Lukas Christen, PhD
Expected Key Learning Outcomes
Definition and the reason human milk need to be pasteurised
Discussion of the most commonly used pasteurisation method and its effect on human milk
Negative aspects of alternative pasteurisation methods
Human milk is beneficial to the preterm infant due to its bioactive components, such as immunological and developmental proteins, digestive enzymes, and cellular components , , . However, many mothers are unable to produce a milk supply adequate to meet their preterm infants’ needs while in the neonatal intensive care unit (NICU) . Efforts to initiate lactation and/or increase milk volume are most desirable but next best is the use of donor human milk , , .
Worldwide, milk banks are providing donor human milk to NICUs, where recipient safety is the highest priority. However, safety is approached very differently between and within countries. For example, all (but one) hospitals in Norway use unpasteurised donor human milk in NICUs. Use of raw donor human milk has a long tradition but requires strict control and frequent donor screening during the donating period. Additionally, all milk is analysed for bacteria and destroyed if it contains pathogens or a total bacterial count of >100,000 colony-forming units (CFU)/mL. Milk containing a count of <10,000 CFU/mL is used for the smallest preterm infants . Interestingly, Norway has one of the lowest incidence rates of necrotising enterocolitis and late onset sepsis , which may be partly due to the use of raw not pasteurised donor human milk.
Most milk bank and NICU guidelines require donor human milk to be pasteurised , , . This process prevents disease transmission from donor to preterm infant by eliminating bacteria and viruses , , . The most common pasteurisation method used worldwide is a heat treatment called Holder pasteurisation , , , . This treatment reduces vegetative bacteria sufficient to meet milk banking guidelines (MBG). However, the process results in significant loss of activity of important bioactive components , , .
Since most MBGs require pasteurisation as a main safety step, a treatment is required to increase the pasteurised donor human milk quality, which might improve the health outcomes of preterm infants. The alternative must meet the same safety standard as Holder pasteurisation when reducing micro-organisms in human milk but increase its bioactive component retention.
Pasteurisation is a process whereby food, usually a liquid, is partially sterilised, making it safe for consumption and extending its shelf-life. Thermal treatments are most common in the food industry, although other treatments that reduce nutritional damage but maintain taste, smell and appeal during processing are of increasing interest . Alternative pasteurisation methods have been studied using bovine milk but the results are not always applicable to human milk. This is primarily because the main focus of the dairy industry is to extend the shelf-life while maintaining food safety and optimising processing costs. The dairy industry inactivates digestive enzymes to increase shelf life of the milk. However, in human milk, retention of digestive enzymes such as bile salt stimulated lipase (BSSL) may be a high priority to improve preterm infants’ nutrition , . High retention of immunological and developmental components in human milk is desirable to enhance the infants’ immature immune system and support physiological development. Thus, the selection criteria for pasteurisation methods differ for human and bovine milk.
Pasteurisation methods include chemical, biological, physical, and separation methods (▶Fig. 18.1). Since adding chemical or biological agents to human milk is generally considered not to be safe or appropriate, processing should be restricted to physical and separation methods.
This method requires human milk to be heated at a specific temperature for a set time. The two major thermal pasteurisation methods are the low temperature, long time (LTLT) and the high temperature, short time (HTST) treatments. Both methods equally destroy the most heat resistant of the non-spore-forming pathogenic organisms, Myco-bacterium tuberculosis and Coxiella burnetii .
Ultrahigh temperature (UHT), whereby milk is heated at > 135 °C for 1–2 seconds, is considered to be a sterilisation method and therefore has to be distinguished from pasteurisation.
Heat alters a wide variety of biological components within micro-organisms, thus inactivating them by multiple mechanisms. The main effects of heat are DNA strand breaks, enzymes inactivation and protein coagulation. Cell death is also caused by thermally-induced membrane damage, which results in loss of nutrients and ions. A minor cause of thermal inactivation is ribosome degradation and ribonucleic acid (RNA) hydrolysis . Many mechanisms of inactivation may be an advantage when inactivating a broad spectrum of micro-organisms. However, a major disadvantage of thermal pasteurisation is that its enzyme inactivation and protein coagulation is not specific to microorganisms but also affects bioactive components in human milk.
