Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Inactivation of Yersinia enterocolitica by nitrite and nitrate in food M. De Giusti & E. De Vito To cite this article: M. De Giusti & E. De Vito (1992) Inactivation of Yersinia enterocolitica by nitrite and nitrate in food, Food Additives & Contaminants, 9:5, 405-408, DOI: 10.1080/02652039209374091 To link to this article: http://dx.doi.org/10.1080/02652039209374091
Published online: 10 Jan 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [Universite Laval]
Date: 20 November 2015, At: 01:34
FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 5, 4 0 5 - 4 0 8
Inactivation of Yersinia enterocolitica by nitrite and nitrate in food M. DE GIUSTI and E. DE VITO
Downloaded by [Universite Laval] at 01:34 20 November 2015
Dept. Medicina Sperimentale, Cattedra di Igiene, Università di Roma La Sapienza, Rome, Italy The antimicrobial effects of sodium nitrite and sodium and potassium nitrate against Yersinia enterocolitica were investigated in solution and in treated pork meat. Potassium nitrate and sodium nitrate showed only feeble antimicrobial activity in cultures; no antimicrobial activity was detected with sodium nitrite. Conversely, all three salts displayed apparent antimicrobial activity in pork meat, possibly due to selective effects on competitive flora. Keywords: Yersinia enterocolitica, meat, antimicrobial additives
Introduction
Studies concerning the epidemiology of Yersinia enterocolitica human infection are still in progress but the importance of this food-borne pathology is taking on a more definite outline. In recent years the isolation frequency of Yersinia enterocolitica has dramatically increased with regard not only to number of cases but also to countries, especially European, reporting isolations. The foods most frequently involved in the spreading of yersiniosis are meat (particularly pork), raw and pasteurized milk, and fresh vegetables. Diseased persons, pigs and probably dogs are the most important infection reservoir. Y. enterocolitica has been isolated in water (drinking water, recreational water) and is widespread within the animal kingdom, with pig farming being the major reservoir for strains associated with human disease. Food-borne and water-borne epidemics have been described and familiar outbreaks suggest that person to person transmission is important. However, in most sporadic cases the acquisition and transmission pathways of infection remain unknown. Y. enterocolitica is a psychrotrophic microorganism and can grow until 7°C. This ability explains the isolation of Y. enterocolitica from frozen food (Bucci and Maini 1987), refrigerated milk (Greenwood et al. 1990) and meat (Cox et al. 1990). The recovery of Y. enterocolitica in food, usually eaten without thermal treatment, underlines the need to study the antimicrobial activity of the main additives used in food preservation. Method
The antimicrobial activity toward Y. enterocolitica of sodium nitrite and sodium and potassium nitrate was studied. 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
M. de Giusti and E. de Vito
Downloaded by [Universite Laval] at 01:34 20 November 2015
406
These additives are commonly added to preserved meat at the maximum permitted level of 150 mg/kg and 250 mg/kg. This activity was studied using a Y. enterocolitica strain isolated from porcine lymphnodes, serotype 0:3, biotype 4, lysotype 8. The activity of sodium and potassium nitrate and sodium nitrite was studied in vitro using four different concentrations containing 5, 10, 15 and 20mg/ml in relation to bacterial concentrations of 104, 103, 102, 10 cfu/ml at 10 min, 60 min, 24 h and 48 h of contact. After these times, the Y. enterocolitica survival was tested by means of subculture in Y. selective medium (CIN UNIPATH) incubated at 32°C for 48 h. Sodium nitrite and sodium and potassium nitrate were also tested in pork mince experimentally contaminated with Y. enterocolitica suspension having a final concentration in meat around 10 000, 1000 and 100 CFU/g. Meat was treated with 250, 150 and 100 mg/kg of sodium nitrite and 350, 250 and 150 mg/kg of sodium nitrate and potassium nitrate before contamination.
Table 1. In vitro test results. y. enterocolitica (cfu/ml)
Additive concern rations (mg/ml)
Time
104
103
102
10
10 min 60 min 24 h 48 h
Y Y Y —
Y Y Y —
Y Y — —
10
10 min 60 min 24 h 48 h
Y Y — —
Y Y Y — Y Y Y —
Y Y Y —
Y Y — —
15
10 min 60 min 24 n 48 h
Y Y
Y Y
Y Y
Y
10 min 60 min 24 h 48 h
Y Y
10 min 60 min 24 h 48 h
Y Y Y Y
10 min 60 min 24 h 48 h
Y Y Y Y
KNO 3 5
20
NaNO 3 5
10
I Y Y
Y Y Y Y Y Y Y Y
Y Y
Y —
Y Y Y Y
Y Y Y
Y Y Y Y
Y Y Y Y
Y: Presence of Y. enterocolitica detected.
