APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1991, p. 3355-3360
Vol. 57, No. 11
0099-2240/91/113355-06$02.00/0
Effect of the Lactoperoxidase System on Listeria monocytogenes Behavior in Raw Milk at Refrigeration Temperatures PILAR GAYA, MARGARITA MEDINA,* AND MANUEL NUNEZ
Departamento de Producci6n
y
Tecnologia de Alimentos, CIT-INIA, Apartado 8111, 28080 Madrid, Spain
Received 3 May 1991/Accepted 9 September 1991
Activity of raw milk lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system on four Listeria monocytogenes strains at refrigeration temperatures after addition of 0.25 mM sodium thiocyanate and 0.25 mM hydrogen peroxide was studied. The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8°C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested. D values in activated-LP system milk for the four strains tested ranged from 4.1 to 11.2 days at 4°C and from 4.4 to 9.7 days at 8°C. The lactoperoxidase level in raw milk declined during a 7-day incubation, the decrease being more pronounced at 8°C than at 4°C and in control milk than in activated-LP system milk. The thiocyanate concentration decreased considerably in activated-LP system milk at both temperatures during the first 8 h of incubation. LP system activation was shown to be a feasible procedure for controlling development of L. monocytogenes in raw milk at refrigeration temperatures.
The lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system is a natural antimicrobial system present in milk (33). Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide, yielding short-lived oxidation products (27), hypothiocyanite ions (2, 20), and higher oxyacids (5) being mainly responsible for the antibacterial effect of the system. Lactoperoxidase constitutes about 1% of whey proteins (30), with a reported mean value of 0.87 U/ml in milk from Friesian cows (25). Thiocyanate ion is derived from glucosinolates and cyanogenic glucosides present in food (31), concentrations in bovine milk being in the range of 1 to 7 ppm (6, 17). Hydrogen peroxide can be supplied exogenously or generated by lactic acid bacteria from milk microflora or starter cultures. Preservative action of the LP system in milk after addition of thiocyanate and hydrogen peroxide has been demonstrated previously (4, 6, 23). Different workers (3, 7, 26, 32, 42) agree on its bactericidal activity against gram-negative bacteria such as Campylobacter jejuni, Salmonella spp., Escherichia coli, and Pseudomonas spp. However, only a bacteriostatic effect has been recorded for gram-positive bacteria of the genera Streptococcus and Bacillus (25-27, 40, 43). The high occurrence of Listeria monocytogenes in raw milk (11, 15, 19, 24, 38) and its abilities to grow at refrigeration temperatures (12, 34, 41) and to withstand a 10% NaCl concentration (35) and a pH level of 5.0 (9), together with recent outbreaks of food-borne listeriosis (16), point to L. monocytogenes as a pathogen of major concern in milk and
of incubation has recently been described (14). The use of the LP system decreases thermal resistance of L. monocytogenes in raw milk at subpasteurization temperatures (22). The purpose of the present work was to evaluate the activity of the raw milk LP system, after addition of thiocyanate and hydrogen peroxide, on the behavior of L. monocytogenes at refrigeration temperatures and to evaluate the stability of LP system components during milk storage.
MATERIALS AND METHODS
Organisms. Four isolates of L. monocytogenes, strains Scott A and 5069 (obtained from R. G. Crawford, Food and Drug Administration, Cincinnati, Ohio), ATCC 19119, and NCTC 11994, were used as test organisms. They were designated Lm 1, Lm 2, Lm 3, and Lm 4, respectively. Stock cultures were maintained at 4°C in slants of tryptic soy agar (Difco Laboratories, Detroit, Mich.) with 0.6% yeast extract added. Before the experiments, they were grown in tryptic soy-0.6% yeast extract broth and incubated at 37°C for 24 h. A second transfer was done to the same broth incubated at 20°C for 48 h. Sample preparation. L. monocytogenes strains were inoculated to a concentration of approximately 104 CFU/ml into 250-ml screw-cap flasks containing 100 ml of refrigerated raw milk from Holstein cows, before activation of the LP system. The LP system was activated by adding to flasks 1 ml of a 25 mM aqueous solution of sodium thiocyanate (Merck, Darmstadt, Germany) and then 1 ml of a 25 mM aqueous solution of hydrogen peroxide (30% [wt/vol] aqueous solution; Merck) previously filter sterilized through 0.22-,umpore-size filters. Activated and control flasks were incubated at 4°C for 7 days, 8°C for 7 days, or 20°C for 3 days. L. monocytogenes counts, total viable counts, milk pH, and lactoperoxidase and thiocyanate concentrations were monitored daily in duplicate experiments. The stability of lactoperoxidase in sterile milk was tested by adding 0.50 U of lactoperoxidase (Boehringer Mannheim, Mannheim, Germany) per ml to ultrahigh-temperaturetreated milk which was incubated at 20°C for 3 days. Microbiological analyses. L. monocytogenes was enumerated on duplicate plates of Listeria selective medium, Ox-
dairy products. Biological inhibitors such as lysozyme (21), organic acids (1), and bacteriocins produced by strains of lactic acid bacteria have been described as being effective against L. monocytogenes (8, 18, 28, 29). Investigations of antimicrobial activity of artificially added LP systems on L. monocytogenes in broth and sterile milk (10, 13, 37) demonstrated a bacteriostatic effect which was dependent on temperature and culture medium. A bactericidal effect of the LP system on L. monocytogenes in raw milk at 35°C during the first 4 h *
Corresponding author. 3355
GAYA ET AL.
3356
APPL. ENVIRON. MICROBIOL.
TABLE 1. Log counts of four L. monocytogenes strains in control and activated-LP system raw milk during storage at 4°C Log counts of L. monocytogenes strains in milk"
Length of incubation (days)
8b 1 2 3 4 5 6 7
Lm 1
Lm 4
Lm 3
Lm 2
C
A
C
A
C
A
C
A
4.08 3.82c 4.03 4.03 3.97 3.94 3.97 4.12c
4.05 3.94 3.83c,d 3.76
3.90' 3.87' 3.80c 3.79C
3.81c 3.64cd 3.74C
3.80c 3.88c 3.84c 3.85C 3.81c 3.94 3.90c 3.67C
3.80c 3.85c
3.30c,d
3.78c 3.76c 3.73C 3.70c
3.60c
3.83c 3.79C 3.80c 3.75C 4.07
3.33c.d
3.14d
3.35c.d 3.05c.d
3.57c,d
3.76c 3.92 4.01
3.00c,d
4.12C
3.12c.d 3.31c,d
4.17c 4.70c
3.48c.d 3.54c,d 3.32c,d 3.65c,d
3.66c,d 3.31c,d 3.43cd 2.46c,d 3.18c,d
2.80c,d
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 11994. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value for the same strain at 0 h after inoculation of milk. d Data are significantly (P < 0.01) different from the respective value (same strain, same days of incubation) in control milk.