MBGs define thermal pasteurisation by holding temperature and holding time. However, the full temperature profile is not specified. The time taken to heat or cool the milk depends on many variables, including milk volume, heat exchange surface-to-milk volume ratio, heat transfer rate of human milk (depends on milk density and composition), and heat transfer rate of the bottle (depends on bottle wall thickness and glass/plastic material) , . Milk can therefore be treated differently even when using the same MBG, which can lead to pasteurised donor human milk of differing quality in terms of bioactive components.
The most common pasteurisation method for human milk is the LTLT or Holder method, which is used in milk banks worldwide. Bottled human milk is heated in a water bath at 62.5 °C for 30 minutes , , . This method can reduce vegetative bacteria by 5 log10  but Bacillus endospores are very heat resistant and not inactivated by this method . Common viruses found in human milk are also eliminated, including human immunodeficiency virus type I (HIV-1) , cytomegalovirus (CMV)  and human T-lymphotropic virus type I (HTLV-1) . However, HTLV-1 studies were not in human milk. Unfortunately, Holder pasteurisation inactivates a wide range of human milk components , , .
Increasing bioactive component retention with Holder pasteurisation has been investigated. Two independent studies suggested reducing holding temperature and/or holding time. Changing parameters to 62.5 °C for 5 minutes, 56 °C for 15 minutes, or 57 °C for 30 minutes increased the retention of immunological proteins to above 90% while reducing vegetative bacteria by 2-log10, 2-log10, and 3-log10, respectively , . However, bacterial reductions do not meet the requirements of all MBG, and the essential enzyme BSSL is likely to be lost completely with such parameter changes.
HTST is a treatment whereby milk is heated to 72 °C for 15 seconds. A short treatment time is preferred by the dairy industry to reduce energy consumption while maintaining better milk colour and flavour compared with LTLT pasteurisation.
HTST pasteurisation has shown to eliminate HIV, hepatitis B virus, hepatitis C virus  and CMV . However, some researchers reported no change in BSSL, lactoferrin, and secretory immunoglobulin A (sIgA) while others found a reduction in immunological proteins and complete BSSL inactivation , , , . One study found differences in immunoglobulin retention when milk was treated at different flow rates, and concluded that this was due to variations in heating apparatus, sample size, conditions before/after pasteurisation, and analyses . Future studies should track pre- and post-bacterial counts, temperature profiles, and biomarkers such as alkaline phosphatase (ALP) to reduce such discrepancies.
Overall, optimising bioactive component retention, e.g., lactoferrin, lysozyme, and sIgA, is possible by altering pasteurisation temperature and time. However, BSSL retention is not possible with thermal pasteurisation as its degradation starts at around 45 °C, which is below the bacterial inactivation temperature . As such, cold pasteurisation is necessary to protect enzymes such as BSSL.
Liquids placed under a high pressure result in damage to cell membranes of micro-organisms. Bacterial reduction in milk similar to that after thermal pasteurisation can be achieved by applying a pressure of 400 MPa for 15 minutes or 500 MPa for 3 minutes . Reduction of vegetative bacteria occurs above 100 MPa depending on bacterial species and food treated. Gram-negative bacteria are generally more pressure sensitive than gram-positive bacteria . Spores tend to have a high-pressure tolerance of over 1200 MPa , although the pressure can be reduced with additional heat application. Pressure-damaged proteins differ from heat-denatured proteins, which may have nutritional and biological consequences . Additionally, pressures above 230 MPa reduce casein micelle size in bovine milk, changing their viscosity and turbidity . High pressure also causes alterations in crystallisation behaviour  and a phase change in bovine milk fat . Along with low reductions of Escherichia coli, such alterations to bovine milk indicate that pressure pasteurisation is unlikely to be a suitable method for human milk.
