Lactoperoxidase System in Milk

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Lactoperoxidase System in Milk

• OSMAN ÖZER
• 506051507

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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

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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

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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

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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

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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 µg/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

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• Hydrogen peroxide (H2O2) is the third component
  of the LP system.Many lactobacilli, lactococci,
  and streptococci produce sufficient H2O2 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,

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• 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

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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

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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

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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.

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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

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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 106 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

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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

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Stability

• LP is deactivated during storage at pH 3 with
  partial denaturation at ltpH 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

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ANTIMICROBIAL ACTIVITY

• LP is an enzyme with a primary function to
  oxidize thiocyanate at the expense of H2O2 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

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REACTIONS
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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

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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

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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

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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
  ºC and 1.85 log units at 8 ºC during the first 24
  h.
• The LP system showed a bacteriostatic effect
• against E. coli in South African goat milk
  kept at 30 ºC

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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

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STUDIES

• The LP system exhibited a bactericidal effect
  against L. monocytogenes in Saanen and South
  African Indigenous
• goat milk kept at 30 ºC
• 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

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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 H2O2, and supplementing milk with SCN-,
  makes the LP system bactericidal

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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

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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.

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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

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CONCLUSION

• Before industrial applications can be achieved in
  food, alternative natural methods of achieving
  suitable SCNK and H2O2 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.

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TESEKKÜRLER!!