TYPE Review

PUBLISHED 01 November 2024

DOI 10.3389/fnut.2024.1468886

Donor human milk: the influence

OPEN ACCESS of processing technologies on its
nutritional and microbial
EDITED BY
Alessandro Iellamo,
FHI 360, United States

REVIEWED BY
Debashree Roy,
composition
Massey University, New Zealand
Andre Leke,
Centre Hospitalier Universitaire (CHU)
Ruth Conboy-Stephenson 1,2,3, R. Paul Ross 2,3, Alan L. Kelly 4 and
d’Amiens, France Catherine Stanton 1,3*
*CORRESPONDENCE 1
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland, 2 School of Microbiology,
Catherine Stanton
University College Cork, Cork, Ireland, 3 APC Microbiome Institute, University College Cork, Cork,
[Link]@[Link]
Ireland, 4 School of Food and Nutritional Sciences, University College Cork, Cork, Ireland
RECEIVED 22 July 2024
ACCEPTED 03 October 2024
PUBLISHED 01 November 2024 Human milk is regarded as the gold standard nutrition for newborn infants, providing
CITATION all nutrients required for adequate growth and development from birth to 6 months.
​ onboy-Stephenson R, Ross RP, Kelly AL and
C In addition, human milk is host to an array of bioactive factors that confer immune
Stanton C (2024) Donor human milk: the
influence of processing technologies on its
protection to the newborn infant. For this reason, the supply of human milk is
nutritional and microbial composition. crucial for premature, seriously ill, or low birth weight infants (<1,500 g). When a
Front. Nutr. 11:1468886. mother’s own milk is unavailable, donor human milk is the recommended alternative
doi: 10.3389/fnut.2024.1468886
by the World Health Organization. Prior to consumption, donor human milk
COPYRIGHT
undergoes pasteurization to ensure the eradication of bacterial agents and prevent
© 2024 Conboy-Stephenson, Ross, Kelly and
Stanton. This is an open-access article the transfer of potentially pathogenic organisms. Currently, Holder Pasteurization,
distributed under the terms of the Creative a heat-based treatment, is the widely adopted pasteurization technique used by
Commons Attribution License (CC BY). The milk banks. Holder pasteurization has demonstrated degradative effects on some
use, distribution or reproduction in other
forums is permitted, provided the original of milk’s biologically active factors, thus depleting critical bioactive agents with
author(s) and the copyright owner(s) are known functional, protective, and beneficial properties, ultimately reducing the
credited and that the original publication in immunoprotective value of donor human milk. As a result, alternative strategies
this journal is cited, in accordance with
accepted academic practice. No use, for the processing of donor human milk have garnered much interest. These
distribution or reproduction is permitted include thermal and non-thermal techniques. In the current review, we describe
which does not comply with these terms. the effects of Holder pasteurization and alternative milk processing technologies
on the nutritional and bioactive properties of milk. In addition, the capacity of
each technique to ensure microbial inactivation of milk is summarized. These
include the most extensively studied, high-temperature short-time and high-
pressure processing, the emerging yet promising techniques, microwave heating
and UV-C irradiation, and the lesser studied technologies, thermoultrasonication,
retort processing, pulsed electric field, and gamma irradiation. Herein, we collate
the findings of studies, to date, to allow for greater insight into the existing gaps
in scientific knowledge. It is apparent that the lack of a cohesive standardized
approach to human milk processing has resulted in contrasting findings, preventing
a direct comparative analysis of the research. We conclude that donor human milk
is a unique and valuable resource to the health sector, and although substantial
research has been completed, persistent data disparities must be overcome to
ensure optimal nutrition for the vulnerable newborn preterm infant group, in
particular.

KEYWORDS

donor human milk, Holder pasteurization, non-thermal, bioactive compounds,

high-pressure processing, ultraviolet-C irradiation, microwave heating,
high-temperature short-time

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1 Introduction published recommendations on milk banking procedures; these

recommendations align closely with those of the Human Milk Bank
Milk is a unique species-specific fluid produced by mammals Association of North America, the National Institute for Health and
post-partum. Human milk is the established optimum nutrition for Care Excellence (NICE), and The Perron Rotary Express Bank
newborn infants, contributing to the newborn infant’s health and Australia (19, 22–25).
development with long-lasting effects across the lifespan. Human milk Following the donation, milk undergoes a series of processing
has been linked with reduced incidence of infections in infants, steps including pooling, pasteurization, and bacteriological screening
including necrotizing enterocolitis (NEC), diarrhea, and respiratory (see Figure 1) (26). For pooling, milk from multiple donors is thawed
diseases, and is associated with reduced rates of infant morbidity and and pooled to create a uniform batch of donor milk with a relatively
mortality (1–3). Human milk is a rich source of macro- and standard level of macronutrients. Although no official criteria exist
micronutrients, an array of bioactive components and microbes (4). regarding the number of donors per batch, Colaizy et al. (27) suggest
The mammary gland confers the unique ability to adjust milk that three to five donors per pool is sufficient to reduce variability and
composition during lactation to the requirements of the growing optimize macronutrient content. Once pooled, milk undergoes
infant. As a result, human milk composition varies based on a number pasteurization and bacterial screening. The microbiological quality
of maternal and environmental factors, including gestational age, stage and safety of DHM are a primary concern for human milk banks. In
of lactation, stage of milk (fore vs. hind), age, diet, geographical addition to the naturally occurring microorganisms of breast milk,
location, and diurnal variations (5). DHM is at risk of exogenous contamination during the handling and
Although fresh mothers’ own milk is regarded as the optimal food processing steps (28, 29).
for newborn infants (6), donor human milk (DHM) is recommended Holder pasteurization (HoP), also known as low-temperature
by the World Health Organization (WHO) when mothers’ own milk long-time pasteurization, is performed to ensure the inactivation of
is not available (7, 8). DHM is particularly beneficial to infants born potentially harmful bacterial agents and viruses. HoP is the standard
prematurely, with a very low birth weight (<1,500 g) or a serious illness preservation technique adopted by milk banks worldwide (22),
(9, 10). Preterm and low birth weight infants are at increased risk of subjecting milk samples to a temperature of 62.5°C for 30 min.
serious health complications, including NEC, due to an Following treatment, milk is rapidly cooled and frozen awaiting use.
underdeveloped immune system (11, 12). For these infants, DHM Current guidelines regarding bacterial screening by milk banks vary.
contributes to the optimal recovery, growth, and health of the NICE recommends that samples should be screened prior to
newborn and reduces the risk of negative medical outcomes (13–16). pasteurization and discarded if total microbial counts exceed 105
However, increasing evidence suggests that there is lower availability CFU mL−1 (24). Furthermore, counts of 104 CFU/mL or higher of
of favorable immunoprotective compounds in DHM than in fresh Enterobacteriaceae or Staphylococcus aureus are unacceptable. Post-
milk as a result of the collection, storage, and processing of DHM (17). pasteurization, no level of bacterial growth in milk is permissible.
Preterm infants are the primary recipients of DHM, highlighting
the pivotal role of milk banks in healthcare settings (30). Current
1.1 Milk banks—current handling practices research endeavors to better understand the impact of handling,
and techniques processing, and storage procedures on DHM composition and address
any subsequent nutrient gap. In particular, optimizing the
Human milk banks provide an essential service to infants who are pasteurization method used to ensure minimal loss of bioactive
unable to access their mothers’ own milk. Milk banks are responsible factors of milk while preserving its microbial safety is desirable.
for the collection, processing, storage, and distribution of
DHM. Currently, there are 282 human milk banks supplying DHM in
Europe, with a further 18 planned (18). Similar to Europe, there has 2 The effect of holder pasteurization
been an upsurge in the number of milk banks in the USA, with 11 on the composition of donor human
banks in 2012 which increased to 32 as of 2023 (19). Furthermore, in milk
the USA, a number of for-profit milk banks have also been established.
However, for the purpose of this review, we will only focus on 2.1 Macronutrient composition of human
non-profit milk banks. As the number of milk banks increases, so too milk
does the number of hospitals and patients using their services. An
analysis of surveys conducted by the Centers for Disease Control and Human milk is a tailor-made liquid food that provides complete
Prevention on Maternity Practices in Infant Nutrition between 2007 nutrition to the developing infant (31). The nutritional constituents of
and 2011 demonstrated an increase in the use of DHM by intensive milk originate from three key sources, the lactocytes, dietary origins,
care units from 25.1% in 2007 to 45.2% in 2011 (20). Indeed, a 2015 and maternal stores (32). The macronutrients in human milk
survey notes a 74% increase in neonatal facilities using DHM constitute lipid, carbohydrate, and protein. Human milk composition
compared with 2011 (9). consists of approximately 87% of water, 3.8% of fat, 7% of lactose, and
The women who become donors donate on a volunteer basis if 1% of protein (33, 34).
they have excess milk supply. Donors undergo health evaluation
including behavioral and serological screening to prevent the transfer 2.1.1 Lipids
of infectious diseases (21). Although no international legislation exists In milk, 98–99% of the lipid content is comprised of triglycerides
for donor screening and milk processing, a number of guidelines have in the form of saturated fatty acids (SFA), monounsaturated fatty acids
been developed. The European Milk Banking Association has (MUFA), and polyunsaturated fatty acids (PUFA) (16). Triglycerides

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Conboy-Stephenson et al. 10.3389/fnut.2024.1468886

FIGURE 1
The handling and processing steps of human milk in a hospital milk bank setting [created with [Link]].

are composed of a glycerol molecule with three fatty acids attached. main carbohydrate in milk (56), accounting for 40% of the energy
These fatty acids can be combined in over 45 different positions and supply (57) and 60–70% of the total osmolarity of human milk (16).
combinations (31). The remaining lipid portion of milk includes In addition, lactose facilitates the absorption of minerals and is
cholesterol, phospholipids, gangliosides, and sphingolipids, located associated with the innate immune response (58, 59). Unlike other
within the milk fat globular membrane (16). The lipid content of macronutrients, lactose levels remain relatively constant over
human milk accounts for approximately 50% of milk’s energy supply 12 months of lactation, with a reported mean concentration of 61.4 g/L
and is linked with improved neurodevelopment, immune function, (60). HMOs are a group of structurally diverse unconjugated glycans
and metabolism in the infant (33, 35). unique to human milk (61). After lipids and lactose, they are the third
To date, several studies have investigated the effect of HoP on the most abundant solid component of breast milk, constituting 10–15 g/L
lipid content of DHM. The majority of findings suggest the retention of mature milk (62). Over 200 structures of HMOs have been
of DHM lipid content following HoP (36–42). Pitino et al. noted that identified in human milk to date (63). HMOs have an important role
the fatty acid profile of milk is preserved following HoP (43). Indeed, in the immune system due to their anti-adhesive properties against
no modifications to the fatty acid composition of DHM were observed pathogenic microbes, their promotion of a Bifidobacterium-rich gut
post-HoP (36, 38, 44–48). However, an increase in the proportion of microbiome, and their modulation of intestinal cell responses (62, 64).
medium-chain fatty acids alongside a reduction in oleic acid was There is consensus regarding the effects of HoP on DHM
reported (49). In contrast, some studies have reported reductions in carbohydrate levels. Adhisivam et al. observed no significant reduction
the total lipid content of DHM of 3.5–8.9% (50–53) with a subsequent in carbohydrate levels following HoP (54). Indeed, despite one report
loss in total energy of 2.8–2.9% (50, 51). Similarly, an even greater of a slight reduction (0.92%) in lactose levels (53), the stability of
decrease in fat and energy of 25 and 16%, respectively, was carbohydrate levels post-HoP has been further demonstrated
documented in DHM from a South Indian milk bank (54). In elsewhere (37, 39–42, 50, 52, 65–67). Additionally, other milk
addition, Capriati et al. (55) noted a reduction in the triglyceride carbohydrates were preserved following HoP, including myo-inositol
content of DHM of 21%. A reduction in the total lipid content of (65, 66), glucose (51, 65, 66), glycosaminoglycans (68), and
DHM is unexpected, and the reason for these reported reductions oligosaccharides (67, 69).
remains unclear.
2.1.3 Proteins
2.1.2 Carbohydrates Accounting for just 1%, the protein content of human milk is low
Human milk consists of many complex carbohydrates, of which but has high bioavailability (70). Human milk proteins can
the major and most studied are lactose and human milk be categorized into three main classes, whey protein, caseins, and
oligosaccharides (HMOs). Lactose is a disaccharide sugar and is the mucin proteins (71). Whey protein is the predominant protein in

