The Lactoperoxidase (LP) system is widely known as a naturally antimicrobial
system present in raw milk. The system has been shown to be active against
both Gram-positive and Gram-negative microorganisms (Naidu, 2000; Marks
et al., 2001). In brief, three components are required for the
LP activation: the lactoperoxidase enzyme, thiocyanate and hydrogen peroxide.
The LP enzyme is abundant in raw milk whereas thiocyanate and hydrogen
peroxide are present at low concentrations. When activated, lactoperoxidase
catalyzes the oxidation of thiocyanate by hydrogen peroxide generating
several short-lived oxidation products in which two important products
namely hypothiocyanite (OSCN-) and hypothiocyanous acid (HOSCN),
exhibit antibacterial activity (Kussendrager and van Hooijdonk, 2000).
In developed countries, the microbiological quality of raw milk is safeguarded
during collection, storage and transportation via refrigeration. In contrast,
this practice becomes an obstacle especially in rural areas of most developing
countries due to limited facilities. The application of the LP-system
has been proposed to be a useful procedure in preserving raw milk quality.
Although the natural LP system in raw milk loses its effect within 2 h
of the milking, its antimicrobial activity can be reactivated by addition
of thiocyanate and hydrogen peroxide (IDF, 1994; Barrett et al.,
1999). As a result, the shelf life of raw milk can be significantly prolonged
by this means (FAO, 1999). In this study, we aim to evaluate the effect
of the LP-system on the growth and survival of bacterial pathogen using
Escherichia coli as a model in raw cow milk. In addition, the Keeping
Quality (KQ) of the raw milk is also examined.
MATERIALS AND METHODS
Pretreatment of Milk Samples
Raw cow`s milk samples were collected from the dairy farm of the Department
of Animal Science, Faculty of Agriculture, Chiang Mai University between
March and April 2006. Prior to any further experiment, the milk samples
were warmed to 37°C for 15 min to activate the LP system immediately.
Inoculation of Milk Samples with E. coli
Milk samples were aseptically divided into four bottles of 200
mL each. Sample A was used as untreated raw milk control; sample B was
subjected to activation of the LP system; whereas sample C and D were
initially inoculated with E. coli ATCC 25922 (~106-107
cfu mL-1 and only sample C was subsequently introduced the
LP system activation.
Activation of the LP System
The activation of the LP system (Sample B and C) was carried out by
addition of 40 mg L-1 of sodium thiocyanate (Fluka, Switzerland)
as a source of thiocyanate anion (SCN-). After 1 min of thorough
mixing, the samples were added with 30 mg L-1 of sodium percarbonate
(Riedel-de Haën, Germany) as a source of hydrogen peroxide (IDF,
1988; Dajanta, 2003) and incubated at 37°C. The samples were then
drawn from each milk sample every 2 h (from 0 to 8 h) to determine Total
Viable Count (TVC) and LP activity whereas the alcohol test was determined
every hour. The experiment was conducted in triplicate.
The milk samples were added with equal volumes of 75% alcohol (neutralized).
The development of the milk clotting was noted and referred to the end
of shelf life.
Determination of Total Viable Count
One milliliter of each milk sample was mixed with 9 mL of sterile
0.1% peptone water (Merck, Germany). Ten fold serial dilutions were then
performed in 0.1% peptone water and 1 mL of appropriate dilutions were
spread onto PCA plates (Merck, Germany) which were then incubated at 37°C
for 48 h. The resulting colonies were counted and expressed as colony
forming units per milliliter (cfu mL-1) of sample.
Lactoperoxidase Activity Assay
The LP activity assay was determined using the modified IDF method
(IDF, 1994) in which the ABTS (2,2`-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic
acid) was used as a substrate. The assay mixture consisted of 2 mL of
ABTS source (2 mM ABTS, 0.2 mM H2O2, 0.1 M phosphate
buffer pH 6.0) and 0.05 mL of diluted milk samples. Absorbance was measured
at 412 nm after 2 min incubation using a spectrophotometer (Biomate 5,
Unicam Co., Ltd., England) adjusted at 25°C. One unit of LP activity
was defined as amount of the enzyme that catalyzes 1 μmol of substrate
per minute under experimental conditions.
RESULTS AND DISCUSSION
The activity of the LP enzyme was assayed every 2 h during the storage
time (Table 1). Although there seemed to be some slightly
fluctuations during the time tested, the LP activity of the four samples
tended to decrease after 8 h of incubation.