ford formulation (Oxoid Ltd., Basingstoke, Hampshire, England), for each decimal dilution, after incubation at 37°C for 24 h. For expression of results, L. monocytogenes counts during incubation were corrected to an inoculum of 104 CFU/ml for all strains. Total viable counts were determined on duplicate plates of plate count agar after incubation at 30°C for 72 h. Chemical analyses. Lactoperoxidase activity was analyzed in duplicate according to the method of Marshall et al. (25) by monitoring the oxidation of ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) (Sigma Chemical Co., St. Louis, Mo.) at 412 nm with a Beckman DU7 spectrophotometer (36). The thiocyanate concentration was evaluated in duplicate after precipitation with 20% (wt/vol) trichloroacetic acid, as the colored complex formed with the ferric nitrate reagent (7). The A460 was measured, and the content of thiocyanate was calculated from a standard curve. Statistical treatment of data. Analysis of variance of data obtained was performed by using program BMDP8V (Department of Biomathematics, University of California at Los Angeles, Los Angeles), and regression analysis was performed by using programs Curve and Regress (Sigstat, Provo, Utah). L. monocytogenes D values were calculated from linear regression equations of decreasing log counts on length of incubation and are expressed in days. The leastsignificant-difference test (39) was used for comparisons of means.
RESULTS AND DISCUSSION Effect of the LP system in raw milk at 4 and 8°C. Behavior of L. monocytogenes strains in the activated-LP system raw milk and control raw milk at 4 and 8°C is shown in Tables 1 and 2, respectively. Analysis of variance revealed highly significant (P < 0.001) effects of LP system activation, L. monocytogenes strain, temperature, and length of incubation on L. monocytogenes counts. In control milk incubated at 4°C, counts of L. monocytogenes Lm 1 remained virtually unchanged throughout incubation. Strain Lm 2 and Lm 3 counts decreased during the first 4 and 3 days, respectively, with growth thereafter. Strain Lm 4 counts were, during most of the incubation period, lower (P < 0.01) than the respective 0-h levels. The logarithmic-linear regression equation (L. monocytogenes log counts on days of storage) was superior to the rest of equations in program Curve for all L. monocytogenes strains in control milk at 4°C. Significant trends of decrease were found only for strains Lm 2 and Lm 3. Equations for all strains and regression significance levels (P) obtained by using program Regress are shown in Table 3. Determination coefficients (r2) were 0.575 for Lm 2 and 0.399 for Lm 3. L. monocytogenes counts in activated-LP system milk incubated at 4°C were for all strains tested significantly (P < 0.01) lower than the respective values in control milk throughout incubation except for the first 1 to 3 days,
TABLE 2. Log counts of four L. monocytogenes strains in control and activated-LP system raw milk during storage at 8°C
Length of incubation (days)
Log counts of L. monocytogenes strains in milka Lm 1
Lm 2
Lm 3
C
A
C
A
C
4.10 4.02 4.04
4.02 3.84c.d 3.62c,d
3.85c 3.89
3.78c 3.75c 3.75c
3.85c
1 2
3.78c 3.83'
3
4.09
3.31c,d
4 5
4.11
2.74c,d
4.14
2.93c,d
6
4.31c
2.87c,d
7
4.29c
2.75cd
8b
3.85c 3.85c
3.92 3.83c 4.29c 4.35c
3.33c,d 3.41c,d 2.57c,d
3.41c,d 3.74c,d
4.06 4.36c 4.47c 4.75c 4.94c
Lm 4
C
A
3.80C
3.87
3.76c
3.78c
3.90 3.90
3.85c
A
3.75c 3.60c,d
3.70c,d 2.91c,d 3.37c,d 2.40c,d
3.58cd
3.89 4.01 4.11
3.17c,d
4.88c
2.93 d
4.lld
5.01c
2.33c,d
3.52c,d
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 1194. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value for the same strain at 0 h after inoculation of milk. d Data are significantly (P < 0.01) different from the respective value (same strain, same days of incubation) in control milk.
LP SYSTEM AGAINST L. MONOCYTOGENES IN RAW MILK
VOL. 57, 1991
3357
TABLE 3. Regression equations of decreasing log counts of four L. monocytogenes strains on days of incubation in activated-LP system
and control milk Storage temp
40C
80C
Strain'a
Results with control milkb
Results with activated-LP system milkb Equation
P
Day
Equation
P
Day
y = 4.061 - 0.214x y = 3.966 - 0.082x
7 5 6 5
y = y y
= 3.902 - 0.089x = 4.039 - 0.245x
0.000 0.000 0.000 0.000
7 5 6 7
y y y y
= = = =
0.201x 0.226x 0.103x 0.255x
0.000 0.000 0.000 0.000
Lm Lm Lm Lm
1 2 3 4
1 4 3 7
Lm Lm Lm Lm
1 2 3 4
0 5 2 0
y y
= 3.912 - 0.060x = 3.902 - 0.016x
0.205 0.000 0.003 0.108
y y
= 3.918 - 0.015x = 3.923 - 0.069x
0.327 0.074
4.066 - 0.147x y = 3.944 - 0.166x
3.974 4.009 3.924 3.957
-
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 11994. b Days are those with decreasing L. monocytogenes log counts. P is the regression significance level. y represents decreasing log counts; x represents days of incubation.