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milk, with a whey protein-to-casein ratio of 90:10 in colostrum and that IR analyzers have a high degree of accuracy for crude protein and
60:40 in mature milk (72). Milk proteins contribute to a wide range of fat analysis (82). In contrast, carbohydrate determination was less
functions, including providing nitrogen and amino acids for tissue precise, with Fourier- and mid-IR analysis reporting lower
growth and maintenance, antimicrobial activity, immunomodulatory concentrations, while filtered- and near-IR led to increased levels
effects, and aiding digestion (34, 73). Overall, findings indicate that (20–50%) when compared with reference values. These findings
HoP does not reduce the total protein content of DHM. Indeed, a suggest that IR analysis is accurate at determining the protein and fat
number of studies reported the retention of total protein levels content but may not be the optimum choice for carbohydrate
post-HoP (37, 39–42, 50, 51, 74–76). However, similar to the lipid quantification. Furthermore, the correct calibration, validation, and
content, contrasting data on the effect of HoP on the total protein operation of the instrument are critical to ensure consistent accuracy.
content of DHM have been reported. For instance, a reduction in In addition to IR analyzers, laboratory-based analysis methods are
protein levels following HoP of 12.5–13% was demonstrated (77, 54), still widely used and considered favorably. A review of laboratory
with a less but still significant reduction of 2.5–3.9% also being techniques for macronutrient quantification has recently been
reported (52, 53). Importantly, there have been reported changes to performed (83), in addition to a review of methodologies for lactose
the protein profile of HoP-treated DHM in comparison with raw milk analysis (57). These include the Kjeldahl or Dumas method for protein
(76, 78). In particular, Silvestre et al. recorded a significant reduction determination, a combination of solvent extraction and gravimetry for
in lysine levels of DHM following HoP. Lysine is an essential amino lipid analysis, and either high-performance lipid chromatography or
acid found in human milk and can be an indicator of the protein high-performance anion exchange for carbohydrate quantification.
quality of milk. HoP caused a significant loss in the average available Both study design and method of analysis have the potential to impact
lysine of 29.3% when compared with untreated milk (75). These results, while a standardized choice of analytical method and
findings suggest that although HoP may preserve the total protein predetermined selection criteria for the milk sample may reduce the
content, the protein quality of milk may be modified or even inconsistencies noted in findings to date.
diminished. For instance, decreased lysine levels are often used as an Overall, despite some contrasting findings, HoP appears to
indicator of early-stage Maillard reaction (79). Furthermore, a separate adequately preserve the macronutrient portion of DHM and provide
study noted increased production of three key Maillard reaction high nutritive value to the infant.
products, furosine, carboxymethyllysine, and N-epsilon-
carboxyethyllysine, following HoP (80). Taken together, these findings
suggest that HoP-treated DHM may have increased levels of Maillard 2.2 Micronutrients
reaction products, potentially affecting the nutritional value of donor
milk. Future studies should investigate the effect of HoP on the protein Micronutrients in milk refer to essential vitamins and minerals
quality, the potential formation of Maillard reaction products, and any that aid human growth, development, and cell function (84). Vitamin
subsequent health effects on the newborn infant. and mineral intakes are linked with a variety of physiological
Overall, despite the observed heterogeneity within the available functions, including ocular development, hormone synthesis,
data, studies to date, indicate the ability of HoP processing to preserve enzymatic and metabolic functioning, and neurodevelopment
the total lipid, carbohydrate, and protein content of DHM. However, (16, 85).
the potential modification of protein quality warrants further Only a small number of studies have examined the effect of HoP
investigation. Future studies should endeavor to further elicit not only on the mineral content of DHM. HoP did not cause a significant
the effect on macronutrient concentration but also any subsequent reduction in calcium (37, 55). Additionally, phosphorus, iron, copper,
consequences for the activity, quality, and bioavailability of nutritional and zinc levels remained stable following HoP (37). However, a change
constituents. The variability in findings may be due to the absence of in the distribution pattern of zinc following processing was observed,
a systematic approach to study design, sample characterization which could impact zinc bioavailability (37). Usually, zinc
(sample volume, number of donors, and number of freeze–thaw concentration is at its highest in the whey portion of milk with lower
cycles), and the analysis method. The sample storage history, mainly levels in the fat and casein fractions. Goes et al. demonstrated that
freezing, can result in alterations to certain fractions of milk, including although this was the case pre-processing, following pasteurization,
the milk fat globule membrane, leaving components more vulnerable the levels of zinc in the whey portion decreased and an increase
to thermal processing (27). Additionally, a high starting concentration occurred in the fat fraction of milk.
of solid components may impact the efficacy of the analysis method. Findings indicate the potential stability of mineral concentrations
The use of older instruments or varying pasteurizer designs can post-processing. However, only a small number of minerals have been
increase the lag times for heating and cooling, altering the duration of quantified. Future studies should expand to include a wider range of
thermal exposure for milk components. In particular, the choice of the minerals, such as potassium, magnesium, and chloride. Furthermore,
analytical method appears to be a key influential factor, with studies the consequences of any potential modifications to the distribution
choosing from a range of laboratory reference methods or pattern of minerals and other nutrients as a result of pasteurization
commercially available infrared (IR) analyzers. should be further examined.
Milk banks primarily use IR analyzers to assess the nutritional Studies to date indicate a vulnerability of some water-soluble
composition of DHM. IR analyzers use near-, mid-, or Fourier vitamins to thermal processing conditions of HoP. In particular, the
transform IR spectroscopy. Recently, a mid-IR analyzer (Miris Human degradation of vitamin C as a result of HoP leads to losses of up to
Milk Analyzer) was approved by the FDA and is available 36% (44, 46, 86). Similarly, folacin and vitamin B6 underwent
commercially (81). IR analyzers have the advantage of performing significant losses of 31 and 15%, respectively (86). In contrast, other
rapid analysis on low-volume milk samples. Perrin et al. confirmed studies reported the stability of both (74, 87). The variability in

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results is likely due to differing analytical techniques. The retention 2.3.2 Lactoferrin
of riboflavin (B2), biotin (B7), niacin (B3), thiamine (B1), vitamin Lactoferrin is a multifunctional glycoprotein of the transferrin
B2, vitamin B12, and pantothenic acid following HoP has been family with enzymatic activity and antimicrobial, anti-inflammatory,
observed (74, 86, 87). It has been reported by some studies that and anti-infective properties (107, 108). Lactoferrin is best known for
fat-soluble vitamins, including vitamin A (37, 86), vitamin D (86), its role as an iron-binding protein. Indeed, lactoferrin has been shown
and vitamin E (44, 86) are robust to the processing conditions of to bind 20–45% of iron in human milk (108). Lactoferrin is the second
HoP. However, a study by Ribeiro et al. noted a 34% reduction in most abundant protein in human milk (107). Concentrations of
vitamin A with high-performance liquid chromatography following lactoferrin in milk vary according to the stage of lactation, with the
pasteurization (88). A similar reduction of 32.5% in vitamin A was highest levels in colostrum (7 g/L) and declining in mature milk
also reported (89). Vitamin E levels following HoP underwent a (2–4 g/L) (109). The findings reported thus far indicate a degradative
reduction of 17–25.5%, 13–47.3, and 33% in α-, γ- and δ-tocopherols, effect of HoP on the lactoferrin content of DHM. Mayayo et al.
respectively (45, 46). demonstrated that 80% of lactoferrin undergoes denaturation as a
Potential changes to the vitamin concentration and bioavailability result of thermal exposure during HoP (110). Furthermore, the group
of DHM following processing are concerning as low birth weight speculate that lactoferrin degradation occurs as a result of protein
infants, the primary recipients of donor milk, have increased vitamin aggregation caused by disulfide bonds. Separate investigations also
requirements. Interestingly, vitamin A level analysis in both noted a reduction in lactoferrin following HoP of 57–81% (93, 97, 101,
HoP-treated and untreated DHM suggests that neither supply 102, 105, 106). Goulding et al. (111) suggest that the heat stability of
adequate levels, providing only 43.6 and 66%, respectively, of the lactoferrin is influenced by environmental conditions including the
recommended nutrient requirement (88, 89). The clinical implications degree of iron saturation, pH levels, and protein composition.
of reduced vitamin bioavailability in DHM require further Evidently, lactoferrin is poorly preserved during HoP, undergoing
consideration. Evidently, HoP has varying effects on the vitamin thermally induced degradation likely as a result of changes in the
profile of DHM, which are dependent on the vitamins’ ability to protein structure.
withstand thermal exposure. Any future milk processing technique
should endeavor to optimize micronutrient preservation. 2.3.3 Lysozyme
Lysozyme is a major enzyme in human milk with anti-infective
properties (34), contributing to the bacteriostatic capacity of human
2.3 Bioactive milk components milk. Findings to date demonstrate a reduction in the concentration
of lysozyme following HoP (74, 101, 102). Koenig et al. noted
Human milk is a valuable source of bioactive compounds with reductions of 65–85% in colostrum (77). A similar decrease of 60.6%
a range of anti-inflammatory and antimicrobial properties that was reported in mature milk (93). Moreover, the effect on lysozyme
contribute to immune development and gut colonization. These activity was measured in a series of Micrococcus lysodeikticus
include hormones, enzymes, cell signaling molecules, and turbidimetric and lyso-plate assays and determined reductions of
bioactive agents such as immunoglobulins (Igs), lactoferrin, and 36–44% (98, 100, 112). Sousa et al. hypothesized that the loss of both
lysozyme. A number of studies have indicated the negative lysozyme concentration and activity following HoP was due to the
influence of HoP on these bioactive compounds, mainly due to neutral pH environment of milk (7–7.4) (98). Indeed, lysozyme is heat
their proteinaceous structure. stable at acidic pH (3–4) but heat labile at pH >7 (113). It is apparent
that the thermal processing conditions of HoP, likely in combination
2.3.1 Immunoglobulins with the pH environment of milk, result in lysozyme inactivation.
There are five classes of Igs in human milk IgA, IgG, IgM, IgD, and
IgE (90). The most abundant is IgA followed by IgG and IgM, with 2.3.4 Human milk enzymes
mean concentrations of 2.785, 0.059, and 0.036 g/L, respectively, Human milk lipase activity consists of two enzymes, bile salt-
across gestational age ranges (77). Indeed, secretory IgA (sIgA) stimulated lipase (BSSL) and lipoprotein lipase (LPL). BSSL facilitates
accounts for 80–90% of the Ig profile of human milk (91). The Ig the digestion and absorption of fat (114, 115). LPL participates in the
content of milk has a critical role in the protective capacity of human production of milk lipids in the mammary gland (116), and although
milk against infection (92). it has no known function in milk, it is thought to have a protective role
The Ig profile of DHM has been widely studied, and findings during cold storage (117).
suggest significant reductions in Ig levels as a result of HoP. Adhisivam The evidence thus far indicates the near complete destruction of
and investigators reported a reduction in IgA of 30% (54). Similar BSSL following HoP (47, 118, 119). In a study by Hamprecht et al. (74)
reductions in IgA of >20% have been noted in a number of studies (77, <1% of lipase activity was retained. Similarly, Henderson et al. (38)
93–112) and even higher reductions of 50–60% have also been noted a 100% reduction in LPL and BSSL activity. Comparable
reported (65, 101, 102). Furthermore, numerous studies demonstrated findings were reported by Kontopodi et al. in two separate studies
a reduction in sIgA levels (74, 103–105). In addition, a decrease in IgG (101, 102); the group noted low retention of BSSL levels (2–3%) and
levels following HoP has been widely reported (96) although the activity (4–7%) following HoP. The inactivation of BSSL as a result of
extent of loss varied, from 23–34% (98, 106) to 60–79% (54, 77). All thermal processing is not surprising. For instance, Pan et al. indicated
studies to date describe significant reductions in IgM concentration that BSSL remained stable when exposed to 45°C for 30 min (120).
following HoP (65, 77, 96–98, 105). Overall, it is apparent that the However, following an increase in temperature, the stability of BSSL
processing conditions of HoP have severe degradative consequences was significantly reduced at 50°C and completely destroyed at 60°C
on the Ig profile of DHM. (120). It can be concluded that HoP treatment results in the

