richardson. 1967. Effect of type of acid used in direct acidification procedures on moisture, flIIDDess and calcium levels of cheese. J. Dairy Sci. 50:824. 25 Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Backer. 1986. Listeria mollocytogellu in Milch and Milchprodukten, Dtsch. Molk. Ztg. 41:1358, 1359, and 1362. 26 Yousef, A. E., M. A. EI-Shenawy, and E. H. Marth. 1989. Inactivation and injury of Listeria monocytogenes in a minimal medium as affected by benzoic acid and incubation temperature. J. Food Sci. 54:650. 27 Yousef, A. E., E. T. Ryser, and E. H. Marth. 1988. Methods for improved recovery of Listeria monocytogenes from cheese. Appl. Environ. Microbiol. 54:2643.
and the Food Research Institute University of Wisconsin-Madison Madison 53706 ABSTRACT
Unrestricted or minimally restricted growth of Listeria monocytogenes strain V7 occurred 1) at 13"C in tryptose broth with .125 or .25% gluconic acid or .1 to .3% glucono-delta-lactone, 2) at n"C in milk with .125 to 1.0% gluconic acid or .5 or 1.0% gluconoOIt""IV'lV"l
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Figure 6. Behavior of Listeria monocytogenes at 13'C
9
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--
15
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Figure 7. Behavior of Listeria monocytogenes at 13'C
in milk containing gluconic acid.
in broth containing glucono-delta-Iactone.
or .5%, GA did not affect the lag phase, but GT were increased to 110 h as concentration of acid was increased to .5%. Addition of .75% GA prolonged the lag phase to 2 d followed by growth with a GT of 14.5 b. Presence of 1.5% GA inhibited growth of the pathogen, but after 4 wk of incubation (the end of the experiment) the population increased slightly.
complete, inactivation of the pathogen. Complete inactivation after 3 wk of incubation was accomplished by 3.0% GDL (Figure 8). Survival of Listeria monocytogenes During Preparation of Cottage Cheese Curd
Cottage cheese curd was prepared from pasteurized skim milk inoculated to contain ca. 2.5
Behavior of Listeria monocytogenes In the Presence of Glucono-Delta-Lactone at 13'C
L. monocytogenes grew in unacidified broth (control) after less than 1 d with a GT of 3.6 h. Presence of .1 % GDL did not affect bacterial growth, whereas .2% delayed onset of growth to 1 d followed by a GT of 6.5 h. At .3%, GDL delayed onset of growth to 2 d, followed by a GT of 14 h. Concentrations of .4 or .5% GDL inhibited growth for up to 12 d followed by some increase in numbers. Presence of .75 or 1.0% of the lactone completely inhibited growth of the pathogen with some inactivation as the incubation progressed (Figure 7). Incubation of milk containing .5 or 1.0% GDL at 13°C allowed growth of L. monocytogenes after a lag phase of 2 and 3 d; GT were 7.2 and 10.3 h, respectively. The pathogen was inhibited for 2 wk followed by a small increase in numbers when milk contained 1.5% GDL. Presence of 2.0 or 2.5% GDL completely inhibited growth with some, but not Iournal of Dairy Science Vol. 73. No.6. 1990
10 8
6
E ......
~ 4 U 01
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2 0 0
3
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---0---
6 9 12 15 3 4 Days Weeks Control ---..-.5% ----a-- 1.0% 1.5% ~ 2.0% - - 0 - - 2.5% --....-
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Figure 8. Behavior of Listeria monocytogenes at 13'C in milk containing glucono-delta-Iactone.
1435
GLUCONIC ACID CONTROLS USTERIA
7,..---------------6
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]=Log numbers plated on TA
_1)
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_____ 2
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o
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4
3
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Figure 9. Survival of Listeria monocytogenes during preparation of cottage cheese curd made with rennet, HeI, or gluconic acid. 0) Immediately after cutting, I) after temperature was increased to 48.9·C, 2) after temperature was increased to 54.4"C, 3) after temperature was increased to S7.2·C, 4) after 15 min of cooking, and 5) after 30 min of cooking. TA Tryptose agar, MLA McBride Listeria agar, and TSA = tryptose-salt agar.
=
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2
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4
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Figure 10. Survival of Listeria monocytogenes in the whey during preparation of cooked cottage cheese curd made with rennet, HCI, or gluconic acid. Symbols and lines
are the same as in Figure 9.