depending on the strain. Counts of Lm 1 decreased until the end of incubation, whereas growth of Lm 2, Lm 3, and Lm 4 started after 5, 6, and 5 days, respectively. Nevertheless, log counts of L. monocytogenes strains in activated-LP system milk after 7 days at 4°C were 0.70 to 1.07 units lower than the respective values in control milk. As with control milk, the logarithmic-linear regression equation was superior for all strains in activated-LP system milk, according to program Curve. Highly significant (P < 0.001) regressions of decreasing L. monocytogenes log counts on days of storage at 4°C were obtained for all strains tested by using program Regress (Table 3), with r2 of 0.704, 0.488, 0.452, and 0.463 for strains Lm 1 to Lm 4, respectively. For strains Lm 2 and Lm 3, with significant trends of decrease in both activated-LP system and control milk, the period with decreasing log counts was longer and the slope value was higher in the former (Table 3). D values of L. monocytogenes at 4°C in activated-LP system milk, calculated from these regression equations, were 6.8, 6.0, 11.2, and 4.1 days for Lm 1 to Lm 4, respectively. Although at 8°C, counts of L. monocytogenes Lm 2 and Lm 3 in control milk suffered an initial decrease, all strains were present in significantly (P < 0.01) higher levels after 6 to 7 days of incubation at this temperature than in milk at 0 days. Increases in log counts after 7 days at 8°C ranged from 0.29 for Lm 1 to 1.01 for Lm 4. The logarithmic-linear regression equation again performed the best, according to program Curve. However, no significant regression equations were obtained (Table 3), indicating the absence of significant trends of decrease in control milk at 8°C. As previously seen for 4°C, L. monocytogenes counts in activated-LP system milk incubated at 8°C were for all strains tested significantly (P < 0.01) lower than the respective values in control milk except for the first 1 or 2 days of incubation, depending on the strain. Counts of Lm 1 and Lm 4 decreased to the end of incubation, but development of Lm 2 and Lm 3 began after 5 and 6 days, respectively. Log counts of L. monocytogenes strains in activated-LP system milk after 7 days at 8°C were 0.61 to 2.68 units lower than the respective log counts in control milk. At 8°C, the logarithmic-linear regression also yielded the best-fitting equations for all L. monocytogenes strains in activated-LP system milk, according to program Curve. Highly significant (P < 0.001) regressions of L. monocytogenes log counts on days of storage at 8°C were obtained for all strains tested by using program Regress, with r2 of 0.564, 0.407, 0.335, and 0.607 for strains Lm 1 to Lm 4, respectively. L. monocytogenes D
values in activated-LP system milk at 8°C, calculated from these regression equations, were 5.0, 4.4, 9.7, and 4.5 days for Lm 1 to Lm 4, respectively. Denis and Ramet (10) reported a bactericidal effect of the LP system against L. monocytogenes ATCC 19111, with a higher D value at 4°C (8 days) than at 15°C (5 days) in ultrahigh-temperature-treated milk. A reduction of L. monocytogenes NCTC 11994 counts by two log cycles after 6 days at 10°C was achieved with sterile ultrahigh-temperaturetreated milk with an added LP system by Earnshaw and Banks (13) who, however, considered the activity of the LP system only bacteriostatic. The inhibition of L. monocytogenes by an LP system (0.37 U/ml) artificially added to milk at 20°C has been demonstrated by Siragusa and Johnson (37). An extended lag period of 12 to 36 h versus 9 h in control milk and log counts approximately 3 log cycles lower in activated-LP system milk than in control milk after 36 and 68 h at 20°C were obtained by these authors with L. monocytogenes Scott A. In our experiment, mean decreases in L. monocytogenes log counts of 0.36 after 1 day and 0.89 after 3 days were achieved at 20°C by activation of the LP system, with a D value ranging from 2.0 days for Lm 2 to 4.6 days for Lm 1 (data not shown). A bactericidal effect of the LP system on L. monocytogenes in raw milk at 35°C was recently reported (14), with decreases of approximately one log cycle after 4 h. Highly significant (P < 0.001) effects of LP system activation, temperature, and length of incubation on total viable counts were revealed by analysis of variance. Log total viable counts in milk were 5.29 before inoculation and LP system activation. A mean increase of 2.77 log units was found with control milk after 7 days at 4°C (Table 4), whereas in activated-LP system milk, total viable counts remained fairly constant during the first 5 days and increased by approximately 1 log unit from day 5 to day 7. At 8°C, total viable counts increased in control milk 3.24 log units after 7 days (Table 5), but in activated-LP system milk, they were stabilized during the first 2 days and increased by 2.35 log units from day 2 to day 7. Similar results were obtained by Zajac et al. (44) with raw milk at refrigeration temperatures by repeated activation of the LP system. Milk pH at refrigeration temperatures (Tables 4 and 5) was not significantly influenced by any of the effects investigated, according to analysis of variance. After 7 days, pH in control milk decreased 0.16 unit at 4°C and 0.43 unit at 8°C, whereas in activated-LP system milk, the respective decreases were only 0.01 and 0.12 unit. L. monocytogenes growth or sur-
3358
GAYA ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 4. Log total viable counts, pH, lactoperoxidase, and thiocyanate in control and activated-LP system raw milk during storage at 40C' Length of incubation
Total viable count
pH
(days)
C
A
C
A
C
8b
5.50c 5.64c 6.32c 6.80c 7.44c
5.17d 5.13d 5.20d 5.1d 5.06c,d 5.45d 5.83cd 6.52c,d
6.71 6.70 6.74 6.81 6.69 6.66 6.60 6.55
6.69 6.69 6.75 6.82 6.74
0.63c 0.60c 0.59C 0.60C 0.56c 0.52C 0.49c
1 2 3 4 5 6 7
7.72c
7.96c 8.06c
SCN-
LP
6.75 6.64 6.70
A
0.62C 0.58c
A
6.38 4.35 3.65c 3.66c
0.60c 0.65d
4.89
0.63c,d
4.32
0.59C,d 0.62c,d
5.02
18.09c, 17.2215.4715.9414.8314.3316.04-
4.10
16.15c,d
0.55"
0.48c
C
a Lactoperoxidase (LP) is expressed in units per milliliter, and thiocyanate (SCN-) is expressed in parts per million. C, control raw milk; A, activated-LP system raw milk. b Lenth of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value at 0 h. d Data are significantly (P < 0.01) different from the respective value (same days of incubation) in control milk.
vival should not be influenced by milk pH decreases recorded in the present work. According to our results, the LP system exhibited a bactericidal activity against L. monocytogenes in raw milk at refrigeration temperatures. This bactericidal activity was dependent on the strain of L. monocytogenes, as shown by analysis of variance and by the D values obtained, which points out different strain sensitivities to the LP system. Factors other than the LP system may be involved in L. monocytogenes death, as shown by the fact that strains Lm 2 and Lm 3 decreased significantly in control milk at 4°C, although at a slower rate than in activated-LP system milk. These factors would be partly responsible for the variability not explained by the regression equations of the different L. monocytogenes strains in activated-LP system milk, in which r2 were in the range of 0.335 to 0.704. Lower L. monocytogenes counts in activated-LP system milk cannot be ascribed to cell injury caused by the LP system followed by a failure to recover on selective media. In an analogous work with the same L. monocytogenes strains which is being carried out at our laboratory (45), with goat's milk with total viable counts before L. monocytogenes inoculation considerably lower than those due to the inoculum, no significant differences throughout milk incubation were recorded for Listeria counts on plate count agar and on Listeria selective medium, Oxford formulation. El-
Shenawy et al. (14) compared Listeria counts on TA and TA-plus-5.5% NaCl media and concluded that no cell injury by the LP system occurred. Stability of lactoperoxidase and thiocyanate in refrigerated milk. The lactoperoxidase level in milk before activation of the LP system was 0.70 U/ml. Analysis of variance detected significant effects (P < 0.001) of LP system activation, temperature, and length of incubation on the lactoperoxidase concentration. Lactoperoxidase was more stable at 4°C than at 8°C and in activated-LP system milk than in control milk (Tables 4 and 5). The level of lactoperoxidase added to sterile ultrahigh-temperature-treated milk did not decrease during incubation for 3 days at 20°C (data not shown). This fact and the finding that the regression of log total counts on the lactoperoxidase level after 6 or 7 days at 4°C or 8°C was highly significant (r2 = 0.854; P < 0.01) suggest the involvement of milk microflora in lactoperoxidase inactivation. The level of thiocyanate (Tables 4 and 5) was also significantly influenced (P < 0.001) by LP system activation and length of incubation, with no significant differences between temperatures. The thiocyanate concentration in milk was 5.37 ppm before and 27.33 ppm after activation of the LP system. A decrease of approximately 10 ppm was detected at both temperatures during the first 8 h of incubation in activated-LP system milk, the resulting level remaining virtually constant thereafter. Minimal changes in the thiocy-
TABLE 5. Log total viable counts, pH, lactoperoxidase, and thiocyanate in control and activated-LP system raw milk during storage at 80C' Length of incubation
Total viable count
pH
LP
(days)
C
A
C
A
C
8b
5.49c 5.82c
5.22d 5.15d 5.22d 5.48c,d 5.88cd
6.69 6.75 6.74 6.81 6.58 6.52 6.38 6.28
6.66 6.73 6.76 6.86 6.66 6.68 6.65 6.59
0.62c 0.49c
0.60c 0.62C 0.56C 0.5OC 0.48c 0.45c
1 2 3 4 5 6 7
6.90c 7.46c 8.00c 8.23c
8.36c 8.53c
6.38"
7.25c,d 7.57c,d
SCN-
C
A
0.59c 0.46c
7.34 4.60
15.8417.69-
0.53c,d 0.60C
3.77
15.14c,d
3.84 4.41 4.68 4.63 3.96
16.7515.41 14.85-
A
0.54c 0.60c d
0.57c,d 0.52 d
15.8614.61
a Lactoperoxidase (LP) is expressed in units per milliliter, and thiocyanate (SCN-) is expressed in parts per million. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value at 0 h. d Data are significantly (P < 0.01) different from the respective value (same days of incubation) in control milk.