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TABLE 1 Influence of Holder Pasteurization on the cytokine content of

denaturation of BSSL. BSSL is particularly important for newborn donor human milk.
infants, due to their limited secretion of pancreatic lipase and bile
salts. Indeed, pasteurization of DHM results in a reduction of lipid Cytokine Effect Volume Pooled Reference
absorption of 30% in preterm infants (113). Discernibly, a loss of BSSL pasteurized (number
(mL) of
interferes with the lipid metabolism of the neonate and is a serious
donors)
limitation of HoP.
– 119 Yes (4) (49)
Fewer studies have assessed the impact of HoP on other enzymes,
including amylase and alkaline phosphatase (ALP); however, reports IL-2 – <10 No (65)

indicate their reduction. Amylase contributes to the digestion of ↓ (47%) <50 No (125)
starch; its presence in human milk negates the limited salivary and – 119 Yes (4) (49)
pancreatic amylase activity in the first few months of life (121). Similar
IL-4 – <10 No (65)
to milk lipases, amylase is relatively heat-labile (113). However,
↓ (>70%) <50 No (125)
amylase appears more resistant to HoP processing with a reduction of
6–15% (38, 122). ALP, due to its thermal resistance, is often used as a – 119 Yes (4) (49)
marker of adequate pasteurization (123, 124). Indeed, two studies IL-5 – <10 No (65)
noted the complete inactivation of ALP post-HoP (74, 102). Evidently, ↓ (>45%) <50 No (125)
thermal exposure during pasteurization induces structural
– 20 Yes (6) (45)
modifications, which can result in a loss of enzyme activity,
functionality, and concentration. The degree of denaturation is – 119 Yes (4) (49)
IL-12
dependent on the optimum conditions of the enzyme. – <10 No (65)

↓ (42%) <50 No (125)

2.3.5 Cell signaling molecules – 119 Yes (4) (49)
Cell signaling molecules in DHM are important regulators of the
IL-13 – <10 No (65)
inflammatory and immune response. Cell signaling molecules include
cytokines, chemokines, and growth factors (GF). The influence of HoP ↓ (>90%) Not specified No (99)

on a variety of cytokines has been assessed with inconsistent results, – 20 Yes (6) (45)
IL-17
see Table 1. The majority of findings to date suggest interleukin (IL)-2, – <10 No (65)
IL-4, IL-5, IL-12, and IL-13 are not influenced by HoP (45, 49, 65);
MIP-1β ↓ <10 No (65)
however, contrasting results have been noted (99, 125). It appears that
HoP has no effect on IL-17 (45, 65). Both macrophage inflammatory EPO ↓ Not specified No (126)

protein (MIP)-1β and erythropoietin (EPO) were reduced by HoP (65, ↓ 119 Yes (4) (49)
126). Similarly, a reduction in IL-1β IL-6, IL-10, and tumor necrosis IL-1β ↓ (>70%) <50 No (125)
factor (TNF)-α was observed (45, 49, 99, 125, 126), although – <10 No (65)
Espinosa-Martos et al. noted no effect of pasteurization on these
↓ (75%) 20 Yes (6) (45)
cytokines (65). Similarly, a reduction post-HoP of interferon (IFN)-γ
was reported (49, 125) but no effect was noted elsewhere (45). ↓ Not specified No (99)
IL-6
Interestingly, an increase in the concentration of IL-7, IL-8, and ↓ (>50%) <50 No (125)
monocyte chemotactic protein (MCP)-1 following pasteurization has – <10 No (65)
been recorded (45, 49, 65, 99). However, no increase in IL-8 and
↓ (24%) 20 Yes (6) (45)
MCP-1 was reported (65, 125), although high levels of IL-8 retention
↓ 119 Yes (4) (49)
occurred (89%) (125).
Similarly, HoP has variable effects on milk GFs (see Table 2). No ↓ (>75%) <50 No (125)
IL-10
effect of HoP on the following GFs has been reported, Transforming ↓ Not specified No (126)
GF (TGF)-β1, TGF-β2, and epidermal GF (EGF) (65, 99, 126, 127). In – <10 No (65)
contrast, reductions in hepatocyte GF (HGF), insulin-like GF (IGF)-1,
– Not specified No (99)
IGF-2, IGFBP-2, and IGFBP-3 were recorded (49, 127), while
increased levels of granulocyte–macrophage colony-stimulating factor ↓ (95%) 20 Yes (6) (45)

(GM-CSF) following pasteurization were reported (65). It appears as ↓ 119 Yes (4) (49)
though the effect of HoP on both cytokines and GFs is molecule- TNF-α ↓ Not specified No (99)
dependent. The mechanism by which cytokines and GFs are impacted
↓ (>80%) <50 No (125)
by HoP is unknown but may be dependent on their protein structure.
– <10 No (65)
Espinosa-Martos et al. (65) attributed changes in concentration to
their release from cellular or fat compartments into the aqueous ↓ 119 Yes (4) (49)
fraction following heat exposure. IFN- γ ↓ (>60%) <50 No (125)
Overall, findings indicate a vulnerability of some cell signaling – 20 Yes (6) (45)
molecules to thermal pasteurization conditions; however, due to the
IL-7 ↑ <10 No (65)
limited and contrasting data a total effect cannot be determined. Due
to the essential role of these molecules in the inflammatory response, (Continued)

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TABLE 1 (Continued)
result of HoP (131). Smyczynska et al. demonstrated an 82-fold
Cytokine Effect Volume Pooled Reference decrease in whole material miRNA reads and a 302-fold decrease in
pasteurized (number miRNA exosome reads following HoP. This significant reduction
(mL) of prevented the group from further analyzing and characterizing the
donors) effects of HoP on the miRNA composition and function in
↑ (41%) 20 Yes (6) (45) DHM. However, in a subsequent study, Lamberti et al. observed a
↑ 119 Yes (4) (49) significant modification in the diversity of miRNA content of
IL-8 ↑ Not specified No (99)
HoP-treated milk exosomes, noting 33 differential miRNAs (132). The
differential miRNAs were implicated in five key pathways associated
– <10 No (65)
with immune and cellular function, highlighting the potential for
– <50 No (125) alterations to the immunomodulatory activity of DHM as a result of
↑ (62%) 20 Yes (6) (45) processing. As it stands, the impact of HoP on the miRNA composition
MCP-1
– <10 No (65)
of DHM remains relatively unexplored. Further investigations are
required to establish the full effect of HoP, however, based on the
(↑) A significant increase in cytokine level occurred.
(↓) A significant reduction in cytokine level occurred. findings to date a shift in the abundance and diversity of miRNA as a
(–) No significant effect on cytokine concentration was observed. result of HoP has been observed.
(%) The percentage reduction or increase is noted in brackets.

2.3.7 Milk hormones

TABLE 2 Influence of Holder pasteurization on the growth factor Milk hormones are non-nutritive bioactive compounds (69).
composition of human milk.
More recently, links between hormones and infant health and
Growth factor Effect Reference development, particularly in metabolic health, have been reported
(133, 134).
TGF- β1 – (99, 126)
A small number of studies have assessed the response of milk
TGF- β2 – (65, 99)
hormones to HoP treatment. Marousez et al. demonstrated a
EGF – (99, 126, 127) significant effect of HoP on a variety of metabolic hormones. They
HGF ↓ (49) reported a decrease of 63, 41, 11, 100, 41, and 83% in insulin,
nesfatin-1, cortisol, leptin, apelin, and GLP-1, respectively (69),
IGF-1 ↓ (39%) (127)
whereas adiponectin levels remained unchanged following treatment.
IGF-2 ↓ (10%) (127)
Ley et al. (51) also noted a reduction in insulin of 46% and, unlike the
IGFBP-2 ↓ (19%) (127) above study, a decline in adiponectin of 33%. Furthermore, melatonin,
IGFBP-3 ↓ (7%) (127) a key hormone in the regulation of the sleep/wake cycle, was
GM-CSF ↑ (65)
unaffected by HoP (135). The potential for alterations in the levels of
milk hormones as a result of processing raises concern due to their
(↓) A significant reduction in growth factor level occurred.
(–) No significant effect on growth factor concentration was observed. roles in energy regulation and the metabolic development of infants.
(↑) A significant increase in growth factor occurred. The potential for long-term effects on the development and
physiological function should be assessed.
Although HoP-treated milk retains a significant portion of the
minor alterations may influence the immunoprotective value of nutritive value of DHM, HoP also depletes a range of biologically
DHM. Further evaluation of changes in the balance of pro- and anti- active milk components. The loss of irreplaceable bioactive agents,
inflammatory cells following HoP is necessary, as a shift could including Igs, lactoferrin, and BSSL is a substantial limitation to the
significantly alter the clinical value of DHM. use of HoP. The affected components have known functional and
beneficial properties for the newborn. It is apparent the thermal
2.3.6 MicroRNA conditions of HoP negatively disrupt the structure of bioactive
Human milk is a rich source of microRNAs (miRNAs), defined milk proteins. This shortcoming calls for the development and
as short (19–24 nucleotides), non-coding segments of RNA, that implementation of alternative techniques with less
act as post-transcriptional regulators of gene expression (128). degradative outcomes.
miRNAs are located within extracellular vesicles called milk
exosomes. Milk exosomes encapsulate miRNAs, protecting them
from degradation by RNases, digestion, or low pH, facilitating 2.4 Holder pasteurization and microbial
their transport to target cells and tissues via the bloodstream (115, inactivation
129). Although the extent of miRNA activity is still under
investigation, evidence suggests a role in cell functionality and Although bacterial levels in milk vary, starting bacterial counts of
modulation of genes involved in physiological processes, such as 2.7–4.1 log10 CFU/mL and 2.6–5.2 log10 CFU/mL in DHM have been
metabolism, neurocognitive development, and immune function reported (65, 66). Furthermore, the predominant bacteria were of the
(90, 130). Staphylococcus, Streptococcus, Lactobacillus, and Bacillus genera. The
There have been relatively few studies assessing the impact of HoP primary objective of HoP is to remove any pathogenic microbes that
on miRNAs. A recent study noted that miRNA, within whole milk could potentially infect an infant upon ingestion. A number of studies
material and milk exosomes, undergoes significant degradation as a have demonstrated the efficacy of HoP at eliminating potentially