Y. enterocolitica (cfu/ml)
Additive concentrations (mg/ml)
Time
104
103
102
10
15
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y — —
20
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y —
10 min 60 min 24 h
Y Y Y
Y Y Y
Y Y Y
Y Y Y
10
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y Y
15
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y Y
20
10 min 60 min 24 h 48 h
Y
Y Y Y Y
Y Y Y Y
Y Y
NaNO 2 5
Y Y Y
Y Y
Yersinia enterocolitica inactivation
407
The contaminated meat was maintained for 24 h, 48 h and 7 days at 4°C and then tested for the presence of Y. enterocolitica by blending 25 g of pork mince in ITC Broth and incubating at 32°C for 24 h. The isolation was performed using Y. selective medium (CIN) incubated at 32°C for 48 h.
Downloaded by [Universite Laval] at 01:34 20 November 2015
Results and discussion
The in vitro results (table 1) show a feeble antimicrobial activity of KNO3 depending on contact time and on Y. enterocolitica concentrations. With NaNCh antimicrobial activity was observed at higher additive concentrations and the lowest Y. enterocolitica concentration only. NaNC>2 displayed no detectable antimicrobial activity under these conditions. The results from experimental contamination of pork (table 2) show that after 24 h admissible concentrations of NaNC>2 are effective, even on higher Y. enterocolitica concentrations. On the other hand, NaNC>3 was ineffective at the lowest concentration for all bacterial concentrations and KNO3 showed a low effect at the highest Y. enterocolitica concentration. Y. enterocolitica was not isolated from bacteriological negative controls. After 48 h and 7 days every additive seemed effective at all concentrations tested. It is difficult to compare the results obtained in vitro with those using treated pork. In fact, the low activity in vitro agrees with other studies on the effectiveness of nitrite and nitrate toward Gram-negative flora, on the other hand, the good effect in pork seems to be in contrast with other published studies. An explanation for this result can be found in the considerable presence of competitive bacterial Gram-negative flora in all meat samples examined
Table 2. Test with pork results. Y. enterocolitica (cfu/g) Additives concentrations (mg/kg)
104
103
102
KNO3 350 250 150
— Y Y
—
—
—
—
NaNO 3 350 250 150
— — Y
— — Y
— — Y
—
—
NaNO 2 250 150 100
— —
Y: Y. enterocolitica present at 24 h.
408
M. de Giusti and E. de Vito
{Streptococcus marcescens, Streptococcus liquefaciens, Escherichia coli, Proteus mirabilis). References BUCCI, G., and MAINI, P., 1987, Isolamento di Yersinia spp. in provincia di Ferrara con particolare riferimento al biennio 1984-1985. L'Igiene Moderna, 88, 189-202. CROCI, L., DE FELIP, G., GIZZARELLI, S., OREFICE, L., and TOTI, L., 1988, Yersinia enterocolitica:
considerazioni sul ruolo nella contaminazione degli alimenti. Estratto da: Igiene e Sanità Pubblica, XLIV, 11-12, 369-387. DARIS, F., ROMANO, R., and NEDOCLAN, G., 1989, Dinamica di crescita di Yersinia enterocolitica in
alimenti conservati in ambiente refrigerato L'Igiene Moderna, 91, 337-352. GRENWOOD, M. H., HOOPER, W. L., and RODHOUSE, J. C., 1990, The source of Yersinia spp. in
pasteurized milk. Epidemiology and Infection, 104, 345-350. HARMON, M. C., SWAMINATHAN, B., and FORREST, J. C., 1990, Isolation of Y. enterocolitica and
related species from porcine samples obtained from an abattoir. Journal of Applied Bacteriology, 56, 421-427.
Downloaded by [Universite Laval] at 01:34 20 November 2015
WAUTERS, G., GOOSSENS, V., JANSSENS, M., and VANDEPITTE, J., 1988, New enrichment method from
isolation of pathogenic Yersinia enterocolitica serogroup 0:3 from pork. Applied and Environmental Microbiology, 54, 851-854.