=
x 1()6 cfulml L. monocytogenes/ml. Bovine rennet, HCI, or GA were used separately to coagulate the milk. The skim milk was free of L. monocytogenes before inoculation. After curd foonation, the pathogen was more concentrated in curd than in whey (Figures 9 and 10). Immediately after cutting rennet curd, the pathogen was recovered from curd or whey at a level similar to that initially in milk. With acid treatments, recovery of the organism was .5 and 1.5 orders of magnitude less in curd or whey, respectively, than the initial count of milk. After the temperature was raised to 48.9°C, numbers of the pathogen in rennet curd or rennet whey had not changed, whereas a further reduction in numbers (from that observed initially) occurred in curd and whey prepared with HCI or GA. After the temperature was raised to 54.4°C, the pathogen started to grow in the rennet curd, but numbers in rennet whey decreased somewhat. There was a decrease of 1 or 2 orders of magnitude, respectively, in curd made with HCI or GA, but there were no appreciable differences in numbers of the pathogen in accompanying wheys when tested by plating on TA. After the temperature was raised to 57.2°C,
numbers of the pathogen in rennet curd increased further but continued to decrease in rennet whey. Numbers in both types of acid curd decreased further; reduction in numbers in acid whey was greater than in acid curd. After 15 min of cooking, numbers of the bacterium in rennet curd decreased to the initial population; whereas the decrease in w~ey was about 1 order of magnitude. After the same treatment, there was a decrease of 3 to 4 orders of magnitude in population of acid curd with the reduction being greater in GA than in HCI curd. The pathogen was still detected (10 cfu/ml) in whey of HCI curd, whereas it was not detectable in GA whey with our method. At the end of cooking (30 min at 57.2°C) numbers decreased by about 1.5 and 2.5 orders of magnitude, respectively, in curd or whey from the rennet treatment. After completion of the cooking process, the pathogen was not completely eliminated from curd made with HCI, but it could not be detected in whey. No viable cells of L. monocytogenes could be detected in GA curd or GA whey when the cooking process was complete. Listeria monocytogenes was not recovered from GA curd after cold enrichment in TB for up to 8 wk. However, the pathogen was recovered through cold enrichment (after 6 wk) of Journal of Dairy Science Vol. 73,
No.6, 1990
1436
EL-SHENAWY AND MARnI
HCI whey taken at the end of cooking and (after 4 wk) from GA whey taken after 15 min of cooking. Numbers of L. monocytogenes in curd or whey plated on TA, MLA, or TSA were a~ proximately the same until immediately after cutting the curd After the temperature was increased to over 48.9°C, differences in counts obtained with the different media began to increase. Differences in numbers of the bacterium when plating was on TA and MIA were always small, but this was not true between TA or MLA and TSA. DISCUSSION
Gluconic acid and GDL are the most common acid and acidogen products suggested for use in making certain kinds of cheese. Our results indicate that GA and GDL at various concentrations can affect behavior of L. monocytogenes. The effect depended, in part, on incubation temperature. The pathogen was more susceptible to effect of GA or GDL at the higher (35°C) rather than lower (13°C) temperature. For example, .75% GA in TB completely inactivated the pathogen after 5 d at 35°C, whereas the same concentration or more (1.0%) of the acid only inhIbited growth of the pathogen at 13°C. When the medium was milk, 1.5% GA completely inhIbited growth of the bacterium and caused some decrease in population at 35°C, whereas the same concentration of GA permitted a small increase in numbers at 13°C. The same was true with GDL in TB or milk. As expected, GA became more effective as the concentration was increased; the higher the concentration, the lower the pH and the greater the inhibition. For example, .125% GA in TB at 35°C gave a pH of 5.9 and did not affect growth of the pathogen (Figure 1). However, when the concentration of GA was raised to .25%, the pH was reduced to 5.3, the lag phase was prolonged from 3 to 6 h, and the GT was increased from 54 to 90 min. Listeria monocytogenes was more susceptible to effects of GA or GDL in TB than in milk. For example, .75% GA in TB completely inactivated the bacterium after 5 d at 35°C; however, in milk, 1.5% GA failed to cause complete inactivation at 35°C. This can be explained by differences in the pH achieved in the two media. In the first instance, .75% GA in Journal of Daily Science Vol. 73,
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TB reduced the pH from 7.0 to 4.13, whereas the same concentration of GA in milk reduced the pH from 6.4 to 4.98. The same was true when GDL was used Hydrolysis of lactone was more rapid in TB than in milk (fable I), and the buffering action of milk was greater than of TB. The lactone hydrolyzed rapidly in broth during the first 2 to 3 h, and then only small additional decreases in pH were observed unti16 h (fable 1). Hydrolysis was less rapid in milk; the lactone continued to hydrolyze during the first 5 to 6 h and then only a small further decrease in pH was observed by 24 h. The concentration of GDL required to reduce the pH of TB was less than half that needed to reduce the pH of milk to the same value. This explains why the pathogen was more susceptible to GDL in TB than in milk and why higher concentrations were needed to inhibit or inactivate L. monocytogenes in milk than in TB. Shahamat et al. (23) concluded that inhibition of L. monocytogenes by sodium nitrite depended on the nature of the substrate, pH, and storage temperature. E1-Shenawy and Marth (lO, 11, 12, 13) found that susceptibility of L. monocytogenes to sorbic, benzoic, or propionic acids depended on incubation temperature and pH of the medium either singly or in combination as well as on concentration of preservative. Our data support conclusions made from the earlier observations. To study the effect of GA on survival of L. monocytogenes during preparation of cooked cottage cheese curd, milk was inoculated with the pathogen and then rennet curd was prepared; it served as the unacidified control. We did not prepare the second control, cottage cheese made with a lactic starter culture, because such work was done earlier by Ryser et al. (22). Hydrochloric acid served to prepare curd by lowering th pH via hydrogen ion concentration, and GA served in a similar way, but with undissociated acid molecules present. Mter cutting the curd, lower levels of the pathogen were recovered from curd or whey, resulting from action of the acids rather than rennet. This can be attributed to the low pH needed to coagulate the milk with the acids. Raising the temperature for cooking (S7.2°C) did not appreciably reduce numbers of the pathogen in rennet curd but did reduce them in whey. Evidently cells in curd were afforded greater protection from heat than those in whey.