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harmful bacteria in DHM, both naturally occurring and through studies evaluate the efficacy of HoP or undertake the development of
artificial inoculation. alternative processing techniques.
The efficacy of HoP against naturally occurring microbes in
DHM has been determined (65, 136). Lima et al. (104) noted no
aerobic or coliform growth post-HoP. Additionally, in a study by 3 Alternative thermal techniques
Landers et al. (137), pre-HoP culturing demonstrated that of 303
milk pools, 87% were colonized with coagulase-negative As a result of the degradative effects of HoP on bioactive agents of
Staphylococcus, 16% with Enterococcus, 8% with α-Streptococcus, 4% milk and the inability to fully eradicate bacterial spores, a variety of
with S. aureus, and 61% of samples were colonized with at least one other thermal processing techniques are under investigation. These
Gram-negative rod. Following HoP, 93% of the samples showed no methods alter the processing conditions applied (temperature and
bacterial growth. The persistent bacterial growth was mainly Bacillus duration of exposure) or the mode of thermal application.
growth (5%) and also coagulase-negative Staphylococcus (1%). This
study emphasizes the need for mandatory pasteurization due to the
prevalence and variability of bacterial growth in DHM. Moreover, 3.1 High-temperature short-time
the results to date indicate the efficacy of HoP in eliminating the pasteurization
non-spore-forming bacterial community of DHM. Indeed, Czank
et al. assessed the capacity of HoP to deactivate inoculated bacterial High-temperature short-time (HTST) pasteurization is an
strains (93). HoP was carried out on milk samples with a established, effective, and efficient method of pasteurization in the
concentration of 1×105 CFU/mL of five bacterial strains (S. aureus, commercial dairy industry (139, 140). This method requires the rapid
Enterobacter cloacae, Bacillus cereus, Staphylococcus epidermis, and heating of milk to 72–75°C for a short period of time (5–15 s), usually
Escherichia coli). The tested concentration of bacteria is the using continuous-flow plate heat exchangers. In recent years, HTST
maximum advisable level of growth in DHM, according to NICE has emerged as an alternative to HoP, due to its ability to eradicate the
guidelines (24). The study demonstrated that HoP reduced the microbes of DHM. However, it remains to be established if a reduction
number of bacterial species by at least 99.9%. Furthermore, the in the length of thermal exposure better preserves the nutritional and
group determined the most to least heat-resistant strains were as bioactive factors of DHM.
follows: S. aureus, B. cereus, E. cloacae, E. coli, and S. epidermis.
Similarly, findings by Raptopoulou-Gigi et al. (138) noted the ability 3.1.1 HTST and the nutritional composition of
of HoP to eliminate bacterial growth in samples inoculated with DHM
2×107 of S. aureus and E. coli. HTST conditions (72.5°C/15 s) applied to DHM, with either a
The ability of HoP to eliminate a high concentration of non-spore- laboratory-scale continuous-flow device or water bath immersion, did
forming bacteria has been established, but an ongoing concern for not significantly affect the macronutrient content of the DHM (39,
milk banks is the presence of bacterial spores. A number of studies 122). In addition, no significant changes in the fatty acid profile or
demonstrated the inefficiency of HoP in eliminating bacterial spores, levels of malondialdehyde, an indicator of lipid oxidation, were
particularly B. cereus (65, 66). In a study of 21 DHM samples, 14% of observed (122). Further studies noted the retention of the total protein
samples had B. cereus growth post-pasteurization (66). Indeed, the content of DHM (74, 75, 102) and a better-retained protein profile
enhanced growth of Bacillus following exposure to thermal than HoP-treated milk following HTST (118). Moreover, Silvestre
pasteurization conditions has been observed. Landers et al. reported et al. reported a superior retention of the mean lysine concentration
B. cereus growth in 17 of 303 pooled milk samples post-HoP (137). Of following HTST than HoP, 85.11 and 70.69%, respectively (75),
the 17 samples, 10 were positive for B. cereus growth pre-pasteurization; suggesting improved protein quality with HTST-treated DHM. Again,
however, the remaining 7 were only positive subsequent to HoP these findings highlight the possibility that milk processing techniques
processing. Similarly, Lima et al. (104) reported B. cereus growth in 3 may modify the nutritional quality of DHM to varying degrees despite
of 12 samples post-HoP. Again, pre-HoP culturing noted only one retaining the overall nutrient levels.
sample positive for B. cereus prior to pasteurization. These findings Few studies have examined the effect of HTST on the vitamin
suggest that HoP is not only inefficient at eliminating B. cereus growth micronutrient composition of DHM. The retention of folic acid and
but also poses a risk of increasing B. cereus levels by inducing some vitamins, including vitamins B1, B2, B6, B12, and C, was
sporulation during thermal processing. Clearly, post-processing, reported following HTST (72°C /5–15 s) (74, 141). In contrast,
DHM is at increased risk of B. cereus growth. Martysiak-Żurowska et al. (122) noted a reduction in vitamin C of
The starting levels of bacterial growth in DHM and the potential 50.2%. However, despite the observed reduction in vitamin C, the total
for bacterial spore contamination emphasize the need for routine antioxidant capacity (TAC) of milk was maintained (122, 142).
pre-HoP bacterial screening. However, any post-pasteurization In conclusion, HTST processing maintains the macronutrient
screening must be carried out with caution to reduce the risk of composition of DHM. Moreover, findings suggest that HTST may
further contamination (24). It is evident that although HoP is effective be superior at retaining the protein profile and quality of DHM. The
in eliminating the majority of bacterial growth, this does not extend full effect of HTST on the micronutrients in DHM is yet to
to robust organisms such as spores, in this case, B. cereus. This is a key be established due to the limited number of studies. However, findings
challenge facing the milk banking sector, which is largely reliant on propose some B vitamins and the TAC are retained with HTST
strict post-processing handling and storage protocols to minimize the treatment. Further studies examining a wider range of vitamins and
risk of bacterial sporulation. The inactivation of microbial spores and indeed minerals should be carried out to allow for comparison with
other thermo-tolerant organisms should be considered when future HoP-treated DHM.

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3.1.2 HTST and the bioactive factors in DHM between raw and HTST-DHM (102). It remains unclear, whether
Reports on the Ig content of DHM following HTST are limited, HTST or HoP treatment has a more degradative influence, due to
with the majority of data only reporting on the IgA and sIgA content contrasting findings. However, the effect of HTST on the
of DHM. A number of studies noted high levels of retention. Indeed, bacteriostatic capacity of milk against E. coli is apparent. Further
following HTST (72°C/5–15 s) retention of IgA of 74–84% and sIgA studies are needed to determine which processing technique best
of 79–100% was reported (74, 102, 141, 143), although a depletion in preserves the bacteriostatic properties of milk. A depletion in the
IgA of 36–57% was also documented (97, 144). In addition, a natural bactericidal capacity of DHM following processing is
reduction in IgG of 42% and IgM of 100% was noted following HTST concerning, as those in receipt of DHM are at increased risk of
at 72°C for 15 s (97). The evidence suggests that the Ig content of infection and disease. Minimizing the negative effect of thermal
DHM may incur losses following HTST; however, the extent of IgA processing on the natural bacteriostatic capacity of raw milk should
reduction when compared with HoP suggests that HTST is superior be considered when selecting an ideal technology for
for IgA retention. Clearly, further investigation on the IgG and IgM milk processing.
content following HTST is warranted. Findings suggest that HTST is superior or at the least equivalent
Contrary to the findings of Goldblum et al. (141), a considerable to HoP at retaining some of the bioactive agents of milk. The observed
decrease in lactoferrin levels, of 42–73%, following HTST has been variability in results may be due to the holding time and temperature
widely reported (102, 122, 143). The observed reductions in lactoferrin applied, the chosen method of biochemical analysis, or the apparatus
are likely influenced by the form of lactoferrin and therefore the used for HTST processing. For instance, a wide assortment of
degree of iron saturation (122, 145). Indeed, the iron-depleted (apo) methodologies has been used to perform HTST including plate heat
form of lactoferrin denatures at temperatures higher than 70°C at a exchange (141), water bath immersion (122, 144), and various
pH of ∼7.0 (145). Taken together, the evidence suggests lactoferrin is laboratory-scale continuous-flow devices (74, 143, 146). Indeed, the
not retained following HTST. apparatus used to implement HTST may have differing effects on the
Several studies demonstrate the maintenance of lysozyme temperature and cooling profiles of DHM. In particular, temperature
concentration and activity following HTST (102, 122, 146), and two distribution may vary with non-continuous-flow instruments,
studies reported increased lysozyme activity (141, 144). However, a resulting in portions of the sample being exposed to higher thermal
reduction in lysozyme levels of 80% was reported elsewhere (74). The conditions for increased periods. Evidently, precise temperature
contradictory findings may be caused by different methods of regulation, calibration of instruments, and a uniform approach are
biochemical analysis. For instance, studies noting lysozyme retention critical to gather comparable findings.
used turbidimetric M. lysodeikticus assays (122, 141, 144) or liquid
chromatography–mass spectrometry (LC/MS) (102), whereas 3.1.3 Microbial inactivation with HTST
Hamprecht et al. employed radial immunodiffusion to evaluate The ability of any milk processing technology to effectively
lysozyme levels (74). Overall, the evidence supports the stability of eliminate bacterial growth is critical in order to be considered for a
lysozyme during HTST. As discussed previously, lysozyme is heat milk bank setting. HTST successfully inhibits the naturally occurring
labile at the pH of milk (113), suggesting the shorter exposure time of microbial community in DHM (123, 141, 146). The microbial
HTST processing prevents the destruction of lysozyme. deactivation of DHM following HTST has been further demonstrated
Similar to HoP, a significant depletion in human milk lipase, with inoculation studies. Bacterial spiking studies demonstrated the
particularly BSSL, occurs following HTST (74, 141, 146). Kontopodi efficacy of HTST against E. coli (108 CFU/mL), S. aureus (107–108 CFU/
et al. noted low retention of BSSL levels and activity of 9 and 19%, mL), Streptococcus agalactiae (106 CFU/mL), Listeria monocytogenes
respectively (102). Nonetheless, the observed reductions in (109 CFU/mL), and Cronobacter sakazakii (109 CFU/mL) (146, 148).
HTST-DHM are significantly less than those of HoP-treated DHM However, a critical finding is that HTST was not effective against
(102, 146). In addition, the digestive enzyme α-amylase underwent a naturally occurring B. cereus (123). In this study, HTST conditions of
reduction in activity of 7% following HTST (122). The complete 70–75°C for 5–25 s were tested and none of the temperature–time
destruction of ALP following HTST has been repeatedly demonstrated, combinations applied were capable of B. cereus eradication. For HTST
confirming the efficacy of HTST pasteurization conditions (74, to be a favorable alternative to HoP, inactivation of Bacillus is
102, 143). advisable. Further studies should assess the potential resistance of
Studies on the influence of HTST on cell signaling molecules are B. cereus to HTST conditions.
sparse. However, a reduction in IGFBP-2 and the retention of IGF-1, Overall, HTST appears equally effective to HoP at eliminating the
IGF-2, IGFBP-3, and EGF have been demonstrated (127). These microbial community of DHM; however, both procedures fail to
findings are in contrast to HoP, where in the same study the authors address the ongoing challenge of B. cereus contamination.
noted a significant reduction following HoP in all of the GFs analyzed, Furthermore, although widely adopted by the dairy industry, the
except EGF (127). logistics of implementing HTST in a milk bank setting may
The contents of lactoferrin, lysozyme, and Ig of DHM are likely be challenging. Aside from high costs, HTST equipment requires
contributors to the intrinsic bacteriostatic ability of milk. Indeed, cleaning between each pasteurization cycle, making it labor-intensive.
a negative correlation between a reduction in these three proteins Additionally, in comparison with the dairy sector, much smaller
and the growth rate of bacteria was identified (102). Thus far, volumes are processed by milk banks. Although some of these issues
findings are in agreement that HTST impacts the bacteriostatic may be overcome, Giribaldi et al. (146) designed a small-scale
capacity of milk, with increased levels in the growth rate of E. coli continuous-flow HTST instrument with the ability to process 0.1–10 L
in HTST-treated DHM in comparison with raw milk (102, 147), of milk. Such a device would need to be readily available commercially
although no differences in the growth of S. aureus were observed and undergo further testing. Importantly, the need for cleaning

