APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1991, p. 3355-3360 Vol. 57, No.

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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 monocy-
togenes 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 demon-
strated 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 refriger-
ation 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
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 antimicro-
bial activity of artificially added LP systems on L. monocy-
togenes 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
of incubation has recently been described (14). The use of
the LP system decreases thermal resistance of L. monocy-
togenes 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 inoc-
ulated 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 sys-
tem. 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 solu-
tion; Merck) previously filter sterilized through 0.22-,um-
pore-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 moni-
tored 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-temperature-
treated milk which was incubated at 20°C for 3 days.
Microbiological analyses. L. monocytogenes was enumer-
ated on duplicate plates of Listeria selective medium, Ox-
3356 GAYA ET AL. 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 Lm 1 Lm 2 Lm 3 Lm 4
(days)
C A C A C A C A
8b 4.08 4.05 3.90' 3.81c 3.83c 3.78c 3.80c 3.80c
1 3.82c 3.94 3.87' 3.64cd 3.79C 3.76c 3.88c 3.85c
2 4.03 3.83c,d 3.80c 3.74C 3.80c 3.73C 3.84c 3.66c,d
3 4.03 3.76 3.79C 3.57c,d 3.75C 3.70c 3.85C 3.31c,d
4 3.97 3.33c.d 3.60c 3.30c,d 4.07 3.48c.d 3.81c 3.43cd
5 3.94 3.14d 3.76c 3.00c,d 4.12C 3.54c,d 3.94 2.46c,d
6 3.97 3.35c.d 3.92 3.12c.d 4.17c 3.32c,d 3.90c 3.18c,d
7 4.12c 3.05c.d 4.01 3.31c,d 4.70c 3.65c,d 3.67C 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, En-
gland), 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-eth-
ylbenzthiazoline-6-sulfonic acid) (Sigma Chemical Co., St.
Louis, Mo.) at 412 nm with a Beckman DU7 spectrophotom-
eter (36). The thiocyanate concentration was evaluated in
duplicate after precipitation with 20% (wt/vol) trichloroace-
tic 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 (De-
partment of Biomathematics, University of California at Los
Angeles, Los Angeles), and regression analysis was per-
formed 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 least-
significant-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 incuba-
tion on L. monocytogenes counts.
In control milk incubated at 4°C, counts of L. monocyto-
genes Lm 1 remained virtually unchanged throughout incu-
bation. 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
Log counts of L. monocytogenes strains in milka
Length of
incubation Lm 1 Lm 2 Lm 3 Lm 4
(days)
C A C A C A C A
8b 4.10 4.02 3.85c 3.78c 3.85c 3.80C 3.87 3.76c
1 4.02 3.84c.d 3.89 3.75c 3.78c 3.78c 3.90 3.85c
2 4.04 3.62c,d 3.85c 3.75c 3.83' 3.75c 3.90 3.70c,d
3 4.09 3.31c,d 3.85c 3.33c,d 4.06 3.60c,d 3.89 2.91c,d
4 4.11 2.74c,d 3.92 3.41c,d 4.36c 3.52c,d 4.01 3.37c,d
5 4.14 2.93c,d 3.83c 2.57c,d 4.47c 3.58cd 4.11 2.40c,d
6 4.31c 2.87c,d 4.29c 3.41c,d 4.75c 3.17c,d 4.88c 2.93 d
7 4.29c 2.75cd 4.35c 3.74c,d 4.94c 4.lld 5.01c 2.33c,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.
VOL. 57, 1991 LP SYSTEM AGAINST L. MONOCYTOGENES IN RAW MILK
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 Strain'a Results with control milkb
temp Day Equation
40C Lm 1 1 y = 4.061 - 0.214x
Lm 2 4 y = 3.966 - 0.082x
Lm 3 3 y = 3.912 - 0.060x
Lm 4 7 y = 3.902 - 0.016x
80C Lm 1 0
Lm 2 5 y = 3.918 - 0.015x
Lm 3 2 y = 3.923 - 0.069x
Lm 4 0
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 activat-
ed-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 equa-
tions 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 respec-
tive 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 logarith-
mic-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
3357
Results with activated-LP system milkb
P Day Equation P
0.205 7 y = 4.066 - 0.147x 0.000
0.000 5 y = 3.944 - 0.166x 0.000
0.003 6 y = 3.902 - 0.089x 0.000
0.108 5 y = 4.039 - 0.245x 0.000
7 y = 3.974 - 0.201x 0.000
0.327 5 y = 4.009 - 0.226x 0.000
0.074 6 y = 3.924 - 0.103x 0.000
7 y = 3.957 - 0.255x 0.000
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. mono-
cytogenes NCTC 11994 counts by two log cycles after 6 days
at 10°C was achieved with sterile ultrahigh-temperature-
treated 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 demon-
strated 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 acti-
vation, 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 Total viable count pH LP SCN-
incubation
(days) C A C A C A C A
8b 5.50c 5.17d 6.71 6.69 0.63c 0.62C 6.38 18.09c,
1 5.64c 5.13d 6.70 6.69 0.60c 0.58c 4.35 17.22-
2 6.32c 5.20d 6.74 6.75 0.59C 0.55" 3.65c 15.47-
3 6.80c 5.1d 6.81 6.82 0.60C 0.60c 3.66c 15.94-
4 7.44c 5.06c,d 6.69 6.74 0.56c 0.65d 4.89 14.83-
5 7.72c 5.45d 6.66 6.75 0.52C 0.63c,d 4.32 14.33-
6 7.96c 5.83cd 6.60 6.64 0.49c 0.59C,d 5.02 16.04-
7 8.06c 6.52c,d 6.55 6.70 0.48c 0.62c,d 4.10 16.15c,d
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 re-
corded 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. monocyto-
genes inoculation considerably lower than those due to the
inoculum, no significant differences throughout milk incuba-
tion 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 involve-
ment of milk microflora in lactoperoxidase inactivation.
The level of thiocyanate (Tables 4 and 5) was also signif-
icantly 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 Total viable count pH LP SCN-
incubation
(days) C A C A C A C A
8b 5.49c 5.22d 6.69 6.66 0.62c 0.59c 7.34 15.84-
1 5.82c 5.15d 6.75 6.73 0.49c 0.46c 4.60 17.69-
2 6.90c 5.22d 6.74 6.76 0.60c 0.53c,d 3.77 15.14c,d
3 7.46c 5.48c,d 6.81 6.86 0.62C 0.60C 3.84 16.75-
4 8.00c 5.88cd 6.58 6.66 0.56C 0.54c 4.41 15.41
5 8.23c 6.38" 6.52 6.68 0.5OC 0.60c d 4.68 14.85-
6 8.36c 7.25c,d 6.38 6.65 0.48c 0.57c,d 4.63 15.86-
7 8.53c 7.57c,d 6.28 6.59 0.45c 0.52 d 3.96 14.61
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, prevent-
ing growth and reducing significantly L. monocytogenes
numbers during refrigerated storage of raw milk. LP system
activation was shown to be a feasible procedure for control-
ling the development of L. monocytogenes in refrigerated
raw milk.
ACKNOWLEDGMENT
We thank Maximo de Paz for statistical assistance.
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