VOL. 57, 1991
LP SYSTEM AGAINST L. MONOCYTOGENES IN RAW MILK
anate concentration were detected in control milk at both temperatures. A bactericidal effect of an activated LP system in raw milk on L. monocytogenes strains has been detected in the present work. D values at refrigeration temperatures, 4.1 to 11.2 days, at 4°C and 4.5 to 9.7 days at 8°C, were high. The bactericidal activity persisted, however, for 5 days, preventing growth and reducing significantly L. monocytogenes numbers during refrigerated storage of raw milk. LP system activation was shown to be a feasible procedure for controlling the development of L. monocytogenes in refrigerated raw milk. ACKNOWLEDGMENT We thank Maximo de Paz for statistical assistance. REFERENCES 1. Ahamad, N., and E. H. Marth. 1989. Behavior of Listeria monocytogenes at 7, 13, 21 and 35°C in tryptose broth acidified with acetic, citric or lactic acid. J. Food Prot. 52:688-695. 2. Aune, T. M., and E. L. Thomas. 1977. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80:209-214. 3. Beumer, R. R., A. Noomen, J. A. Marls, and E. H. Kampelmacher. 1985. Antibacterial action of the lactoperoxidase system on Campylobacter jejuni in cow's milk. Neth. Milk Dairy J. 39:107-114. 4. Bjorck, L. 1978. Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45: 109-118. 5. Bjorck, L., and 0. Claesson. 1980. Correlation between concentration of hypothiocyanate and antibacterial effect of the lactoperoxidase system against Escherichia coli. J. Dairy Sci. 63:
919-922. 6. Bjorck, L., 0. Claesson, and W. Schulthess. 1979. The lactoperoxidase/thiocyanate/hydrogen peroxide system as a temporary preservative for raw milk in developing countries. Milchwissenschaft 34:726-729. 7. Bjorck, L., C. G. Rosen, V. Marshall, and B. Reiter. 1975. Antibacterial activity of the lactoperoxidase system in milk against pseudomonads and other gram-negative bacteria. Appl. Microbiol. 30:199-204. 8. Carminati, D., G. Giraffa, and M. G. Bossi. 1989. Bacteriocinlike inhibitors of Streptococcus lactis against Listeria monocytogenes. J. Food Prot. 52:614-617. 9. Conner, 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. 52:59-63. 10. Denis, F., and J.-P. Ramet. 1989. Antibacterial activity of the lactoperoxidase system on Listeria monocytogenes in trypticase soy broth, UHT milk and French soft cheese. J. Food Prot. 52:706-711. 11. Dominguez Rodriguez, L., J. F. Fernandez Garayzabal, J. A. Vazquez Boland, E. F. Rodriguez Ferri, and G. Suarez Fernandez. 1985. Isolation de microorganisms du genre Listeria a partir de lait cru destine a la consomation humaine. Can. J. Microbiol.
31:938-941. 12. Donnelly, C. W., and E. H. Briggs. 1986. Psychrotrophic growth and thermal inactivation of Listeria monocytogenes as a function of milk composition. J. Food Prot. 49:994-998. 13. Earnshaw, R. G., and J. G. Banks. 1989. A note on the inhibition of Listeria monocytogenes NCTC 11994 in milk by an activated lactoperoxidase system. Lett. Appl. Microbiol. 8:203-205. 14. El-Shenawy, M. A., H. S. Garcia, and E. H. Marth. 1990. Inhibition and inactivation of Listeria monocytogenes by the lactoperoxidase system in raw milk, buffer or a semi-synthetic medium. Milchwissenschaft 45:638-641. 15. Farber, J. M., G. W. Sanders, J. I. Speirs, J.-Y. D'Aoust, D. B. Emmons, and R. McKellar. 1988. Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated raw
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milk. Int. J. Food Microbiol. 7:277-286. 16. Griffiths, M. W. 1989. Listeria monocytogenes: its importance in the dairy industry. J. Sci. Food Agric. 47:133-158. 17. Harnulv, B. G., and C. Kandasamy. 1982. Increasing the keeping quality of milk by activation of its lactoperoxidase system. Results from Sri Lanka. Milchwissenschaft 37:454-457. 18. Harris, L. J., M. A. Daeschel, M. E. Stiles, and T. R. Kiaenhammer. 1989. Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes. J. Food Prot. 52:384-387. 19. Hayes, P. S., J. C. Feeley, L. M. Graves, G. W. Ajello, and D. W. Fleming. 1986. Isolation of Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 51:438-440. 20. Hoogendoorn, H., J. P. Piessens, W. Scholtes, and L. A. Stoddard. 1977. Hypothiocyanite ion: the inhibitor formed by the system lactoperoxidase-thiocyanate-hydrogen peroxide. Caries Res. 11:77-84. 21. Hughey, V. L., and E. A. Johnson. 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53:2165-2170. 22. Kamau, D. N., S. Doores, and K. M. Pruitt. 1990. Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Appl. Environ. Microbiol. 56:2711-2716. 23. Kamau, D. N., and M. Kroger. 1984. Preservation of raw milk by treatment with hydrogen peroxide and by activation of the lactoperoxidase (LP) system. Milchwissenschaft 39:658-661. 24. Lovett, J., D. W. Francis, and J. M. Hunt. 1987. Listeria monocytogenes in raw milk: detection, incidence, and pathogenicity. J. Food Prot. 50:188-192. 25. Marshall, V. M. E., W. M. Cole, and A. J. Bramley. 1986. Influence of the lactoperoxidase system on susceptibility of the udder to Streptococcus uberis infection. J. Dairy Res. 53:507514. 26. Marshall, V. M. E., and B. Reiter. 1980. Comparison of the antibacterial activity of the hypothiocyanite anion towards Streptococcus lactis and Escherichia coli. J. Gen. Microbiol. 120:513-516. 27. Oram, J. D., and B. Reiter. 1966. The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of group N streptococci. Biochem. J. 100:373-381. 28. Pucci, M. J., E. R. Vedamuthu, B. S. Kunka, and P. A. Vandenbergh. 1988. Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0. Appl. Environ. Microbiol. 54:2349-2353. 29. Raccach, M., R. McGrath, and H. Daftarian. 1989. Antibiosis of some lactic acid bacteria including Lactobacillus acidophilus toward Listeria monocytogenes. Int. J. Food Microbiol. 9:2532. 30. Reiter, B. 1985. Lactoperoxidase system of bovine milk, p. 123-141. In K. M. Pruitt and J. 0. Tenovuo (ed.), The lactoperoxidase system: chemistry and biological significance. Immunology series no. 27. Marcel Dekker, Inc., New York. 31. Reiter, B., and G. Harnulv. 1984. Lactoperoxidase antibacterial system: natural occurrence, biological functions and practical applications. J. Food Prot. 47:724-732. 32. Reiter, B., V. M. E. Marshall, L. Bjorck, and C.-G. Rosen. 1976. Nonspecific bactericidal activity of the lactoperoxidase-thiocyanate-hydrogen peroxide system on milk against Escherichia coli and some gram-negative pathogens. Infect. Immun. 13:800807. 33. Reiter, B., A. Pickering, J. D. Oram, and G. S. Pope. 1963. Peroxidase-thiocyanate inhibition of streptococci in raw milk. J. Gen. Microbiol. 33:xii. 34. Rosenow, E. M., and E. H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21 and 35°C. J. Food Prot. 50:452-459. 35. Seeliger, H. P. R., and D. Jones. 1986. Listeria, p. 1235-1245. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 36. Shindler, J. S., and G. Bardsley. 1975. Steady-state kinetics of
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38.