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between cycles is still required. Evidently, aside from the need for following microwave heating at 62.5°C for 5 min (154). High levels
further studies, the practicality and feasibility of downscaling HTST of retention were observed for lactoferrin, sIgA, and vitamin C of
for clinical use warrants further consideration. approximately 60-92%. Furthermore, the retention of TGF-β2 was
documented. However, the study noted a significant depletion of
approximately 91% in basal lipase activity following microwave
3.2 Retort processing heating. In addition, a reduction in lysozyme concentration and
activity of approximately 56 and 30%, respectively, occurred.
Retort processing is a commercial food sterilization technique that These findings demonstrate the potential of microwave heating in
applies heat (115–121°C) and pressure (15–20 psi) to a product for a milk banks with high levels of bioactive factors retained. However, the
set holding time before subsequent cooling (149). Recently, retort choice of temperature and length of treatment is critical. Indeed, when
processing has been utilized for the preservation of DHM, producing Martysiak-Żurowska et al. increased the temperature of microwave
a shelf-stable product that is currently available to neonatal intensive heating to 66°C for 3 min, a significant loss in all of the bioactive
care units in the USA (104). components was observed (154). These findings indicate that
In all studies to date, retort processing of DHM was carried out at microwave heating at 60–65°C allows for optimal retention of
121°C, at 20 psi, for 5 min using water immersion (40, 104, 150, 151). bioactive factors of DHM when the treatment duration is short
Retort processing preserved the gross macronutrient composition of (15–30 s), whereas an increased treatment time of 5 min may deplete
DHM (40). However, a reduction in lysine and thiamine of 20 and levels of lysozyme and lipase.
80%, respectively, was observed. As noted previously, a reduction in More recently, when microwave heating with HTST conditions of
lysine could indicate a modification in the protein quality, despite no 72.5°C for 15 s was applied the macronutrient content and fatty acid
observed change in total protein levels. A significant reduction in profile of DHM were maintained (122). Furthermore, the levels of
bioactive proteins of milk following retort processing occurs in lactoferrin and lysozyme were unchanged post-processing. However,
lactoferrin, IgA, IgG, and IgM of 84% (150, 151), 88–100% (104, 150, a reduction in vitamin C and α-amylase of 42.6 and 6%, respectively,
151), 77–100% (150, 151), and 100% (150), respectively. In addition, was observed. The retention of lactoferrin and lysozyme is in contrast
lysozyme was reduced by 54–89% (104, 151), although the retention to previous findings at 66°C for 3 min and 70°C for 10 s (153, 154).
of lysozyme was reported elsewhere (150). Furthermore, retort The contrast in findings calls for further assessment of microwave
processing results in almost the complete degradation of vascular heating at increased temperatures (>66°C) on DHM to allow for an
endothelial GF (VEGF) and TGF-β2, whereas TGF-β1 is maintained optimal time–temperature combination to be determined.
(150). A clear advantage of retort processing is the complete microbial Critically, the ability of microwave heating at 60°C for 30 s to
inactivation of DHM, including B. cereus (104). eradicate the natural microbiota of DHM and inactivate ALP has
However, it is apparent that the efficiency of microbial inactivation been demonstrated (124). Moreover, microwave heating achieved a
is at a significant nutritional cost. The bioactive properties of DHM complete reduction of Salmonella typhimurium and S. aureus
are at risk of almost complete destruction. A further advantage inoculated at concentrations of 106 CFU/mL (124). Despite the
associated with retort processing is the ability to be packaged and encouraging findings, the data on the capacity of microwave heating
subsequently stored at room temperature where cold storage facilities for microbial inactivation are lacking. Further assessment of the
are lacking. Despite these advantages, the nutritional and therapeutic inhibition of thermo-resistant microbes and the pasteurization
value of shelf-stable DHM is significantly diminished and may require conditions applied is needed. Once the optimal treatment parameters
fortification post-processing, suggesting it is an inferior alternative to for microbial inactivation have been established, subsequent analysis
HoP-treated DHM. on the macro- and micronutrients of DHM should be performed.
Overall, findings to date on microwave heating are encouraging
but preliminary. A key advantage of microwave heating is its ease of
3.3 Microwave heating operation and accessibility within a milk bank setting. Furthermore,
microwave heating requires little space and is time-saving, cost-
Despite the small number of studies, microwave heating has effective, and low energy. However, the non-uniform transfer of heat
emerged as a promising novel pasteurization method. Microwave during microwave heating may pose a challenge, resulting in the
heating relies on the generation of microwaves, which penetrate the creation of cold spots in the milk (152). Further evaluation of the
food product, where they are rapidly absorbed and dissipate energy in dielectric properties of human milk and its subsequent interaction
the form of heat (152). with microwave heating is required to ensure consistent results.
Leite et al. assessed the influence of microwave heating at 60°C, Indeed, a stirring mechanism or the equivalent should be implemented
65°C, and 70°C for 30 s, 15 s, and 10 s, respectively, on certain to prevent this from occurring. In addition, strict monitoring of time
bioactive factors in DHM (153). The group noted that at all and temperature is essential.
temperature and time combinations, microwave heating did not alter
the concentration of oligosaccharides or fatty acids in
DHM. Furthermore, microwave heating at 60°C for 30 s maintained 3.4 Thermo-ultrasonication
82, 88, 95, and 87% of IgA, IgG, IgM, and lactoferrin, respectively.
Similarly, no significant reduction in the Ig or lactoferrin content was The combination of ultrasonication (20–100 kHz) and moderate
observed following processing at 65°C for 15 s. However, microwave heat (>50°C) to induce bacterial inactivation in foodstuffs is known
heating at 70°C for 5 s did cause significant reductions in IgG, IgM, as thermo-ultrasonication (TUS). Ultrasonication occurs following
and lactoferrin levels of 37–76%. Comparable results were reported the formation and cavitation of microscopic bubbles. Bubbles are