Power-ultrasound (20–100 kHz) is an emerging technology in food preservation , , . Power-ultrasound creates cavitation, that is the formation, growth, and implosive collapse of bubbles in liquids . Pressure changes from these implosions create shock waves that disrupt bacterial cell membranes resulting in cell lysis , .
Studies using bovine milk and fruit juices have shown that ultrasonication can eliminate various food-borne pathogens at least as well as thermal pasteurisation , . One study showed that the growth of Trichophyton mentagrophytes was significantly reduced after ultrasonication. Feline herpes virus (an enveloped virus) was also significantly reduced, although there was no effect on feline calicivirus (a non-enveloped virus), suggesting damage to the viral envelope .
Additionally, a study has shown that ultrasonication of human milk can reduce E. coli while keeping the BSSL retention > 90%. However, waste heat increased the human milk temperature to above 50 °C. However, to protect BSSL, human milk must be kept at a low temperature during sonication .
Synergistic effects between ultrasound and other processing technologies are used to optimise food quality or reduce treatment time and energy , , . Thermo-ultrasonication appears promising due to improved energy efficiency and bacterial reduction .
Thermo-sonication of human milk has been found to inactivate E. coli and Staphylococcus epidermidis by 3 log10 with a greater retention of sIgA (91%), lysozyme (80%), lactoferrin (77%), and BSSL (45%) than with Holder pasteurisation. Thermosonication also reduced mean particle size of human milk fat globules from 4.6 µm to 0.6 µm after 5 minutes. However, the effect of smaller milk fat globules and on fat absorption by preterm infants is unknown .
Ultraviolet (UV) is part of the electromagnetic spectrum and subdivided into UV-A (320– 400 nm), UV-B (280–320 nm), UV-C (200–280 nm) and vacuum-UV (100–200 nm). UV-C 250–270 nm has the most germicidal effect and is capable of destroying micro-organisms, such as bacteria, viruses, protozoa, yeasts, moulds, and algae , . At this wavelength DNA bases, mainly pyrimidine and purine, absorb UV-C energy promoting chemical reactions. Common products of these reactions are pyrimidine dimers, other pyrimidine adducts, and pyrimidine hydrates, sometimes involving cross-links with proteins and breaks in DNA strands . Such DNA damage prevents micro-organisms from reproducing and eliminates risk of disease . Bacteria and viruses are inactivated at a similar UV-C dosage but protozoa and fungi need up to a 4-fold and 10-fold greater dosage, respectively .
UV-C is commonly used in surface sterilisation of fruit and vegetables, and treatment of water for drinking and swimming pools. The depth of UV-C penetration in liquid is dependent on soluble solids and suspended matter , , . UV-C treatment of opaque liquids such as human milk is impeded due to its fat and casein content. Consequently, at 254 nm, the absorption coefficient for milk is greater (300 cm–1) than for beer (20 cm–1) or water (0.1 cm–1) . However, a turbulent flow of opaque liquids (e.g., fruit juice or bovine milk) around a UV-C source can enable its UV-C treatment , . Turbulence apparently results in transport and exposure of micro-organisms to photons at the interface between the opaque liquid and UV source.
However, UV-C can damage human milk components by direct oxidation (type 1 photo-oxidation), where amino acids absorb the light, and by indirect oxidation (type 2 photo-oxidation), where reactive oxygen species damage human milk components . Little UV-C-induced protein damage in protein solutions and bovine milk was found compared to damage caused by thermal pasteurisation , . However, UV-C irradiation has also been shown to cause loss of apo-α-lactalbumin milk protein structure and function potentially limiting its use for milk pasteurisation .
A human milk study showed that UV-C irradiation can reduce vegetative bacteria by 5-log10, achieving a higher protein retention than with the Holder method. BSSL and ALP activity were not reduced and retention rates of lactoferrin, lysozyme, and sIgA were 87%, 75%, and 89%, respectively. No changes in fatty acid profile or bacteriostatic property of human milk were reported , .