39.
40.
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lactoperoxidase with ABTS as chromogen. Biochem. Biophys. Res. Commun. 67:1307-1312. Siragusa, G. R., and M. G. Johnson. 1989. Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanateH202 antimicrobial system. Appl. Environ. Microbiol. 55:28022805. Slade, P. J., and D. L. Collins-Thompson. 1988. Comparison of two-stage and direct selective enrichment techniques for isolating Listeria spp. from raw milk. J. Food Sci. 53:1694-1695, 1702. Steel, R. G. D., and J. H. Torrie. 1980. In C. Napier and J. W. Maisel (ed.), Principles and procedures of statistics, a biometrical approach. McGraw-Hill International Book Co., Singapore. Thomas, E. L., K. A. Pera, K. W. Smith, and A. K. Chwang. 1983. Inhibition of Streptococcus mutans by the lactoperoxi-
APPL. ENVIRON. MICROBIOL.
dase antimicrobial system. Infect. Immun. 39:767-778. 41. Wilkins, P. O., R. Bourgeois, and R. G. E. Murray. 1972. Psychrotrophic properties of Listeria monocytogenes. Can. J. Microbiol. 18:543-551. 42. Wray, C., and J. McLaren. 1987. A note on the effect of the lactoperoxidase system on salmonellas in vitro and in vivo. J. Appl. Bacteriol. 62:115-118. 43. Zajac, M., L. Bjorck, and 0. Claesson. 1981. Antibacterial effect of the lactoperoxidase system against Bacillus cereus. Milchwissenschaft 36:417-418. 44. Zajac, M., J. Gladys, M. Skarzynska, G. Harnulv, and L. Bjorck. 1983. Changes in bacteriological quality of raw milk stabilized by activation of its lactoperoxidase system and stored at different temperatures. J. Food Prot. 46:1065-1068. 45. Zapico, P., P. Gaya, M. Medina, and M. Nuniez. Unpublished data.
Vol. 57, No. 11
0099-2240/91/113355-06$02.00/0
Effect of the Lactoperoxidase System on Listeria monocytogenes Behavior in Raw Milk at Refrigeration Temperatures PILAR GAYA, MARGARITA MEDINA,* AND MANUEL NUNEZ
Departamento de Producci6n
y
Tecnologia de Alimentos, CIT-INIA, Apartado 8111, 28080 Madrid, Spain
Received 3 May 1991/Accepted 9 September 1991
Activity of raw milk lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system on four Listeria monocytogenes strains at refrigeration temperatures after addition of 0.25 mM sodium thiocyanate and 0.25 mM hydrogen peroxide was studied. The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8°C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested. D values in activated-LP system milk for the four strains tested ranged from 4.1 to 11.2 days at 4°C and from 4.4 to 9.7 days at 8°C. The lactoperoxidase level in raw milk declined during a 7-day incubation, the decrease being more pronounced at 8°C than at 4°C and in control milk than in activated-LP system milk. The thiocyanate concentration decreased considerably in activated-LP system milk at both temperatures during the first 8 h of incubation. LP system activation was shown to be a feasible procedure for controlling development of L. monocytogenes in raw milk at refrigeration temperatures.
The lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system is a natural antimicrobial system present in milk (33). Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide, yielding short-lived oxidation products (27), hypothiocyanite ions (2, 20), and higher oxyacids (5) being mainly responsible for the antibacterial effect of the system. Lactoperoxidase constitutes about 1% of whey proteins (30), with a reported mean value of 0.87 U/ml in milk from Friesian cows (25). Thiocyanate ion is derived from glucosinolates and cyanogenic glucosides present in food (31), concentrations in bovine milk being in the range of 1 to 7 ppm (6, 17). Hydrogen peroxide can be supplied exogenously or generated by lactic acid bacteria from milk microflora or starter cultures. Preservative action of the LP system in milk after addition of thiocyanate and hydrogen peroxide has been demonstrated previously (4, 6, 23). Different workers (3, 7, 26, 32, 42) agree on its bactericidal activity against gram-negative bacteria such as Campylobacter jejuni, Salmonella spp., Escherichia coli, and Pseudomonas spp. However, only a bacteriostatic effect has been recorded for gram-positive bacteria of the genera Streptococcus and Bacillus (25-27, 40, 43). The high occurrence of Listeria monocytogenes in raw milk (11, 15, 19, 24, 38) and its abilities to grow at refrigeration temperatures (12, 34, 41) and to withstand a 10% NaCl concentration (35) and a pH level of 5.0 (9), together with recent outbreaks of food-borne listeriosis (16), point to L. monocytogenes as a pathogen of major concern in milk and
of incubation has recently been described (14). The use of the LP system decreases thermal resistance of L. monocytogenes in raw milk at subpasteurization temperatures (22). The purpose of the present work was to evaluate the activity of the raw milk LP system, after addition of thiocyanate and hydrogen peroxide, on the behavior of L. monocytogenes at refrigeration temperatures and to evaluate the stability of LP system components during milk storage.