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produced as a result of pressure shifts, following ultrasound wave 90% retention of sIgA, lactoferrin, and lysozyme following processing
propagation during processing. These bubbles collapse rapidly and at 57°C for 30 min with an experimental pasteurizer (93). Similarly,
with force, producing heat and shockwaves. The consequence of this pasteurization at 40–55°C for 30 min retained >60, >80, and > 80% of
is cellular membrane disruption and subsequent cell death (155). IgA, lactoferrin, and lysozyme, respectively (159). The observed
Thus far, the limited data indicate that the bioactive properties of difference in protein retention, despite the lower thermal conditions
DHM undergo moderate reduction following TUS, the extent of which of the latter study, may be due to the pasteurizer design. The
is mainly determined by ultrasound power (W) and also exposure time. experimental pasteurizer used by Czank et al. was capable of precise
For instance, the treatment of DHM at 40 W for 9 min maintained the temperature and time regulation.
IgA, lactoferrin, and lysozyme content, although a reduction in BSSL was In contrast, Zhang et al. used a bottle immersion technique.
observed (101). In comparison, when the power was increased to 60 W Although this method more closely reflects the technique applied in
for a reduced period of 6 min, TUS retained IgA and lactoferrin but milk banks, it does not have the same capacity for temperature
reduced lysozyme and BSSL by more than 40 and 70%, respectively monitoring. Both groups concluded that the temperature applied
(101). Moreover, following TUS at 60 W for 9 min, a significant reduction during processing, not the length of exposure, was the critical factor
in all the examined bioactive proteins occurred and levels of retention for protein retention. Indeed, a 1°C increase in temperature caused a
were comparable to those observed post-HoP (101). As indicated by the reduction in the retention of all three proteins; however, a
authors, the degree of bioactive protein retention decreased as the power proportionate reduction in exposure time had no effect (93).
of ultrasound increased. In a separate study, high levels of insulin Moreover, processing at 62.5°C for 5 s resulted in a much lower
retention (97%) have been reported, with a lower retention rate of 67% retention of lactoferrin (32%) and lysozyme (72%) but a similar
following HoP (156). It appears that TUS has the potential to better retention of IgA (83.2–95%) (143, 160). These findings further suggest
preserve some bioactive properties of DHM; however, this is likely that the temperature applied during pasteurization is the key factor
dependent on the choice of power, time, and temperature applied. influencing bioactive protein retention.
The ability of TUS to cause microbial deactivation of inoculated Importantly, exposure to temperatures >50°C reduced the
strains of S. epidermis, E. coli, and S. aureus has been previously endogenous bacteria in DHM (3.15–4.78 log10 CFU/mL) to
observed (157, 158). Indeed, Kontopodi et al. demonstrated the ability undetectable levels (159). Indeed, pasteurization at 57°C for 30 min
of TUS (40–60 W, 6–9 min) to cause a > 6-log CFU/mL reduction of was sufficient to eradicate inoculated bacteria at a concentration of 1
inoculated E. coli, S. epidermis, and E. cloacae in DHM (101). These × 105 CFU/mL, including B. cereus (93). In contrast, bacterial growth
findings were further supported by Mank et al. (156) who recorded a persisted following processing at 62.5°C for 5 s (143, 160). Additionally,
5-log reduction in the same bacterial strains following TUS at 60 W ALP retention of 6–27.3% suggests that 5 s of treatment is inadequate
for 6 min. Overall, TUS appears an effective technique for the for DHM pasteurization. Collectively, the findings demonstrate the
microbial inactivation of DHM, with significant reductions of inactivation of bacterial contamination and the improved retention of
inoculated pathogenic strains reported to date. However, the efficacy bioactive properties of DHM when processed at 55–57°C for 30 min.
of TUS against bacterial spores is yet to be determined. In particular, Further examination of microbial inactivation at these processing
further evaluation of TUS at 40 W is advisable, due to the optimal conditions is merited. In particular, the inclusion of heat-resistant
retention of bioactive proteins observed at this pasteurization condition. bacteria is necessary, as studies of treatment at higher temperatures
Uniquely, TUS presents the opportunity to reduce thermal for longer periods have noted the persistence of Bacillus spores (66,
exposure of DHM and consequently may produce superior 104, 123). The clinical potential of the improved retention of bioactive
DHM. Evidently, although the findings to date are encouraging, factors needs to be balanced with microbial safety.
additional studies are required to establish the efficiency of TUS and
identify the optimal treatment parameters for DHM pasteurization. 3.5.2 Treatment at 80–100°C
In particular, the macro- and micronutrient composition of DHM Multiple experiments with high-intensity treatment conditions of
following TUS remains unknown. 80–100°C have been performed. As a whole, findings demonstrate the
poor preservation of bioactive factors at this increased temperature
range. Low levels of retention of 0–22% in IgA, IgG, and IgM were
3.5 Experimental temperature and time observed following treatment at 80–100°C (97, 143, 144). Similarly, a
durations reduction of 78–85% in lactoferrin occurred when processed at
81–88°C for 5 s and levels were undetectable following 100°C for
A number of studies have carried out pasteurization at a range of 5 min (97, 143). Lysozyme demonstrated the greatest resistance to
temperatures and holding times to assess the effects on the nutritional thermal exposure, with no significant reduction following processing
and microbial components of human milk. Due to the experimental at 80°C for 15 s (144). However, when the length of treatment
processing conditions chosen, these experiments do not fit within the increased to 10 min, a ∼ 25% reduction was observed, which further
parameters of previously discussed thermal techniques and are increased following processing at 90°C (144).
presented below, categorized according to thermal exposure. The Not surprisingly, the microbial inactivation of DHM occurs
results from these studies may provide greater insight into the optimal following pasteurization at high temperatures. A > 5-log reduction in
conditions for thermal processing of DHM. C. sakazakii and L. monocytogenes and a > 3-log reduction in
Enterococcus faecalis were achieved with pasteurization at 81.5°C for
3.5.1 Use of temperatures <62.5°C 5 s (143).
Various studies reduced the pasteurization temperature or holding Evidently, DHM processing at increased holding temperatures
time below that of HoP (<62.5°C, <30 min). Czank et al. reported a results in a significant decrease in the key bioactive components of

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DHM. Moreover, at higher temperatures, the length of exposure can and distributed evenly throughout the product. The primary
further exacerbate the observed degradation. This is evident with the adjustable conditions that influence the efficacy of HPP are the
level of lysozyme retention decreasing with an extended processing pressure selected (MPa), the holding time, and the temperature (166).
time, despite the same temperature being applied (144). Overall, there As pressurization does not disrupt covalent bonds, the loss of nutrients
is no demonstrated benefit to the treatment of DHM at these high- and bioactive compounds is minimal. Instead, pressure impacts
temperature conditions. A similar microbial inactivation has been weaker bonds (Van der Waals forces, electrostatic interactions, and
achieved with lower temperatures, allowing for greater retention of hydrogen bridges) resulting in a loss of cellular function, membrane
bioactive components of milk. disruption, and subsequent microbial inactivation (165).

3.5.3 Treatment >100°C 4.1.1 HPP and nutritional composition

Heating temperatures of >100°C are commonly applied in the Findings to date indicate that HPP at 300–600 MPa for 5–10 min
dairy industry, e.g., in cases of sterilization (∼121°C) or ultra-high does not cause any change in the macronutrient content of DHM (41,
temperature (UHT) (135–150°C) (161, 162). The extreme 42, 167). Despite this, a 2.9% reduction in total carbohydrate levels
temperatures used by the dairy industry, when applied to DHM, have following HPP at 500 MPa for 8 min was reported (41). However,
an expected negative impact on bioactive properties of milk. Indeed, further supporting the stability of the carbohydrate portion of DHM,
less than 20% of IgA and lactoferrin were retained following Marousex et al. (80) noted the retention of HMOs following HPP at
pasteurization at 127°C for 15 s (159). Similarly, following UHT, the 350 MPa. Similarly, a reduction was reported in some n-3 series
Ig content of DHM was undetectable and an 84% reduction in PUFAs following HPP for 6 min at 600 MPa (45). However, others
lactoferrin occurred (150). Again, lysozyme was the most resistant to note the retention of the lipid profile of DHM following HPP
high-temperature processing, with approximately <40% retention (300–600 MPa), with no reported changes to the fatty acid composition
following 127°C for 15 s (159) and no observed reduction following or the triacylglycerol profile (43, 44, 47, 167). Furthermore, following
UHT (142°C, 6 s) (150). HPP, the total protein profile of DHM was unchanged (76) and more
Furthermore, an effect that has not been seen at lower processing closely resembled that of raw milk than HoP-treated milk (78).
temperatures is the impact of sterilization conditions on the lipid Overall, these findings indicate the retention of the macronutrient
fraction of DHM. Processing at 120°C for 30 min decreased the composition of milk following HPP.
available lipid content by ∼10% due to the formation of surface skin The influence of HPP on the micronutrient content of DHM has
and deposit on the inside of the sterilization container (36). been limited to vitamin composition, with varying findings.
Additionally, sterilization reduced the fatty acid content of DHM, Tocopherols underwent significant reductions following HPP at
specifically linolenic and arachidonic acid (36). Whether these effects 600 MPa of 21–27%, 44–47%, and 25–33%, in α-, δ-, and γ-tocopherol,
occur at sterilization and UHT temperatures, with a reduced respectively (45), whereas pressurization at 400 MPa retained α- and
processing time, is not known. The degradation of bioactive properties δ-tocopherol but reduced γ-tocopherol by 26–29% (45). In contrast,
of DHM and its potential influence on the lipid fraction emphasize a separate study reported the retention of tocopherol levels following
that the current techniques used by the dairy industry are not suitable HPP at 400–600 MPa for 5 min (44). Similarly, contradictory reports
for adoption by milk banks. of vitamin C stability have been recorded. Despite a reported reduction
in vitamin C content of 75% following HPP at 500 MPa for 8 min (41),
the retention of vitamin C following processing at 400–600 MPa for
4 Non-thermal processing techniques 5 min has also been demonstrated (44). Folate was unchanged by HPP
at 500 MPa for 8 min (41). In addition, one study has reported on the
Accumulating evidence illustrates the degradation of various carotenoid content of DHM; both β-carotene and lycopene, important
DHM components with prolonged thermal exposure. The destruction anti-oxidants, are preserved following HPP at 450–600 MPa for
of the heat-labile components produces DHM with a reduced 10–15 min (47). However, lutein and zeaxanthin underwent
therapeutic value. As a result, various non-thermal strategies are now reductions of ∼60%.
being assessed for DHM pasteurization. Collectively, the findings suggest that the effect of pressurization
on the vitamin composition of DHM is distinct for each vitamin.
Furthermore, the extent of vitamin reduction seemingly increases
4.1 High-pressure processing with an increase in pressure. The impact of holding time remains
unclear but may account for the observed discrepancy in vitamin C
In recent years, high-pressure processing (HPP) has been shown retention. It is evident that an investigation of a wider variety of
as a safe, effective, and alternative method of preservation in the food vitamins and indeed minerals is required to fully ascertain the
industry. HPP is widely used in a number of foodstuffs, including influence of HPP on the micronutrient composition of DHM and any
vegetable, meat, and dairy products, due to its ability to reduce subsequent clinical significance.
microbial load while retaining high nutritional value and protecting
the organoleptic characteristics of foods (163, 164). Furthermore, 4.1.2 HPP and bioactives
HPP-treated food is viewed as minimally processed, lending to a high To date, there is promising evidence of Ig retention following
level of consumer acceptability (165). During HPP, food is vacuum- HPP. Indeed, a number of studies noted the retention of IgG, IgM, and
packed and placed into a pressure vessel containing a pressure- IgA following HPP at 200–450 MPa for 2.5–30 min (94, 96, 98, 101,
transmitting fluid. It is then subjected to isostatic pressure, typically 168). However, a significant depletion in Ig is observed with an
400–600 MPa at 4–45°C (164). Pressure is delivered instantaneously increased pressure of 600 MPa (76, 94, 96, 101, 144, 169). For instance,

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TABLE 3 Influence of high-pressure processing on the levels of cell

a reduction in IgA, IgM, and IgG of 20–26%, 59–60%, and 35–40%, signaling molecules in human milk.
respectively, occurred subsequent to HPP at 600 MPa for 15–30 min
(98). Although a reduced holding time of 2.5 min maintained IgA Effect Processing Reference
levels, losses of 21% in IgM and IgG still occurred. Clearly, the choice conditions
of pressure and exposure time are influential factors. Cytokine
This was further illustrated by Permanyer et al., who noted that, IL-6 – 400–600 MPa, 3–6 min (45, 99)
as pressure increased, an additional reduction in IgA occurred; 100%
– 400–600 MPa, 3–6 min (45, 99)
retention at 400 MPa, 87.9% at 500 MPa, and 69.3% at 600 MPa (94). IL-8
↑ 600 MPa, 5 min (99)
Similarly, Kontopodi et al. (101) observed a greater reduction in IgA
levels with increased treatment time during HPP at 600 MPa, with – 400–600 MPa, 3–6 min (45, 99)
TNF-α
55% retention after 3 min and 47% after 5 min. On the whole, these ↓ 500–600 MPa, 5 min (99)
findings suggest that HPP can retain the Ig profile of DHM but the - 400–600 MPa, 3–6 min (45, 99)
selection of processing parameters is a critical determinant. In IL-10
↓ 400-500 MPa, 5 min (99)
particular, as the pressure or time exposure increases, so too does the
risk of Ig loss. Further studies should ascertain the optimum holding MCAF ↓ (22–33%) 400–600 MPa, 3–6 min (45)
time for HPP treatment, with findings to date suggesting that HPP at ↓ (90–95%) 400–600 MPa, 3 min (45)
IFN-γ
<500 MPa is suitable for Ig retention. – 400–600 MPa, 6 min (45)
The retention of lactoferrin following HPP at 350–600 MPa with
400 MPa, 3 min
assorted holding times has been demonstrated (76, 101, 170, 171). – (45)
Conversely, a reduction of 25% following HPP at 500 MPa for 8 min 600 MPa, 6 min
IL-17
was reported (41). Similar reductions of 35–44% were observed 400 MPa, 6 min
↓ (84–100%) (45)
following HPP depending on the combination of pressure, holding, 600 MPa, 3 min
and resting times applied (169). Importantly, the optimal variant
Growth Factor
group tested, HPP at 200 MPa for 10 min followed by a 10 min resting
↓ (64%) 600 MPa, 10 min (167)
period and 400 MPa for 10 min (200 + 400 MPa), did not cause a
significant reduction in lactoferrin. Evidently, the HPP specifications ↓ (61%) 100 MPa + 600 MPa (167)
applied will influence the degree of lactoferrin denaturation. For HGF ↓ (57%) 200 + 600 MPa (167)
example, following 15 min of HPP at 300, 400, 500, or 600 MPa, a
– 200 + 400 MPa (167)
reduction in lactoferrin of 9, 23, 34, and 48%, respectively, occurred
↓ (21%) 450 MPa, 15 min (171)
(110). Furthermore, the majority of reduction in lactoferrin occurred
within the first 5 min of treatment, with only a further 8% loss EGF – 400–600 MPa, 5 min (97)
incurred between 7 and 30 min of treatment (110). Despite the TGF-β1 – 400–600 MPa, 5 min (97)
varying outcomes, the evidence suggests the superior retention of TGF-β2 – 400–600 MPa, 5 min (97)
lactoferrin following HPP as opposed to HoP treatment (41, 169,
(↑) A significant increase in level of cytokine/growth factor occurred.
171). Unlike HoP, which results in the aggregation of disulfide bonds (↓) A significant reduction in level of cytokine/growth factor occurred.
and denaturation of lactoferrin, HPP does not impact covalent bonds (–) No significant effect on cytokine/growth factor concentration was observed.
(110, 171, 172), thus better preserving the lactoferrin content of (%) The percentage reduction/increase is noted in brackets.