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Inactivation of Yersinia enterocolitica by nitrite and nitrate in food M. De Giusti & E. De Vito To cite this article: M. De Giusti & E. De Vito (1992) Inactivation of Yersinia enterocolitica by nitrite and nitrate in food, Food Additives & Contaminants, 9:5, 405-408, DOI: 10.1080/02652039209374091 To link to this article: http://dx.doi.org/10.1080/02652039209374091
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 9
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [Universite Laval]
Date: 20 November 2015, At: 01:34
FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 5, 4 0 5 - 4 0 8
Inactivation of Yersinia enterocolitica by nitrite and nitrate in food M. DE GIUSTI and E. DE VITO
Downloaded by [Universite Laval] at 01:34 20 November 2015
Dept. Medicina Sperimentale, Cattedra di Igiene, Università di Roma La Sapienza, Rome, Italy The antimicrobial effects of sodium nitrite and sodium and potassium nitrate against Yersinia enterocolitica were investigated in solution and in treated pork meat. Potassium nitrate and sodium nitrate showed only feeble antimicrobial activity in cultures; no antimicrobial activity was detected with sodium nitrite. Conversely, all three salts displayed apparent antimicrobial activity in pork meat, possibly due to selective effects on competitive flora. Keywords: Yersinia enterocolitica, meat, antimicrobial additives
Introduction
Studies concerning the epidemiology of Yersinia enterocolitica human infection are still in progress but the importance of this food-borne pathology is taking on a more definite outline. In recent years the isolation frequency of Yersinia enterocolitica has dramatically increased with regard not only to number of cases but also to countries, especially European, reporting isolations. The foods most frequently involved in the spreading of yersiniosis are meat (particularly pork), raw and pasteurized milk, and fresh vegetables. Diseased persons, pigs and probably dogs are the most important infection reservoir. Y. enterocolitica has been isolated in water (drinking water, recreational water) and is widespread within the animal kingdom, with pig farming being the major reservoir for strains associated with human disease. Food-borne and water-borne epidemics have been described and familiar outbreaks suggest that person to person transmission is important. However, in most sporadic cases the acquisition and transmission pathways of infection remain unknown. Y. enterocolitica is a psychrotrophic microorganism and can grow until 7°C. This ability explains the isolation of Y. enterocolitica from frozen food (Bucci and Maini 1987), refrigerated milk (Greenwood et al. 1990) and meat (Cox et al. 1990). The recovery of Y. enterocolitica in food, usually eaten without thermal treatment, underlines the need to study the antimicrobial activity of the main additives used in food preservation. Method
The antimicrobial activity toward Y. enterocolitica of sodium nitrite and sodium and potassium nitrate was studied. 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
M. de Giusti and E. de Vito
Downloaded by [Universite Laval] at 01:34 20 November 2015
406
These additives are commonly added to preserved meat at the maximum permitted level of 150 mg/kg and 250 mg/kg. This activity was studied using a Y. enterocolitica strain isolated from porcine lymphnodes, serotype 0:3, biotype 4, lysotype 8. The activity of sodium and potassium nitrate and sodium nitrite was studied in vitro using four different concentrations containing 5, 10, 15 and 20mg/ml in relation to bacterial concentrations of 104, 103, 102, 10 cfu/ml at 10 min, 60 min, 24 h and 48 h of contact. After these times, the Y. enterocolitica survival was tested by means of subculture in Y. selective medium (CIN UNIPATH) incubated at 32°C for 48 h. Sodium nitrite and sodium and potassium nitrate were also tested in pork mince experimentally contaminated with Y. enterocolitica suspension having a final concentration in meat around 10 000, 1000 and 100 CFU/g. Meat was treated with 250, 150 and 100 mg/kg of sodium nitrite and 350, 250 and 150 mg/kg of sodium nitrate and potassium nitrate before contamination.
Table 1. In vitro test results. y. enterocolitica (cfu/ml)
Additive concern rations (mg/ml)
Time
104
103
102
10
10 min 60 min 24 h 48 h
Y Y Y —
Y Y Y —
Y Y — —
10
10 min 60 min 24 h 48 h
Y Y — —
Y Y Y — Y Y Y —
Y Y Y —
Y Y — —
15
10 min 60 min 24 n 48 h
Y Y
Y Y
Y Y
Y
10 min 60 min 24 h 48 h
Y Y
10 min 60 min 24 h 48 h
Y Y Y Y
10 min 60 min 24 h 48 h
Y Y Y Y
KNO 3 5
20
NaNO 3 5
10
I Y Y
Y Y Y Y Y Y Y Y
Y Y
Y —
Y Y Y Y
Y Y Y
Y Y Y Y
Y Y Y Y
Y: Presence of Y. enterocolitica detected.
