Introduction
The biological properties of milk promote the development of microorganisms which may compromise its quality. Consequently, the use of techniques for preserving the milk matrix from its collection until processing is necessary. While refrigeration on the farm and refrigerated transport of milk from the farm to the processing plant has become the norm in the commercial dairy industry, this option is not always available in the informal sector.
The application of the so-called lactoperoxidase system (LPS) has been suggested, particularly in raw milk preservation, in situations where prompt refrigeration is difficult, and especially in developing countries and during extended or continuous failure of the electricity supply.
What is lactoperoxidase
Lactoperoxidase (LP) is a member of the peroxidase family, a group of natural enzymes that are widely distributed in nature and found in secretions of mammary glands (colostrum and milk), salivary and lacrimal glands. It is an enzyme that is synthesised in the greatest quantity by the mammary gland, having the function of protecting the glands against bacterial pathogens.
Its action alters the metabolism of bacteria and causes lesions or changes in the various structures of the bacterial cell such as the cell wall, the active and passive transport systems, glycolytic enzymes, and nucleic acids, which consequently cause interference with the microorganism’s ability to multiply.
Activation of lactoperoxidase
According to a fact sheet of the International Dairy Federation (2013) lactoperoxidase acts as a catalyst, oxidizing thiocyanate ions in the presence of hydrogen peroxide into hypothiocyanous acid. The acid dissociates in milk and the hypothiocyanate ions react with sulphydryl groups to inactivate the metabolic enzymes of bacteria. This prevents bacteria from multiplying and potentially extends the acceptable quality of raw milk.
Regarding the antibacterial effect, different groups of micro-organisms show a variable degree of sensitivity to the LPS, which may have a bactericidal or bacteriostatic effect depending on factors such as type of microorganism, pH, temperature, incubation time, and cell density. The difference in sensitivity to the LPS can probably be explained by the differences in the cell wall structure and its properties. The antimicrobial activity can cause lesions or modifications in the various structures of the microbial cell (as referred to above), leading to the death, or inhibiting the growth, of microorganisms.
Gram-negative catalase-positive organisms, such as pseudomonads, coliforms, salmonellae, and shigellae, are inhibited by the LP system. Depending on the medium, pH, temperature, incubation time, and cell density, these microorganisms may be killed. It has been shown that the LP system can increase the storage times of raw milk by delaying the growth of psychrotrophs (Wolfson and Sumner, 1993).
Gram-positive bacteria are more resistant. The LPS can however be both bactericidal or bacteriostatic toward S. aureus, an important cause of bovine mastitis. Listeria monocytogenes is a troublesome pathogen in the dairy industry, so much so that the consumption of milk and contaminated products can result in foodborne listeriosis. L. monocytogenes can be found in raw milk, poorly pasteurised milk, and its derivatives. The risk of listeriosis is amplified by the ability of L. monocytogenes to grow at low temperatures and its relative resistance to heat when compared to other bacteria. The system can also be bactericidal or bacteriostatic against L. monocytogenes.
The antibacterial activity of the LP system on the growth and survival of L. monocytogenes in UHT milk and French soft cheese has been determined experimentally. In UHT milk, the presence of the LP system either inhibited growth or completely inactivated inoculated cells. Complete inactivation occurred at different times depending on initial inoculum concentration and storage temperature. In another study, research workers determined that the LP system, using the inherent milk lactoperoxidase, effectively inhibited the growth of L. monocytogenes and S. aureus at 35 and 37°C, respectively.
Prolonging the shelf-life of milk produced in the informal sector
The natural lactoperoxidase system in raw milk is effective for about two hours. Adding a pre-packaged activator (IDF 2013) containing thiocyanate and a source of hydrogen peroxide such as sodium percarbonate activates and extends the effects of the natural lactoperoxidase system (LPS) in raw milk. Where refrigeration is not possible, the addition of the pre-packaged activator increases the acceptable quality of raw milk for about 24 hours at 15°C or between 6 and 8 hours at 30°C, allowing smallholders sufficient time to store and/or transport their milk to a central depot for processing.
The IDF fact sheet warns that while the addition of a chemical activator to raw milk may be the only choice for some small dairy producers located in rural areas, these chemicals must be used correctly. They should not be used to disguise poor-quality milk and should only be added at safe levels.
The antimicrobial agents of the LPS delay milk deterioration, thus preserving the microbiological quality of the milk. The method can be applied to the raw milk of several species, although the system’s effectiveness depends on the type of microbiological contamination, the number of microorganisms, and the milk temperature during its use.