DHM. Overall, HPP can maintain lactoferrin, but this is dependent

on the pressure applied and to a lesser degree, the holding time. A similar variation in GF retention following HPP has been
Although lysozyme is more robust to thermal processing than recorded. For instance, HGF levels were either retained or underwent
other bioactive proteins, a degradative effect of heat treatment has a reduction of 57–64% following HPP depending on the conditions
been observed (98, 168). In contrast, HPP at 200–600 MPa at various applied (169). Furthermore, a 21% reduction in HGF levels occurred
holding times of 2.5–30 min retains the lysozyme levels of DHM (41, following HPP at 450 MPa for 15 min (173). The retention of EGF,
98, 101, 168, 170, 171). Collectively, the available data substantiate the TGF-β1, and TGF-β2 following HPP has been recorded (99). Evidently,
stability of lysozyme during the HPP of DHM. HPP has variable effects on the cytokine and GF levels of DHM,
The influence of HPP on cell signaling molecules is still in its depending on the HPP conditions applied and the cell signaling
infancy with a small number of studies performed and contrasting molecule in question. It is apparent from the conflicting data that
data gathered. The content of cytokines and growth factors in DHM further research is needed to determine the appropriate conditions for
following HPP is presented in Table 3. In brief, there is consensus on cytokine and GF retention. However, the highest retention levels across
the stability of IL-6, IL-8, IL-12, and TNF-α following HPP at 400 MPa the widest range of molecules appear to be HPP at 400 MPa for <5 min.
(45, 99), despite a reported reduction in IL-10 of 31–42% following It is unlikely that a complete lack of effect on cell signaling molecules
HPP at 400–600 MPa (45). Franch et al. only noted a reduction in is achievable. However, HPP with optimal pasteurization parameters
IL-10 when a pressure of 600 MPa was applied (99). Similar disparate may retain key molecules involved in the inflammatory process and
findings on TNF-α retention were reported following HPP at 600 MPa ensure a preferable balance of pro- and anti- inflammatory molecules.
(45, 99). A reduction of 22–33% in monocyte chemotactic and Only one study to date has evaluated the miRNA portion of DHM
activating factor occurred following HPP at 400–600 MPa (45). In following HPP treatment. Smyczynska et al. noted that, unlike HoP,
addition, IFN-γ and IL-17 underwent varying losses, depending on HPP did not decrease the miRNA reads of DHM (131). Nevertheless,
the time–pressure combination applied (45). HPP did result in changes to the composition of the miRNA fraction.

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In particular, reduced levels of miRNA-29 and miRNA-148a-3p were 30 min of treatment was required. These findings indicate the potential
observed. On the other hand, miRNA-30d-5p appeared highly for pressure-resistant microorganisms.
resistant to pressurized conditions. The authors suggest a protective However, subsequent studies have reported the inactivation of
effect of milk exosomes during pressurization. The consequences of S. aureus and E. coli, following HPP. For instance, 15 min of HPP at
compositional changes on miRNA functionality and subsequent 500 MPa was sufficient to reduce S. aureus by 5-log CFU/mL (175). In
immune responses are not known. The limited available data suggest addition, a complete reduction in S. aureus, E. coli, L. monocytogenes,
that HPP may be less detrimental to the miRNA content of human C. sakazakii, and B. cereus was recorded after HPP at 450 MPa for
milk than HoP. However, the modification of DHM miRNA following 15 min, with a starting concentration of 105–7 CFU/mL (173).
milk processing and the potential implications this may have for the Furthermore, a > 7.8-log reduction in S. epidermis and E. cloacae was
recipient requires further study. reported following HPP at 400–600 MPa for 5 min (101). Clearly,
The degradative effect of thermal processing on human milk despite the pressure-resistant capacity of some microbes, when
lipases has been widely reported. Therefore, the demonstrated potential optimal HPP parameters are applied, microbial deactivation is
of HPP to retain high levels of milk lipases is an encouraging prospect. achieved. Moreover, a 5-log reduction is the maximum necessary
Wesolowska et al. reported a 79–87% retention of milk lipase activity reduction if the acceptable growth limits according to milk banking
following HPP at 450 MPa and 200 + 400 MPa (47, 173), whereas HPP guidelines are followed (24). However, the available data on the
at 600 MPa resulted in <17% retention in lipase activity (47). However, microbial safety of DHM following HPP remain limited in comparison
further supporting the maintenance of milk lipase following HPP, LPL with the extensive studies carried out on HoP-treated
was preserved following HPP at 400 MPa for 5 min (171). Furthermore, DHM. Accordingly, future studies should further endeavor to confirm
BSSL is retained following exposure to pressures of 400–600 MPa for the capacity of HPP at 400–600 MPa to eradicate microbial growth
5 min (171), 8 min (41), and 1.5–30 min (101). and determine the minimum treatment time required particularly
The influence of HPP on the hormonal constituents of DHM focusing on those strains with demonstrated pressure resistance and
indicates the retention of some key metabolic hormones. Indeed, bacterial spores, mainly B. cereus.
both leptin and insulin were retained when processed at 350 MPa
(69), 450 MPa (173), and 200 + 400 MPa (169). Similarly, nesfatin-1, 4.1.4 Novel HPP conditions
cortisol, and GLP-1 were stable after processing at 350 MPa (69). In Alternative approaches to HPP of DHM have been tested through
contrast, HPP resulted in a depletion of adiponectin and apelin of an optimized HPP system (176) or by combining pressure treatment
50–85 and 20%, respectively (69, 169, 173). It remains unclear why with extreme temperatures (177, 178). Martysiak-Żurowska et al.
adiponectin and apelin appear more vulnerable to pressurization. combined HPP at 193 MPa with a sub-zero temperature of −20°C
However, as hypothesized by Marousez et al., it may be a result of (177). The group found that HPP at −20°C did not alter the FA
pressure-induced modifications to both the physiochemical composition or the concentration of secondary lipid oxidation
properties of milk and the structural composition of apelin and products. Furthermore, the total vitamin C and TAC levels of DHM
adiponectin (69). Despite these observed reductions, the overall were preserved, although a reduction in ascorbic acid of 11% was
hormonal composition of DHM appears well preserved following observed. The authors hypothesize that extreme temperatures can
HPP, in particular, the higher levels of leptin and insulin when intensify the effect of pressurization, allowing a lower pressure to
compared with HoP-treated DHM. be applied. This study presents the potential for the application of
Despite the modification in the levels of some bioactive factors in lower pressures, enabling greater preservation of bioactive components
DHM, the bacteriostatic ability of milk is preserved following HPP of milk. However, the data are preliminary and must be further
(101, 170, 174). Indeed, DHM processed at 350 MPa retained its substantiated—in particular, the capacity of lower pressure at sub-zero
antimicrobial activity when tested against E. coli and S. agalactiae temperatures to not only retain the nutritional and bioactive content
(170). Similar findings were observed following HPP at 400–500 MPa of milk but also ensure microbial inactivation. In contrast, a study that
and HPP at 200 + 400 MPa against S. aureus and E. coli (101, 174). In combined pressure with high temperatures (178) found that treatment
contrast, a higher pressurization of 600 MPa for 3–5 min did reduce at 65°C and 80°C depleted the α-tocopherol, fatty acid composition,
the inhibition rate of DHM against S. aureus (101). These findings and some cytokines, regardless of the pressure applied. Furthermore,
suggest that DHM processed at <500 MPa preserves the bacteriostatic only a pressure of 300 MPa at 50°C resulted in moderate Ig retention
capacity of DHM. of 48–100%. Evidently, thermal HPP results in disruption to the
nutritional and bioactive components of DHM.
4.1.3 HPP and microbial inactivation Separately, an optimized, novel HPP system was applied to DHM
Multiple studies have demonstrated the ability of HPP at (176). The group designed a HPP system in which the parameters for
400–600 MPa to reduce the endogenous bacteria of DHM to pressure delivery are accounted for, including compression rate,
undetectable levels (42, 94, 168, 171, 175). Importantly, HPP at decompression rate, and application mode. The process parameters
300 MPa for up to 15 min did not eliminate the native bacterial were as follows: pressure (350 MPa), temperature (38°C), application
population of DHM, deeming it unsuitable for application within the rate (1 MPa/s), and application mode (four cycles of 4 min duration
milk bank setting (171, 175). Further studies have assessed the with 5 min latency time between each cycle). Following processing,
bacterial inactivation of inoculated pathogens following HPP. The first high levels of sIgA, lactoferrin, and lipase were retained at 63–64%,
inoculation study was performed by Viazis et al., who reported an 93–97, and 80%, respectively. Critically, the group demonstrated a
8-log reduction in L. monocytogenes and S. agalactiae, after HPP at 6-log CFU/mL reduction of inoculated S. aureus and B. cereus. The
450 MPa for 2 and 4 min, respectively (95). The group also potential of this optimized HPP system to eradicate bacterial spores
demonstrated a 6–8 log reduction in E. coli and S. aureus; however, emphasizes its potential for milk pasteurization. Indeed, 10% of