MATERIALS AND METHODS
Organisms. Four isolates of L. monocytogenes, strains Scott A and 5069 (obtained from R. G. Crawford, Food and Drug Administration, Cincinnati, Ohio), ATCC 19119, and NCTC 11994, were used as test organisms. They were designated Lm 1, Lm 2, Lm 3, and Lm 4, respectively. Stock cultures were maintained at 4°C in slants of tryptic soy agar (Difco Laboratories, Detroit, Mich.) with 0.6% yeast extract added. Before the experiments, they were grown in tryptic soy-0.6% yeast extract broth and incubated at 37°C for 24 h. A second transfer was done to the same broth incubated at 20°C for 48 h. Sample preparation. L. monocytogenes strains were inoculated to a concentration of approximately 104 CFU/ml into 250-ml screw-cap flasks containing 100 ml of refrigerated raw milk from Holstein cows, before activation of the LP system. The LP system was activated by adding to flasks 1 ml of a 25 mM aqueous solution of sodium thiocyanate (Merck, Darmstadt, Germany) and then 1 ml of a 25 mM aqueous solution of hydrogen peroxide (30% [wt/vol] aqueous solution; Merck) previously filter sterilized through 0.22-,umpore-size filters. Activated and control flasks were incubated at 4°C for 7 days, 8°C for 7 days, or 20°C for 3 days. L. monocytogenes counts, total viable counts, milk pH, and lactoperoxidase and thiocyanate concentrations were monitored daily in duplicate experiments. The stability of lactoperoxidase in sterile milk was tested by adding 0.50 U of lactoperoxidase (Boehringer Mannheim, Mannheim, Germany) per ml to ultrahigh-temperaturetreated milk which was incubated at 20°C for 3 days. Microbiological analyses. L. monocytogenes was enumerated on duplicate plates of Listeria selective medium, Ox-
dairy products. Biological inhibitors such as lysozyme (21), organic acids (1), and bacteriocins produced by strains of lactic acid bacteria have been described as being effective against L. monocytogenes (8, 18, 28, 29). Investigations of antimicrobial activity of artificially added LP systems on L. monocytogenes in broth and sterile milk (10, 13, 37) demonstrated a bacteriostatic effect which was dependent on temperature and culture medium. A bactericidal effect of the LP system on L. monocytogenes in raw milk at 35°C during the first 4 h *
Corresponding author. 3355
GAYA ET AL.
3356
APPL. ENVIRON. MICROBIOL.
TABLE 1. Log counts of four L. monocytogenes strains in control and activated-LP system raw milk during storage at 4°C Log counts of L. monocytogenes strains in milk"
Length of incubation (days)
8b 1 2 3 4 5 6 7
Lm 1
Lm 4
Lm 3
Lm 2
C
A
C
A
C
A
C
A
4.08 3.82c 4.03 4.03 3.97 3.94 3.97 4.12c
4.05 3.94 3.83c,d 3.76
3.90' 3.87' 3.80c 3.79C
3.81c 3.64cd 3.74C
3.80c 3.88c 3.84c 3.85C 3.81c 3.94 3.90c 3.67C
3.80c 3.85c
3.30c,d
3.78c 3.76c 3.73C 3.70c
3.60c
3.83c 3.79C 3.80c 3.75C 4.07
3.33c.d
3.14d
3.35c.d 3.05c.d
3.57c,d
3.76c 3.92 4.01
3.00c,d
4.12C
3.12c.d 3.31c,d
4.17c 4.70c
3.48c.d 3.54c,d 3.32c,d 3.65c,d
3.66c,d 3.31c,d 3.43cd 2.46c,d 3.18c,d
2.80c,d
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 11994. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value for the same strain at 0 h after inoculation of milk. d Data are significantly (P < 0.01) different from the respective value (same strain, same days of incubation) in control milk.
ford formulation (Oxoid Ltd., Basingstoke, Hampshire, England), for each decimal dilution, after incubation at 37°C for 24 h. For expression of results, L. monocytogenes counts during incubation were corrected to an inoculum of 104 CFU/ml for all strains. Total viable counts were determined on duplicate plates of plate count agar after incubation at 30°C for 72 h. Chemical analyses. Lactoperoxidase activity was analyzed in duplicate according to the method of Marshall et al. (25) by monitoring the oxidation of ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) (Sigma Chemical Co., St. Louis, Mo.) at 412 nm with a Beckman DU7 spectrophotometer (36). The thiocyanate concentration was evaluated in duplicate after precipitation with 20% (wt/vol) trichloroacetic acid, as the colored complex formed with the ferric nitrate reagent (7). The A460 was measured, and the content of thiocyanate was calculated from a standard curve. Statistical treatment of data. Analysis of variance of data obtained was performed by using program BMDP8V (Department of Biomathematics, University of California at Los Angeles, Los Angeles), and regression analysis was performed by using programs Curve and Regress (Sigstat, Provo, Utah). L. monocytogenes D values were calculated from linear regression equations of decreasing log counts on length of incubation and are expressed in days. The leastsignificant-difference test (39) was used for comparisons of means.
RESULTS AND DISCUSSION Effect of the LP system in raw milk at 4 and 8°C. Behavior of L. monocytogenes strains in the activated-LP system raw milk and control raw milk at 4 and 8°C is shown in Tables 1 and 2, respectively. Analysis of variance revealed highly significant (P < 0.001) effects of LP system activation, L. monocytogenes strain, temperature, and length of incubation on L. monocytogenes counts. In control milk incubated at 4°C, counts of L. monocytogenes Lm 1 remained virtually unchanged throughout incubation. Strain Lm 2 and Lm 3 counts decreased during the first 4 and 3 days, respectively, with growth thereafter. Strain Lm 4 counts were, during most of the incubation period, lower (P < 0.01) than the respective 0-h levels. The logarithmic-linear regression equation (L. monocytogenes log counts on days of storage) was superior to the rest of equations in program Curve for all L. monocytogenes strains in control milk at 4°C. Significant trends of decrease were found only for strains Lm 2 and Lm 3. Equations for all strains and regression significance levels (P) obtained by using program Regress are shown in Table 3. Determination coefficients (r2) were 0.575 for Lm 2 and 0.399 for Lm 3. L. monocytogenes counts in activated-LP system milk incubated at 4°C were for all strains tested significantly (P < 0.01) lower than the respective values in control milk throughout incubation except for the first 1 to 3 days,
TABLE 2. Log counts of four L. monocytogenes strains in control and activated-LP system raw milk during storage at 8°C
Length of incubation (days)
Log counts of L. monocytogenes strains in milka Lm 1
Lm 2
Lm 3
C
A
C
A
C
4.10 4.02 4.04
4.02 3.84c.d 3.62c,d
3.85c 3.89
3.78c 3.75c 3.75c
3.85c
1 2
3.78c 3.83'
3
4.09
3.31c,d
4 5
4.11
2.74c,d
4.14
2.93c,d
6
4.31c
2.87c,d
7
4.29c
2.75cd
8b
3.85c 3.85c
3.92 3.83c 4.29c 4.35c
3.33c,d 3.41c,d 2.57c,d
3.41c,d 3.74c,d
4.06 4.36c 4.47c 4.75c 4.94c
Lm 4
C
A
3.80C
3.87
3.76c
3.78c
3.90 3.90
3.85c
A
3.75c 3.60c,d
3.70c,d 2.91c,d 3.37c,d 2.40c,d
3.58cd
3.89 4.01 4.11
3.17c,d
4.88c
2.93 d
4.lld
5.01c
2.33c,d
3.52c,d
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 1194. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value for the same strain at 0 h after inoculation of milk. d Data are significantly (P < 0.01) different from the respective value (same strain, same days of incubation) in control milk.
