Lactoperoxidase

System in Milk
OSMAN ZER
506051507
CONTENTS
NATURAL ANTIMICROBIALS
LACTOPEROXIDASE
LACTOPEROXIDASE SYSTEM
Occurrence and Biosynthesis
Isolation and Purification
Chemistry and Structure
Stability
ANTIMICROBIAL ACTIVITY
Antimicrobial spectrum
APPLICATIONS IN FOOD INDUSTRY
CONCLUSION

NATURAL ANTIMICROBIALS
Natural antimicrobials include agents found in
plants, microbes, insects, and animals
The antimicrobials isolated from these products
are generally broad-spectrum agents providing
protection against bacteria, fungi, parasites, and
viruses
Antimicrobial substances present in bovine milk
are lactoferrin, lysozyme, lactoperoxidase, and
lactoglobulins



LACTOPEROXIDASE

Lactoperoxidase (LP), a hemoprotein present in
milk, tears, and saliva
The lactoperoxidasethiocyanatehydrogen
peroxide interaction constitutes what is referred
to as the LP system, wherein hydrogen peroxide
serves as a substrate for LP in oxidizing
thiocyanate (SCN-) and iodide ions, resulting in
the generation of highly reactive oxidizing
agents


LACTOPEROXIDASE SYSTEM
The LP system has the ability to inhibit
bacteria, fungi, parasites, and viruses and
thus is considered a broad-spectrum
natural antimicrobial contributing to
protecting the gut of weaning calves from
enteric pathogens, protecting the
mammary gland from disease, and indeed
preserving milk

Occurrence and Biosynthesis

LP is synthesized and secreted by ductal epithelial cells
of the mammary gland and other exocrine glands
The compound constitutes approximately 1% (10 to 30
g/ml) of the whey proteins in the milk
The level of LP in bovine milk is about 20 times higher
than that of human milk and changes constantly during
the postpartum period.
Thiocyanate, which is required for the antimicrobial
activity of the LP system, may be present in significant
amounts in milk, whereas hydrogen peroxide may be
generated by microbial flora, usually bacteria in
mammary gland



Hydrogen peroxide (H
2
O
2
) is the third
component of the LP system.Many lactobacilli,
lactococci, and streptococci produce sufficient
H
2
O
2
under aerobic conditions to activate the LP
system
Hydrogen peroxide may be added or may be
generated by the addition of H2O2 generating
systems such as sodium percarbonate, glucose
oxidase,




Hydrogen peroxide is the only approved
additive for the preservation of milk in the
absence of refrigeration. It maybe added
at a concentration of 100800 ppm.
Hydrogen peroxide is highly toxic for
mammalian cells.
However, at low concentrations and in the
presence of LP and SCN- mammalian
cells are protected from this toxicity

Occurrence and Biosynthesis
In bovine milk, the initial concentration of LP in
colostrum is low, increasing to a peak at 4 to 5
days postpartum, after which it declines to a
level considered relatively high and remains
unchanged at that level during lactation
To combat infections, the concentrations of LP
and SCN increase in milk from infected bovine
udders as compared with normal, healthy
udders


Isolation and Purification

The essential steps involved with isolation
of LP include casein precipitation with
rennet, adsorption of whey proteins
through ion-exchange methods, elution,
fractionation, and final purification

Chemistry and Structure

It has been determined that bovine LP is
comprised of a single peptide chain, with eight
disulfide bonds contributing to the rigidity of the
molecule
The single polypeptide chain contains 612
amino acid residues with a molecular weight of
about 80 kDa
LP is a heme-containing enzyme
The heme structure has been studied in terms of
its electron transfer mechanisms because the
heme is essential for the development of the
oxidationreduction reaction associated with LP
activity.




Chemistry and Structure
The iron content of LP is 0.07%, which
corresponds to 1 iron atom per LP molecule as
part of the heme group
The molecular conformation of LP is thought to
be stabilized by the strong binding of a calcium
ion
Different preparations of natural LP may have
different N-terminal amino acid residues. This
heterogeneity may be a result of variation in
terms of isolation methods




Stability

LP system stored in airtight containers lost only
35% of the initial thiocyanate concentration
during 18 months and that the system was
strong enough to kill 10
6
CFU/ml of four test
organisms.
When the LP system was stored in the presence
of air it lost thiocyanate activity after 7 days, but
after 516 days it was still able to kill inocula of
106 CFU/ml Pseudomonas aeruginosa,
Staphylococcus aureus, and Candida albicans
and Escherichia coli within 2 hours, 4 hours, and
1 week, respectively


Stability
During pasteurization, whole milk loses about
75% of its LP activity, whereas the purified LP
was rendered unstable after15 minutes of
exposure
It is indicated that heat denaturation of LP in
milk, starts at about 70C
The calcium ion concentration influences the
heat sensitivity of LP. The heat stability of LP is
lower under acidic (pH 5.3) conditions and may
be related to the release of calcium from the
molecule