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In this study, numbers of L. monocytogenes were determined with TA and MLA to assess populations of injured and uninjured cells. Differences between C01Dlts obtained with TA and MLA were small for curd or whey receiving any of the treatments (Figures 9 and 10). This is in accord with work of Golden et at (17), who found that MLA is one of the best selective media to recover injured cells of L. monocytogenes. We also used TSA to assess the population of uninjured cells. In earlier studies, this medium was used to assess injury of L. monocytogenes caused by heat (8), acids (1), gamma radiation (14), and extended incubation in a minimal medium at different temperatures and in the presence of benzoic acid (26). In the present study, numbers of L. monocytogenes in curd or whey, immediately after cutting the curd, were approximately the same (Figures 9 and 10), regardless of the medium that was used to enumerate the bacterium. Raising the temperature of curd during cooking to 54.4·C or above was accompanied by differences in C01Dlts of the pathogen when plating was on TA and TSA, indicating injury of L. monocytogenes. The degree of injury was related to the coagulant used and temperature during cooking of the curd (Figures 9 and 10). This can be seen from differences in C01Dlts when rennet (less injury) or acids (HCl or GA) (more injury) served as coagulants. Injury of the pathogen in rennet curd resulted largely from heat alone, whereas injury in acid curd resulted from the combined effects of heat and acid. Injury in whey (Figure 10) was greater than in curd (Figure 9), probably because curd afforded some protection to the bacterial cells. This may explain why the bacterium at one point was recovered by cold enrichment in TB from GA curd but not GA whey. Ryser et al. (22) reported that small numbers of L. monocytogenes survived the cooking process when cottage cheese was manufactured using milk inoculated with 1()4 to lOS L. monocytogenes and coagulated with acid from lactic acid bacteria. The viable survivors initially were f01Dld by cold enrichment and not by direct plating of freshly made cottage cheese curd. Listeria monocytogenes also has been reported to survive during manufacture of Kachkaval cheese made from artificially contaminated ewe's milk (18). In this instance, the bacterium survived a heat treatment of 75 to
76·C. Our results indicate that GA more effectively aided in inactivation of L. monocytogenes during cooking of cottage cheese curd than did HCl. This may be explained by the antimicrobial action of GA, which is related both to pH and to degree of dissociation. Undissociated organic acids are more readily soluble in the bacterial cell membrane and thus are more bactericidal than are dissociated acids (21). Although the bacterium was not completely eliminated from cooked HCI curd, as noted earlier when a lactic starter culture was used (22), there were fewer survivors in it than in the rennet curd. Undoubtedly this resulted from the lower pH (4.7) in the former than in the latter, where enzymatic clotting of curd occurred without an appreciable change in pH. In conclusion, these data demonstrate that inhibition or inactivation of L. monocytogenes by GA was affected by 1) temperature: inactivation was more rapid at 35 than at 13"C; 2) concentration of acid or acidogen: the greater the concentration, the lower the pH and the greater the efficacy of the acid or acidogen; 3) form of acid: GA was more effective than GDL; and 4) the medium: the pathogen was more susceptible to GA or GDL in TB than in milk. Our data suggest that using GA and probably GDL at concentrations high enough to coagulate milk for cottage cheese production, when combined with the heat of cooking as done in these experiments, should result in Listeria-free cottage cheese curd. Appropriate hygienic practices then must be used to keep it Listeria-free during the remainder of the processing procedures.
ACKNOWLEDGMENTS Research supported by the College of Agricultural and Life Sciences and by the Center for Dairy Research, University of Wisconsin-Madison.