Y. enterocolitica (cfu/ml)
Additive concentrations (mg/ml)
Time
104
103
102
10
15
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y — —
20
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y —
10 min 60 min 24 h
Y Y Y
Y Y Y
Y Y Y
Y Y Y
10
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y Y
15
10 min 60 min 24 h 48 h
Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y Y
20
10 min 60 min 24 h 48 h
Y
Y Y Y Y
Y Y Y Y
Y Y
NaNO 2 5
Y Y Y
Y Y
Yersinia enterocolitica inactivation
407
The contaminated meat was maintained for 24 h, 48 h and 7 days at 4°C and then tested for the presence of Y. enterocolitica by blending 25 g of pork mince in ITC Broth and incubating at 32°C for 24 h. The isolation was performed using Y. selective medium (CIN) incubated at 32°C for 48 h.
Downloaded by [Universite Laval] at 01:34 20 November 2015
Results and discussion
The in vitro results (table 1) show a feeble antimicrobial activity of KNO3 depending on contact time and on Y. enterocolitica concentrations. With NaNCh antimicrobial activity was observed at higher additive concentrations and the lowest Y. enterocolitica concentration only. NaNC>2 displayed no detectable antimicrobial activity under these conditions. The results from experimental contamination of pork (table 2) show that after 24 h admissible concentrations of NaNC>2 are effective, even on higher Y. enterocolitica concentrations. On the other hand, NaNC>3 was ineffective at the lowest concentration for all bacterial concentrations and KNO3 showed a low effect at the highest Y. enterocolitica concentration. Y. enterocolitica was not isolated from bacteriological negative controls. After 48 h and 7 days every additive seemed effective at all concentrations tested. It is difficult to compare the results obtained in vitro with those using treated pork. In fact, the low activity in vitro agrees with other studies on the effectiveness of nitrite and nitrate toward Gram-negative flora, on the other hand, the good effect in pork seems to be in contrast with other published studies. An explanation for this result can be found in the considerable presence of competitive bacterial Gram-negative flora in all meat samples examined
Table 2. Test with pork results. Y. enterocolitica (cfu/g) Additives concentrations (mg/kg)
104
103
102
KNO3 350 250 150
— Y Y
—
—
—
—
NaNO 3 350 250 150
— — Y
— — Y
— — Y
—
—
NaNO 2 250 150 100
— —
Y: Y. enterocolitica present at 24 h.
408
M. de Giusti and E. de Vito
{Streptococcus marcescens, Streptococcus liquefaciens, Escherichia coli, Proteus mirabilis). References BUCCI, G., and MAINI, P., 1987, Isolamento di Yersinia spp. in provincia di Ferrara con particolare riferimento al biennio 1984-1985. L'Igiene Moderna, 88, 189-202. CROCI, L., DE FELIP, G., GIZZARELLI, S., OREFICE, L., and TOTI, L., 1988, Yersinia enterocolitica:
considerazioni sul ruolo nella contaminazione degli alimenti. Estratto da: Igiene e Sanità Pubblica, XLIV, 11-12, 369-387. DARIS, F., ROMANO, R., and NEDOCLAN, G., 1989, Dinamica di crescita di Yersinia enterocolitica in
alimenti conservati in ambiente refrigerato L'Igiene Moderna, 91, 337-352. GRENWOOD, M. H., HOOPER, W. L., and RODHOUSE, J. C., 1990, The source of Yersinia spp. in
pasteurized milk. Epidemiology and Infection, 104, 345-350. HARMON, M. C., SWAMINATHAN, B., and FORREST, J. C., 1990, Isolation of Y. enterocolitica and
related species from porcine samples obtained from an abattoir. Journal of Applied Bacteriology, 56, 421-427.
Downloaded by [Universite Laval] at 01:34 20 November 2015
WAUTERS, G., GOOSSENS, V., JANSSENS, M., and VANDEPITTE, J., 1988, New enrichment method from
isolation of pathogenic Yersinia enterocolitica serogroup 0:3 from pork. Applied and Environmental Microbiology, 54, 851-854.