The ability of ionising radiation, including electron, x-ray, and gamma irradiation, to reduce microbial load in industrial sterilisation of products is well known. Due to its high energy density, it is highly potent in micro-organism reduction. However, equipment used to generate ionising radiation is not only expensive, but the operator needs specific training as well as sophisticated protection.
Gamma radiation is reported to damage the nutritive quality of bovine milk, with severe effects on vitamin A, C, and E, moderate effects on carotenoids and riboflavin, and little effect on ALP .
Unlike the sterilisation of medical devices and industrial products, use of electron, gamma, and x-ray irradiation for food is limited and not generally recommended for liquid foods.
Microwaves are a non-ionising, heat-generating radiation, which can be used for thermal pasteurisation. Equal heat distribution throughout the liquid is important to ensure effective pasteurisation. However, microwave pasteurisation is known for its non-uniform heat distribution, producing hot and cold spots in the liquid . Flow-through systems are less prone to heterogeneous heat distribution . As such, microwave irradiation is just another thermal method with the same advantages and disadvantages as the Holder pasteurisation method.
This method applies high voltage (20–80 kV/cm) pulses to contaminated liquid for > 1 second. This causes permeability of cell membranes resulting in lysis. The method is effective in inactivating vegetative bacteria, yeast, and moulds  but also reduces lipase by 70–85%, peroxidase by 30– 40%, and ALP by 5% . However, these were not human milk studies and therefore the findings may not be directly applicable. Pulsed electric field technology is still in development and has not been tested for commercial use.
In this technique, a strong magnetic field of 2– 100 T is applied for 25 µs to 2 ms at a frequency of 5–500 kHz. The antibacterial mechanism is unknown but may involve alteration of ion fluxes across the cell membrane . A 2 log10 reduction of vegetative bacteria was achieved in bovine milk and orange juice but bacterial spores were not affected . Currently, the equipment is very expensive and its potential appears to be limited.
Bactofugation is the removal of microbial cells from milk using high centrifugal forces. This method is most efficient against microbial cells of high density, especially bacterial spores (1.2–1.3 g/l) and somatic cells. The method can remove around 98% of anaerobic spore-forming organisms and 95% of aerobic spore-forming organisms. Vegetative bacteria are more difficult to separate due to their much lower density, and reductions of about 89% can be achieved . For dairy milk, bactofugation is used mostly in combination with thermal pasteurisation due to its relatively good effectiveness against spores but weakness in non-spore bacterial removal .
Human milk consists of a large variety of components that range in size. Sizes of targeted microorganisms and human milk components mostly overlap, making it difficult to separate micro-organisms by size without loss of human milk components.
Thermal pasteurisation of human milk is well researched and its impact on micro-organisms and bioactive components is well known. To optimise the retention of bioactive components, the temperature profile can be changed. However, bacterial reduction and bioactive component retention are closely but inversely related to each other. This may be solved using pasteurisation methods where this relationship is less specific. The most promising alternative pasteurisation method is irradiation. By targeting DNA and RNA directly, electron, x-ray, gamma, and ultraviolet irradiations have a different inactivation mechanism to heat treatment. Of the four, ultraviolet irradiation has the lowest energy density making the costs of the treatment device and safety and protection equipment significantly lower than those of other irradiation types. Furthermore, ultraviolet, specifically UV-C, has been shown to be highly germicidal with less damaging effects on bioactive components in human milk than thermal pasteurisation , . These findings show promise to improve the quality of pasteurised donor human milk while maintaining the safety standard of Holder pasteurisation.
Pasteurisation a process whereby food, usually a liquid, is partially sterilized by eliminating bacteria and viruses, making it safe for consumption
The most common pasteurisation method is Holder pasteurisation which reduces vegetative bacteria sufficient to meet safety standards but does result in significant loss of many important bioactive human milk components
Alternative pasteurisation methods can also damage important milk components, are very expensive or may not be reliable enough to safety standards
Ultraviolet irradiation meets safety standards than Holder pasteurisation but shows higher retention of bioactive components; however, this method needs further investigation