Lactoperoxidase Lactoperoxidase System in MilkSystem in Milk

OSMAN ÖZEROSMAN ÖZER

506051507506051507

CONTENTSCONTENTS

NATURAL ANTIMICROBIALSNATURAL ANTIMICROBIALSLACTOPEROXIDASELACTOPEROXIDASE SYSTEMLACTOPEROXIDASE SYSTEM

Occurrence and Biosynthesis Isolation and Purification Chemistry and Structure Stability

ANTIMICROBIAL ACTIVITYANTIMICROBIAL ACTIVITY Antimicrobial spectrum

APPLICATIONS IN FOOD INDUSTRYAPPLICATIONS IN FOOD INDUSTRYCONCLUSIONCONCLUSION

NATURAL ANTIMICROBIALSNATURAL 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 lactoperoxidase–thiocyanate–hydrogen 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 SYSTEMLACTOPEROXIDASE 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 glandsThe compound constitutes approximately 1% (10 to 30 µg/ml) of the whey proteins in the milkThe 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 (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,

Hydrogen peroxide is the only approved additive for the preservation of milk in the absence of refrigeration. It maybe added at a concentration of 100–800 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 moleculeThe single polypeptide chain contains 612 amino acid residues with a molecular weight of about 80 kDaLP is a heme-containing enzymeThe heme structure has been studied in terms of its electron transfer mechanisms because the heme is essential for the development of the oxidation–reduction 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 groupThe molecular conformation of LP is thought to be stabilized by the strong binding of a calcium ionDifferent 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 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

Stability

During pasteurization, whole milk loses about 75% of its LP activity, whereas the purified LP was rendered unstable after15 minutes of exposureIt is indicated that heat denaturation of LP in milk, starts at about 70°CThe 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 10The optimum pH for the LP catalyzed reaction lies between 5 and 6LP 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 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

REACTIONSREACTIONS

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 systemThe 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 killedGram-positive, catalase negative bacteria, such as streptococci and lactobacilli are generally inhibited but not killed by the LP system

STUDIESSTUDIES

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

STUDIESSTUDIES

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 reportedreportedThe LP system was both bactericidal and bacteriostatic against S. aureus in milkS. 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

STUDIESSTUDIES

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

APPLICATIONS IN FOOD APPLICATIONS IN FOOD INDUSTRYINDUSTRY

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 plantsUsing a glucose/glucose oxidase system to generate H2O2, and supplementing milk with SCN-, makes the LP system bactericidal

APPLICATIONS IN FOOD APPLICATIONS IN FOOD INDUSTRYINDUSTRY

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

CONCLUSIONCONCLUSION

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.

CONCLUSIONCONCLUSION

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 peroxideHowever, most regulatory authorities

do not permit these chemicals as food preservatives

CONCLUSIONCONCLUSION

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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