Stability
LP is deactivated during storage at pH 3
with partial denaturation at <pH 4,
whereas there is no deactivation of the
enzyme at values of up to pH 10
The optimum pH for the LP catalyzed
reaction lies between 5 and 6
LP is not inactivated by the gastric juice of
an infant (pH 5) but that pepsin at pH 2.5
inactivated LP



ANTIMICROBIAL ACTIVITY

LP is an enzyme with a primary function to
oxidize thiocyanate at the expense of H
2
O
2

to generate products that kill or inhibit the
growth of many species of microorganisms
With the oxidation of SCN-, the generation
of OSCN- (hypothiocyanate) and HOSCN
(hypothiocyanous acid) are in equilibrium,
and at the pH of maximal LP activity (pH
5.3), they exist in equal quantities

REACTIONS
ANTIMICROBIAL ACTIVITY
The oxidation of sulfhydryl (SH) groups of
microbial proteins by OSCN (hypothiocyanate)
and HOSCN (hypothiocyanous acid) is
considered to be the key to the antimicrobial
action of the LP system
The structural damage to microbial cytoplasmic
membranes through oxidation of SH groups
causes leakage of potassium ions, amino acids,
and peptides into the medium as well as
inhibition of the uptake of glucose, amino acids,
purines, and pyrimidines and subsequent
synthesis of proteins, RNA , and DNA


Antimicrobial spectrum

The LP system could elicit bactericidal activity
on a variety of susceptible microorganisms
including bacteria, fungi and viruses
The molecular mechanism of such inhibitory
effects depend on the type of electron donor,
test media, temperature, and pH and could
range from oxidative killing to blockage of
glycolytic pathways or interference in cytopathic
effects


Antimicrobial spectrum
Different groups of bacteria show a varying
degree of sensitivity to the LP system.
Gram-negative, catalasepositive organisms,
such as pseudomonas, coliforms, salmonellae
and shigellae, are not only inhibited by the LP
system but also, depending on the medium
conditions (pH, temperature, incubation time,
cell density) may be killed
Gram-positive, catalase negative bacteria, such
as streptococci and lactobacilli are generally
inhibited but not killed by the LP system



STUDIES
It was reported that activation of the LP system
in goat milk was bactericidal against
Pseudomonas fluorescens and resulted in mean
decreases in the levels of P. fluorescens by 1.69
log units at 4 C and 1.85 log units at 8 C during
the first 24 h.
The LP system showed a bacteriostatic effect
against E. coli in South African goat milk kept at
30 C


STUDIES
Campylobacter jejuni is a major cause of acute
enteritis in humans and milk has been
associated with several outbreaks of C. jejuni
enteritis.The bactericidal effect of the LP system
against C. jejuni in milk has been reported
The LP system was both bactericidal and
bacteriostatic against S. aureus in milk
S. aureus is a major causative agent of bovine
mastitisand poses a human health problem
since this pathogen can be shed into milk from
mastitic udders


STUDIES
The LP system exhibited a bactericidal
effect against L. monocytogenes in
Saanen and South African Indigenous
goat milk kept at 30 C
Listeria monocytogenes is a pathogen of
major concern to the dairy industry as
food-borne listeriosis has been related to
consumption of contaminated milk and
milk products




APPLICATIONS IN FOOD
INDUSTRY
The most widely recommended industrial
application of the LP system in food
production is in the dairy industry for the
preservation of raw milk during storage
and/or transportation to processing plants
Using a glucose/glucose oxidase system
to generate H
2
O
2
, and supplementing milk
with SCN-, makes the LP system
bactericidal


APPLICATIONS IN FOOD
INDUSTRY
However, other novel applications of the LP system are
being explored. If the LP system is activated immediately
prior to application of approved thermal processes, the
shelf-life of dairy products may be extended significantly
and high-temperature processes may be replaced with
more economical lower temperature treatments.
In addition to energy savings, LP-low temperature
thermal processes may provide better nutrient and/or
quality retention for highly heat-sensitive foods such as
salad dressings, spreads, beverages, dips and desserts

CONCLUSION
Although the LP system was primarily
meant for preservation of raw milk in warm
tropical climates where cooling facilities
are not available, now its application is
growing beyond raw milk preservation and
it is finding its way to commercial
applications.

CONCLUSION
LP system in milk or other food systems
have been based on the use of pure form
of added potassium or sodium thiocyanate
as a thiocyanate source and sodium
percarbonate as a source of hydrogen
peroxide
However, most regulatory authorities
do not permit these chemicals as food
preservatives


CONCLUSION
Before industrial applications can be
achieved in food, alternative natural
methods of achieving suitable SCNK and
H
2
O
2
sources must be developed.

These could include the use of special
animal feed supplements and/or addition
of thiocyanate enriched vegetable extracts
to the milk or food system.









TEEKKRLER!!