LP SYSTEM AGAINST L. MONOCYTOGENES IN RAW MILK
VOL. 57, 1991
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TABLE 3. Regression equations of decreasing log counts of four L. monocytogenes strains on days of incubation in activated-LP system
and control milk Storage temp
40C
80C
Strain'a
Results with control milkb
Results with activated-LP system milkb Equation
P
Day
Equation
P
Day
y = 4.061 - 0.214x y = 3.966 - 0.082x
7 5 6 5
y = y y
= 3.902 - 0.089x = 4.039 - 0.245x
0.000 0.000 0.000 0.000
7 5 6 7
y y y y
= = = =
0.201x 0.226x 0.103x 0.255x
0.000 0.000 0.000 0.000
Lm Lm Lm Lm
1 2 3 4
1 4 3 7
Lm Lm Lm Lm
1 2 3 4
0 5 2 0
y y
= 3.912 - 0.060x = 3.902 - 0.016x
0.205 0.000 0.003 0.108
y y
= 3.918 - 0.015x = 3.923 - 0.069x
0.327 0.074
4.066 - 0.147x y = 3.944 - 0.166x
3.974 4.009 3.924 3.957
-
a Lm 1, Scott A; Lm 2, 5069; Lm 3, ATCC 19119; Lm 4, NCTC 11994. b Days are those with decreasing L. monocytogenes log counts. P is the regression significance level. y represents decreasing log counts; x represents days of incubation.
depending on the strain. Counts of Lm 1 decreased until the end of incubation, whereas growth of Lm 2, Lm 3, and Lm 4 started after 5, 6, and 5 days, respectively. Nevertheless, log counts of L. monocytogenes strains in activated-LP system milk after 7 days at 4°C were 0.70 to 1.07 units lower than the respective values in control milk. As with control milk, the logarithmic-linear regression equation was superior for all strains in activated-LP system milk, according to program Curve. Highly significant (P < 0.001) regressions of decreasing L. monocytogenes log counts on days of storage at 4°C were obtained for all strains tested by using program Regress (Table 3), with r2 of 0.704, 0.488, 0.452, and 0.463 for strains Lm 1 to Lm 4, respectively. For strains Lm 2 and Lm 3, with significant trends of decrease in both activated-LP system and control milk, the period with decreasing log counts was longer and the slope value was higher in the former (Table 3). D values of L. monocytogenes at 4°C in activated-LP system milk, calculated from these regression equations, were 6.8, 6.0, 11.2, and 4.1 days for Lm 1 to Lm 4, respectively. Although at 8°C, counts of L. monocytogenes Lm 2 and Lm 3 in control milk suffered an initial decrease, all strains were present in significantly (P < 0.01) higher levels after 6 to 7 days of incubation at this temperature than in milk at 0 days. Increases in log counts after 7 days at 8°C ranged from 0.29 for Lm 1 to 1.01 for Lm 4. The logarithmic-linear regression equation again performed the best, according to program Curve. However, no significant regression equations were obtained (Table 3), indicating the absence of significant trends of decrease in control milk at 8°C. As previously seen for 4°C, L. monocytogenes counts in activated-LP system milk incubated at 8°C were for all strains tested significantly (P < 0.01) lower than the respective values in control milk except for the first 1 or 2 days of incubation, depending on the strain. Counts of Lm 1 and Lm 4 decreased to the end of incubation, but development of Lm 2 and Lm 3 began after 5 and 6 days, respectively. Log counts of L. monocytogenes strains in activated-LP system milk after 7 days at 8°C were 0.61 to 2.68 units lower than the respective log counts in control milk. At 8°C, the logarithmic-linear regression also yielded the best-fitting equations for all L. monocytogenes strains in activated-LP system milk, according to program Curve. Highly significant (P < 0.001) regressions of L. monocytogenes log counts on days of storage at 8°C were obtained for all strains tested by using program Regress, with r2 of 0.564, 0.407, 0.335, and 0.607 for strains Lm 1 to Lm 4, respectively. L. monocytogenes D
values in activated-LP system milk at 8°C, calculated from these regression equations, were 5.0, 4.4, 9.7, and 4.5 days for Lm 1 to Lm 4, respectively. Denis and Ramet (10) reported a bactericidal effect of the LP system against L. monocytogenes ATCC 19111, with a higher D value at 4°C (8 days) than at 15°C (5 days) in ultrahigh-temperature-treated milk. A reduction of L. monocytogenes NCTC 11994 counts by two log cycles after 6 days at 10°C was achieved with sterile ultrahigh-temperaturetreated milk with an added LP system by Earnshaw and Banks (13) who, however, considered the activity of the LP system only bacteriostatic. The inhibition of L. monocytogenes by an LP system (0.37 U/ml) artificially added to milk at 20°C has been demonstrated by Siragusa and Johnson (37). An extended lag period of 12 to 36 h versus 9 h in control milk and log counts approximately 3 log cycles lower in activated-LP system milk than in control milk after 36 and 68 h at 20°C were obtained by these authors with L. monocytogenes Scott A. In our experiment, mean decreases in L. monocytogenes log counts of 0.36 after 1 day and 0.89 after 3 days were achieved at 20°C by activation of the LP system, with a D value ranging from 2.0 days for Lm 2 to 4.6 days for Lm 1 (data not shown). A bactericidal effect of the LP system on L. monocytogenes in raw milk at 35°C was recently reported (14), with decreases of approximately one log cycle after 4 h. Highly significant (P < 0.001) effects of LP system activation, temperature, and length of incubation on total viable counts were revealed by analysis of variance. Log total viable counts in milk were 5.29 before inoculation and LP system activation. A mean increase of 2.77 log units was found with control milk after 7 days at 4°C (Table 4), whereas in activated-LP system milk, total viable counts remained fairly constant during the first 5 days and increased by approximately 1 log unit from day 5 to day 7. At 8°C, total viable counts increased in control milk 3.24 log units after 7 days (Table 5), but in activated-LP system milk, they were stabilized during the first 2 days and increased by 2.35 log units from day 2 to day 7. Similar results were obtained by Zajac et al. (44) with raw milk at refrigeration temperatures by repeated activation of the LP system. Milk pH at refrigeration temperatures (Tables 4 and 5) was not significantly influenced by any of the effects investigated, according to analysis of variance. After 7 days, pH in control milk decreased 0.16 unit at 4°C and 0.43 unit at 8°C, whereas in activated-LP system milk, the respective decreases were only 0.01 and 0.12 unit. L. monocytogenes growth or sur-
3358
GAYA ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 4. Log total viable counts, pH, lactoperoxidase, and thiocyanate in control and activated-LP system raw milk during storage at 40C' Length of incubation
Total viable count
pH
(days)
C
A
C
A
C
8b
5.50c 5.64c 6.32c 6.80c 7.44c
5.17d 5.13d 5.20d 5.1d 5.06c,d 5.45d 5.83cd 6.52c,d
6.71 6.70 6.74 6.81 6.69 6.66 6.60 6.55
6.69 6.69 6.75 6.82 6.74
0.63c 0.60c 0.59C 0.60C 0.56c 0.52C 0.49c
1 2 3 4 5 6 7
7.72c
7.96c 8.06c
SCN-
LP
6.75 6.64 6.70
A
0.62C 0.58c
A
6.38 4.35 3.65c 3.66c
0.60c 0.65d
4.89
0.63c,d
4.32
0.59C,d 0.62c,d
5.02
18.09c, 17.2215.4715.9414.8314.3316.04-
4.10
16.15c,d
0.55"
0.48c
C
a Lactoperoxidase (LP) is expressed in units per milliliter, and thiocyanate (SCN-) is expressed in parts per million. C, control raw milk; A, activated-LP system raw milk. b Lenth of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value at 0 h. d Data are significantly (P < 0.01) different from the respective value (same days of incubation) in control milk.