The Codex Alimentarius Commission (CAC) provides guidelines that focus on the application of the LPS to avoid milk deterioration during collection and transportation to the processing plant when proper refrigeration is not feasible. Since the adoption of these guidelines, a substantial amount of data on the effectiveness of the LPS has been obtained not only from laboratory and field studies but also from the adoption of large-scale use of the system in commercial milk production in some countries such as Cuba, Colombia, Peru, Venezuela, Cameroon, Kenya, Uganda, Pakistan, and others (CAC 1991). Overall, this data confirms the effectiveness of the LPS in preventing the deterioration of raw milk at room temperature.
Utilising the lactoperoxidase system (LPS) at different temperatures and times within the structure defined by CAC (1991) is subject to:
- the application of the principles of good hygiene practice in milk production that is necessary in order to ensure milk of good microbiological quality;
- The storage temperature of the milk treated with the LPS, determines the inhibitory effect of the treatment.
It is important to note that unlike pasteurisation, the LPS does not make milk safer for consumption; it only preserves the initial quality of the product from the farm until it reaches the processing plant.
Possible application of the lactoperoxidase system
in the commercial dairy sector
Commercially refrigerated and transported milk
In Western Countries, including South Africa, milk is cooled and stored for increasingly long periods because of distribution circumstances. After 2 d, such milk can deteriorate through the multiplication of psychrotrophic organisms (mainly pseudomonads), which produce extremely heat-resistant lipases and proteases which survive pasteurization. These enzymes can render the milk testing positive for the Alizarol test at the processing plant platform or it can lead to spoilage of dairy products such as pasteurised and UHT milk, butter, and cheese.
The bactericidal effects of the activated LPS are greater at low temperatures (0- 5°C). Studies found that observable multiplication of surviving natural milk microflora started after 12 d in LP system-treated milk at these temperatures, compared to 4 d in untreated milk. After 22 d, viable counts in untreated milk reached 106 -107 cells per ml compared to about 101 cells per ml in LPS-treated milk. In another study, results showed that at 4°C the standard plate count in LPS milk remained basically unchanged for at least 104 h, whereas bacterial multiplication in the controls started after 48 h.
Fermented dairy products
Bovine milk lactoperoxidase is relatively heat resistant, with the enzyme being only partially inactivated by short-time pasteurization at 74°C, for 15 seconds leaving sufficient activity to catalyse the reactions between thiocyanate and hydrogen peroxide. The antimicrobial compounds in the LPS may interfere with the activity of lactic acid starter cultures causing problems during the manufacture of fermented products such as cheese, yoghurt etc. The effect of the LP system on the behaviour of a thermophilic starter culture commonly used in the dairy industry for cheesemaking found this starter culture to be very sensitive to the LP system; the activity of the starter culture was strongly reduced. Other researchers however made acceptable varieties of soft and hard cheeses from chemically treated milk. Using the LP system, it was possible to compare cheese yields with products made from untreated and treated 8 d old milk. Chemically treated milk increased yields by 2% as compared to control cheese.
Researchers established some factors that can affect the use of raw milk treated with the LPS for producing fermented dairy products. These included the type of milk, the type of starter cultures used, and their inoculation rate, as well as the concentration of thiocyanate and hydrogen peroxide used to activate the system. The yield of fresh cheese from raw cow’s milk treated with the LPS was significantly higher than that of cheese made from milk without using the system. Related research also determined that the taste of fermented milk and cheese could be improved by LPS action, by changing the balance of the microbial types in the raw milk. Yoghurt made from milk treated with the LPS did not present any significant differences in the chemical composition or sensory properties when compared to control yoghurt. In another study LPS activation of the milk promoted an increase in the yield and sensory quality of cheese. Evidence from studies indicates that the LPS has no negative effects on the quality of cheese and fermented products when using milk that has undergone an appropriate heat treatment after using the system.
Conclusion
It is possible that the LPS can have a significant effect in producing dairy products on an industrial scale, since thermal processes at lower temperatures provide greater nutrient retention in foods that are more sensitive to heat ‒ such as creams and dairy drinks ‒ which translates into final product quality, in addition to energy savings.
It is also important to consider that the current trend of the 21st-century consumer is towards more natural foods. These consumers are careful and attentive to preservation methods and health promotion, thus reaffirming the future potential of the LPS in the production of dairy products.