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2 Anis, S.M.K., and B.G Ladkani. 1988. Effect of kind of acidulaDt and pH level on some characteristics of Iownal of Dairy Science Vol. 73,
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Mozzarella cheese curd made by direct acidification. Egypt. J. Dairy Sci. 16:267. 3 Anonymous. 1985. Listeriosis ontbreak associated with Mexican-style cheese - California. Morbid. Mortal. Weeldy Rep. 34:357. 4 Anonymous. 1985. Recalls of soft ripened and Camembert cheese suspected to be contaminated with Listeria. FDA Enforcement Rep. Sept. 4. FDA, Washington, DC. 5 Anonymous. 1986. Finds Listeria in Brie from Frenchcertified plant. Food Chern. News 27(50):2. 6 Bayoumi, S., and S. Madkor. 1988. The user of GDL in the manufacture of yoghurt. Egypt. J. Dairy Sci. 16:233. 7 Bayoumi, S., and H. Reuter. 1986. The user of GDL in Domiati cheese made from UFmilk concentrate. Minia J. Agric. Res. Dev. 8:271. 8 Dallmier, A. W., and S. E. Martin. 1988. Catalase and superoxide dismutase activities after beat injury of L. monocytogenes. Appl. Environ. Microbiol. 54:581. 9 Deane, D. D., and E. G. Hammond. 1960. Coagulation of milk for cheese making by ester hydrolysis. J. Dairy Sci. 43:1421. 10 EI-Shenawy, M A., and E. H. Marth. 1988. Sodium benzoate inhibits growth of or inactivates Listeria monocytogenes. J. Food Prot. 51:525. 11 EI-Shenawy, M. A., and E. H. Marth. 1988. Inhibition and inactivation of Listeria monocytogenes by sorbic acid. J. Food Prot. 51:842. 12 EI-Shenawy, M. A., and E. H. Marth. 1989. Behavior of Listeria monocytogenes in the presence of sodium propionate. Intern. J. Food Microbiol. 8:85. 13 EI-Shenawy, M A., and E. H. Marth. 1989. Inhibition or inactivation of Listeria mollocytogelles by sodium benzoate together with some organic acids. J. Food Prot 52: 771. 14 El-Shenawy, M. A., A. E. Yousef, and E. H. Marth. 1989. Radiation sensitivity of Listeria mollOcytogenes in broth or in raw ground beef. Lebensm. Wiss. Technol. 22:387. 15 Emstrom, C. A. 1965. Mechanized "pizza cheese" making by means of continnous direct acidification method. Manuf. Milk Prod. J. 56:7. 16 Fleming, D. W., S. L. Cacm, K. R. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audrier, 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.
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17 Golden, D. A., L. R Beuchal, and R. E. Bracket. 1988. Evaluation of selective direct plating media for their suitability to recover uninjured, heat injured, and freezeinjured LiSteria mollOcytogenes from foods. Appl. Environ. Microbiol. 54:1451. 18 Ikonomov, L., and D. Todorov. 1964. Studies of the viability of Listeria mollocytogenes in ewe's milk and dairy products. Vet Med. Nauki, Sofiya 1:23 (Dairy Sci. Abstr. 27:2). 19 Mabbill, L. A., H. R. Chapman, and N.J. Berridge. 1955. Experiments in cheesemaking without starter. J. Dairy Res. 22:365. 20 Rosenow, E. M., and E. H. Marth. 1987. Listeria, listeriosis and dairy foods: A review. Cult Dairy Prod. J. 22(4):13. 21 Ryser, E. T., and E. H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food Prot 51:615. 22 Ryser, E. T., E. H. Marth, and M P. Doyle. 1985. Survival of Listeria mollOcytogenes during manufacture and storage of cottage cheese. J. Food Prot. 48:746. 23 Shahamat, M., A. Seaman, and M. Woodbine. 1980. Influence of sodium chloride, pH and temperature on the inhibitory activity of sodium nitrite on Listeria monocytogelles. Page 227 ill Microbial growth and survival in extremes of environment. J .EL. Corry and G. W. Gould, ed. Academic Press, London, Engl. 24 Shehata, A. E., M Iyer, N. F. Olson, and T. Richardson. 1967. Effect of type of acid used in direct acidification procedures on moisture, flIIDDess and calcium levels of cheese. J. Dairy Sci. 50:824. 25 Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Backer. 1986. Listeria mollocytogellu in Milch and Milchprodukten, Dtsch. Molk. Ztg. 41:1358, 1359, and 1362. 26 Yousef, A. E., M. A. EI-Shenawy, and E. H. Marth. 1989. Inactivation and injury of Listeria monocytogenes in a minimal medium as affected by benzoic acid and incubation temperature. J. Food Sci. 54:650. 27 Yousef, A. E., E. T. Ryser, and E. H. Marth. 1988. Methods for improved recovery of Listeria monocytogenes from cheese. Appl. Environ. Microbiol. 54:2643.