vival should not be influenced by milk pH decreases recorded in the present work. According to our results, the LP system exhibited a bactericidal activity against L. monocytogenes in raw milk at refrigeration temperatures. This bactericidal activity was dependent on the strain of L. monocytogenes, as shown by analysis of variance and by the D values obtained, which points out different strain sensitivities to the LP system. Factors other than the LP system may be involved in L. monocytogenes death, as shown by the fact that strains Lm 2 and Lm 3 decreased significantly in control milk at 4°C, although at a slower rate than in activated-LP system milk. These factors would be partly responsible for the variability not explained by the regression equations of the different L. monocytogenes strains in activated-LP system milk, in which r2 were in the range of 0.335 to 0.704. Lower L. monocytogenes counts in activated-LP system milk cannot be ascribed to cell injury caused by the LP system followed by a failure to recover on selective media. In an analogous work with the same L. monocytogenes strains which is being carried out at our laboratory (45), with goat's milk with total viable counts before L. monocytogenes inoculation considerably lower than those due to the inoculum, no significant differences throughout milk incubation were recorded for Listeria counts on plate count agar and on Listeria selective medium, Oxford formulation. El-
Shenawy et al. (14) compared Listeria counts on TA and TA-plus-5.5% NaCl media and concluded that no cell injury by the LP system occurred. Stability of lactoperoxidase and thiocyanate in refrigerated milk. The lactoperoxidase level in milk before activation of the LP system was 0.70 U/ml. Analysis of variance detected significant effects (P < 0.001) of LP system activation, temperature, and length of incubation on the lactoperoxidase concentration. Lactoperoxidase was more stable at 4°C than at 8°C and in activated-LP system milk than in control milk (Tables 4 and 5). The level of lactoperoxidase added to sterile ultrahigh-temperature-treated milk did not decrease during incubation for 3 days at 20°C (data not shown). This fact and the finding that the regression of log total counts on the lactoperoxidase level after 6 or 7 days at 4°C or 8°C was highly significant (r2 = 0.854; P < 0.01) suggest the involvement of milk microflora in lactoperoxidase inactivation. The level of thiocyanate (Tables 4 and 5) was also significantly influenced (P < 0.001) by LP system activation and length of incubation, with no significant differences between temperatures. The thiocyanate concentration in milk was 5.37 ppm before and 27.33 ppm after activation of the LP system. A decrease of approximately 10 ppm was detected at both temperatures during the first 8 h of incubation in activated-LP system milk, the resulting level remaining virtually constant thereafter. Minimal changes in the thiocy-
TABLE 5. Log total viable counts, pH, lactoperoxidase, and thiocyanate in control and activated-LP system raw milk during storage at 80C' Length of incubation
Total viable count
pH
LP
(days)
C
A
C
A
C
8b
5.49c 5.82c
5.22d 5.15d 5.22d 5.48c,d 5.88cd
6.69 6.75 6.74 6.81 6.58 6.52 6.38 6.28
6.66 6.73 6.76 6.86 6.66 6.68 6.65 6.59
0.62c 0.49c
0.60c 0.62C 0.56C 0.5OC 0.48c 0.45c
1 2 3 4 5 6 7
6.90c 7.46c 8.00c 8.23c
8.36c 8.53c
6.38"
7.25c,d 7.57c,d
SCN-
C
A
0.59c 0.46c
7.34 4.60
15.8417.69-
0.53c,d 0.60C
3.77
15.14c,d
3.84 4.41 4.68 4.63 3.96
16.7515.41 14.85-
A
0.54c 0.60c d
0.57c,d 0.52 d
15.8614.61
a Lactoperoxidase (LP) is expressed in units per milliliter, and thiocyanate (SCN-) is expressed in parts per million. C, control raw milk; A, activated-LP system raw milk. b Length of incubation was 8 h. c Data are significantly (P < 0.01) different from the respective value at 0 h. d Data are significantly (P < 0.01) different from the respective value (same days of incubation) in control milk.
VOL. 57, 1991
LP SYSTEM AGAINST L. MONOCYTOGENES IN RAW MILK
anate concentration were detected in control milk at both temperatures. A bactericidal effect of an activated LP system in raw milk on L. monocytogenes strains has been detected in the present work. D values at refrigeration temperatures, 4.1 to 11.2 days, at 4°C and 4.5 to 9.7 days at 8°C, were high. The bactericidal activity persisted, however, for 5 days, preventing growth and reducing significantly L. monocytogenes numbers during refrigerated storage of raw milk. LP system activation was shown to be a feasible procedure for controlling the development of L. monocytogenes in refrigerated raw milk. ACKNOWLEDGMENT We thank Maximo de Paz for statistical assistance. REFERENCES 1. Ahamad, N., and E. H. Marth. 1989. Behavior of Listeria monocytogenes at 7, 13, 21 and 35°C in tryptose broth acidified with acetic, citric or lactic acid. J. Food Prot. 52:688-695. 2. Aune, T. M., and E. L. Thomas. 1977. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80:209-214. 3. Beumer, R. R., A. Noomen, J. A. Marls, and E. H. Kampelmacher. 1985. Antibacterial action of the lactoperoxidase system on Campylobacter jejuni in cow's milk. Neth. Milk Dairy J. 39:107-114. 4. Bjorck, L. 1978. Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45: 109-118. 5. Bjorck, L., and 0. Claesson. 1980. Correlation between concentration of hypothiocyanate and antibacterial effect of the lactoperoxidase system against Escherichia coli. J. Dairy Sci. 63:
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