Lactoperoxidase activity in raw milk samples during
incubation at 37°C for 8 h
The values shown are mean±SD of three replicates. Means followed
by the different capital and small letter(s) within the same column and
row, respectively are significantly different, p≤0.05 (analyzed using
Duncan`s multiple range test), NS: Not significant (p>0.05) for any
Keeping quality (h) of milk samples incubated at
37°C: Raw milk control (RM), activated LP-system raw milk (RM+LPS),
E. coli inoculated raw milk (RM+E) and E. coli inoculated
raw milk and activated LP-system (RM+E+LPS)
Effect of the LP System on Keeping Quality
According to Barrett et al. (1999), the alcohol stability can
be used as a good indicator of KQ due to its reliable and consistent results.
As shown in Fig. 1, the KQ of untreated raw cow milk
(sample A) was 5 h and in the presence of E. coli (sample
D), the KQ was decreased to 2 h. In contrast, in samples B and C where
the LP system was activated, the KQ values became higher to 8 and 7 h,
respectively. Our data have confirmed that introduction of the LP system
can extend the KQ parameters similar to those reported by Marks et
Effect of the LP System on the Growth and Survival of Microorganisms
in Raw Milk
To further explore the possible use of the LP system whether it could
serve as an alternative to cooling in Thailand, we designed the microbial-contaminated
experiment in which the bacterium E. coli was used as a
spoilage model. Milk samples, after milking, are rich in nutrients and
thus tend to spoil easily by indigenous microorganisms under inappropriate
conditions. Therefore, apart from normal milk samples, we also inoculated
E. coli in the milk samples in order to accelerate the spoiling
rate of the milk. The effect of the LP system on microbial population
was then monitored between 0 and 8 h for both inoculated and uninoculated
milk samples. As shown in Fig. 2, initial total viable
counts of samples A and B (uninoculated samples) were 9.12x104
cfu mL-1 whereas those of samples C and D including the inoculation
of E. coli at the beginning were 1.74x107 cfu
mL-1. Under storage condition (from 0-8 h), although the total
viable counts of the four milk samples reached their peak at the end of
the experiment, their pattern during the storage was slightly different.
In addition, the microbial count of sample D which was inoculated with
E. coli was much higher throughout the period as expected.
On the other hand, the microbial count of samples B and C where the LP
system was activated tended to be lower from the beginning to the end
of the experiment (Fig. 2). Interestingly, it was clearly
observed that, when the LP system was introduced (samples B and C), the
microbial counts were lower than those of the counterpart samples (sample
A and D) throughout the experiment (Table 2).
||Microbial cell count (cfu mL-1)
of raw milk samples after incubation at 37°C for 8 h
|a: Percent reduction in viable count was
calculated as follows: 100 x (viable count in the control milk at
8 h-viable count in the LP activated milk at 8 h)/(viable count in
the control milk at 8 h)
||Total viable count of microorganisms in the active and
inactive LP system raw milk samples incubated at 37°C: Raw milk
control (♦), activated LP-system raw milk (■), E. coli
inoculated raw milk (▲) and E. coli inoculated raw milk
and activated LP-system (●)
The LP system has been shown to inhibit various Gram-positive and Gram-negative
bacteria (reviewed in Naidu, 2000). Its activity has shown to be either
bacteriostatic or bactericidal effect on sensitive strains depending on
environmental conditions and bacterial species. According to El-Agamy
et al. (1992), the LP system is bactericidal to Gram-negative bacteria
and bacteriostatic to Gram-positive bacteria. Farrag et al. (1992)
reported the bactericidal effect against E. coli O157:H7
at 4°C, while at 30°C the growth of pathogen was only inhibited
for 12 h. In addition, it has been shown that the LP system could inhibit
the growth of E. coli and L. innocua at 20°C
for 24 h but did not cause any inactivation after this period (Garcia-Graells
et al., 2000).
This study, part of a programme to improve the KQ of raw cow`s milk quality
in Thailand, clearly showed that the LP system exhibited antimicrobial
effect in raw cow milk. The active LP system decreases the microbial population
in raw milk samples (both untreated and E. coli treated
experiments) under storage conditions determined. Present results also
show that the LP system affects the KQ of the raw milk and thus can be
used to extend its shelf life. It is therefore possible to use the LP
system as an alternative means to preserve raw milk quality during transportation
or in areas with lack of cooling facility.
The authors would like to thank the National Research Council of Thailand
for financial support.