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donated milk samples in this study were contaminated with B. cereus HoP that will better preserve key DHM components. Indeed,
(176). This approach would produce milk high in nutritional value comparative studies noted much lower levels of retention of
with improved microbial safety. Implementation of this HPP system lactoferrin, lysozyme, and IgA following HoP of 9–32%, 41–78, and
should be performed in future studies with further assessment of 40%, respectively (101, 103, 181). In addition, a number of studies
microbial inactivation and an evaluation of its influence on the have reported the retention of BSSL following UV-C at 1,100–
nutritional composition of milk. 5,500 J/L (101, 119, 179). Indeed, a reduction in BSSL of 64.7% was
only observed when a high UV-C dosage of 16,500 J/L was applied,
which further increased to 79.8% at a dosage of 33,000 J/L (119).
4.2 UV-C irradiation These findings demonstrate that BSSL is stable when exposed to
moderate UV-C exposure.
Ultraviolet (UV)-C irradiation is an emerging innovative The retention of immune proteins and antioxidant factors is a
alternative technique for milk pasteurization. UV-C (200–280 nm) likely contributor to the preserved bacteriostatic activity of UV-C-
exposure disrupts the cell’s genetic material, interfering with the cell’s treated DHM. Barbarska et al. noted the preserved bactericidal
ability for DNA transcription and replication, resulting in cell death capacity of DHM following UV-C at 6720 J/L. Indeed, untreated DHM
(101). The penetrative ability of UV-C during milk processing faces caused a 46.6% reduction in E. coli growth, whereas, following UV-C
two challenges. First, the high absorption coefficient of milk due to its irradiation, E. coli growth was reduced by 57.6–62.6%. In comparison,
opaque appearance (300 cm−1 at 254 nm). In comparison, translucent HoP-treated DHM only reduced growth by 12% (174). Retention of
solutions such as water have a much lower absorption coefficient the antimicrobial activity of DHM following UV-C at 2084–4863 J/L
(0.1 cm−1) (179, 180). Second, the total solid concentration of milk, against S. aureus and E. coli was also demonstrated elsewhere (101,
which varies, can further increase the absorption coefficient (181, 103). These findings suggest that UV-C retains not only the bioactive
182). Despite these challenges, the ability of UV-C to penetrate bovine properties but also the subsequent antimicrobial activity of milk.
milk has been achieved by creating a thin film turbulent flow-through A number of studies have demonstrated the efficacy of UV-C in
of milk, ensuring exposure of the whole sample to the UV source (180, ensuring sufficient microbial inactivation of DHM. Christen et al.
182, 183). A similar approach has been adopted by studies assessing were the first to note that UV-C at a dosage of 4,836 J/L results in a
UV-C processing of DHM. 5-log CFU/mL reduction of S. epidermis, S. aureus, E. cloacae, and
Only a small number of studies have reported on the B. cereus (179). Similarly, at the same dosage, a reduction of 5.78–6.95
macronutrient content of DHM following UV-C. Pitino et al. reported log CFU/mL was achieved in DHM inoculated with a concentration
no significant changes in the concentration of carbohydrates, lipid, of 108 CFU/mL S. epidermis, E. cloacae, and E. coli (101). In the same
and protein content of DHM following UV-C treatment (41). study, a reduced UV-C dosage of 3,645 J/L and 2,430 J/L resulted in log
Furthermore, UV-C at 4863 J/L caused no significant changes to the reductions of 4.6–5.9 and 4.3–5, respectively (101). These findings
protein concentration and fatty acid profile of DHM, except for an were further supported by Almutawif et al. (181) who observed
increase in the level of fatty acid C8:0 (101, 179). However, increased a > 5-log reduction in S. aureus when UV-C was applied to DHM at
levels of lipid oxidation end products following UV-C at a low dosage 2259 J/L. In fact, no growth persisted following treatment for 14 days
of 172.9–740 J/L have been reported (184). Similarly, there has been at 4°C and 18 h at 37°C.
minimal reporting on the micronutrient composition of DHM However, a separate study reported that a higher dosage of
following UV-C irradiation. Two studies have recorded reductions in 2,750 J/L resulted in a less than 5-log reduction in C. sakazakii,
vitamin C and folate of 35–72 and 25%, respectively (41, 184). Despite E. faecium, L. monocytogenes, and S. aureus (119). Furthermore, this
this reduction, the TAC of DHM was preserved (184). Overall, the dosage only resulted in a 2.75 log reduction in bacterial spores of the
macronutrient composition of UV-C treated DHM appears stable Bacillus and Paenibacillus species. The study noted that a high UV-C
post-processing. However, the observed increase in lipid oxidation dosage (8,250 J/L) was required for a > 5-log reduction across all
end products calls for further evaluation of the lipid fraction post- strains tested, including spores. Finally, UV-C treatment at 85–740 J/L
processing. Additionally, any potential loss or alteration in the is inadequate for microbial inactivation of DHM, with reductions in
concentration or composition of micronutrients remains E. faecium of <1-log following 550 J/L and < 3-log following 740 J/L
relatively unknown. (119, 184). Evidently, UV-C processing of DHM can produce
As this process is non-thermal, heat-induced denaturation of microbiologically safe milk when the correct parameters are applied.
bioactive compounds does not occur. Studies suggest that UV-C Findings thus far indicate that a UV-C dosage of 4,863 J/L and
preserves key immune factors including lactoferrin, lysozyme, and 8,250 J/L can inactivate the microbial components of DHM, including
IgA. Lactoferrin retention of 90–95% and 80–87% following UV-C bacterial spores.
at 2084–3645 J/L and 4,683–4,863 J/L, respectively, has been Although there have been positive indications for UV-C treatment
reported (101, 103, 181). Similarly, high levels of lysozyme at lower doses (2259–3,645 J/L), further studies are necessary due to
preservation were observed at 84–96% and 75–80%, respectively the contradictory results reported. In particular, the potential survival
(101, 103, 181), although lower levels of lysozyme retention (∼60%) of bacterial spores requires further examination. Furthermore, prior
were reported elsewhere (184). Studies of the Ig profile following to processing the total solid concentration of the milk samples should
UV-C are limited and have focused on the IgA content. IgA levels be quantified. As observed by Christen et al., the required UV-C
were preserved at >80% following UV-C at 2430–4863 J/L (101). dosage applied to milk increases linearly with the total solid
These findings were further supported by sIgA retention of 89–95% concentration of milk (179). This may account for the discrepancies
following UV-C at 2084–4683 J/L (103, 181). Clearly, findings noted in findings to date, in addition to the applied turbulent flow rate
suggest that UV-C may be a suitable non-thermal alternative to and pasteurizer design during UV-C processing.

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As a whole, the research suggests that UV-C at >4,863 J/L results inducing cell death, and consequently pathogen reduction (185–
in a > 5-log reduction in microbial growth, the maximum allowed 187). Despite the approval of gamma irradiation use in certain food
growth in milk banks (24). Importantly, significant retention of the groups by multiple agencies, including the WHO, the European Food
bioactive properties of milk is also achieved with these treatment Safety Authority, and the US Food and Drug Administration (188–
parameters, as summarized in Table 4. The observed variance in 190), consumer hesitancy persists (191). Consequently, studies of this
bacterial reduction at lower dosages suggests further study is required technology for DHM pasteurization are minimal and its applicability
to ascertain the minimal parameters for UV-C treatment of milk. All to milk banks may be viewed unfavorably. Gamma irradiation
studies discussed applied a turbulent flow to milk while undergoing exposure of up to 10 kGy is sufficient to ensure microbial safety,
UV-C to expose DHM evenly and overcome the high absorption retain the nutritional components of foodstuff, and limit radiolysis
coefficient of milk. To optimize treatment parameters, future studies (185, 187). Consequently, 10 kGy has mostly been applied as the
should assess the total solid content of milk prior to processing. In maximum dose in DHM studies to date and the results are
addition, further evaluation of the influence of UV-C on other key presented herein.
factors with biologically active roles, including IgG, IgM, cytokines, Gamma irradiation of DHM has mainly been applied as part of
and hormones, is warranted. a hybrid approach with either freeze-drying or the addition of an
antimicrobial mixture. For instance, Blackshaw et al. studied the
effects of gamma irradiation on freeze-dried DHM. The group
4.3 Gamma irradiation suggest that 2–5 kGy is the optimal exposure range for the retention
of milk’s nutritional properties (192). Indeed, irradiation at 2 kGy
Gamma irradiation is a non-thermal technology that can be used retained the protein profile of DHM and did not increase the levels
to pasteurize foodstuffs. During processing, the product undergoes of lipid oxidation end products. In a separate study, the same group
controlled exposure to gamma rays from a radioactive source, for demonstrated the capacity of irradiation at 5 kGy to completely
example, cobalt-60 or cesium-137. The gamma rays are absorbed by eliminate strains of S. aureus and S. typhimurium inoculated at a
the food product, damaging the genetic material of microbial cells, concentration of 106 CFU/mL (193). Furthermore, the bacteriostatic
properties of DHM were retained following irradiation (193).
Similarly, Balaji et al. (194) used an alternative hybrid approach on
TABLE 4 Percentage retention of bioactive proteins following UV-C
processing of milk. DHM of gamma irradiation at 5 kGy combined with antimicrobial
formulations. The hybrid treatment resulted in a 6-log reduction in
Bioactive UV-C Retention Reference bacteria, including B. cereus. Furthermore, the combined treatment
compound (J/L) (%)
maintained the Ig and lactose levels in DHM. However, protein
2,084 95 (103) hydrolysis and a subsequent increase in peptide concentration
2,259 92 (181) occurred; while this did not impact the in vitro digestibility
2,430 >90 (101)
of protein.
Few studies have assessed the potential of gamma irradiation as
Lactoferrin 3,474 93 (103)
a standalone treatment. That being said, reported reductions of
3,645 >85 (101) 70.8, 18, and 28% in BSSL, IgA, and lactoferrin, respectively,
4,683 87 (103) occurred following gamma irradiation of DHM (119, 138).
4,863 >80 (101) Importantly, both studies applied a high dosage of 25 kGy during
processing. The increased dosage may explain the depletion in IgA,
85 100 (184)
as opposed to its retention following hybrid IR processing. However,
740 ∼60 (184)
the levels of BSSL and lactoferrin have not been assessed elsewhere,
2084 91 (103) and therefore, the influence of a reduced irradiation dosage
2,259 96 (181) remains unknown.
Lysozyme 2,430 >85 (101)
Clearly, there is not enough data to determine the efficacy of
irradiation for DHM processing. Further evaluation of the influence
3,474 84 (103)
of gamma irradiation on the nutritional, bioactive, and microbial
3,645 >80 (101) composition of DHM is required. Future studies should be carried out
4,683 75 (103) at a dosage of approximately 5 kGy to ensure optimal retention and
4,863 >80 (101) allow for comparison with more recent findings. Overall, based on
findings to date and potential consumer perspective issues, gamma
2084 95 (103)
irradiation is an unlikely candidate for milk bank implementation.
2,259 92 (181) However, the initial results highlight the value of combined hybrid
2,430 >90 (101) treatments. Indeed, the combination of gamma irradiation and freeze-
IgA*
3,474 94 (103) drying results in effective microbial inactivation and retention of key
antimicrobial and nutritional properties of DHM. Advantageously,
3,645 >90 (101)
this hybrid approach would allow for the pasteurization of
4,683 89 (103)
pre-packaged powdered DHM with an extended shelf life.
4,863 >80 (101) Additionally, the use of antimicrobial formulations may contribute to
Includes IgA and sIgA.
*
increased shelf stability of DHM. The applicability of freeze-drying or

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Conboy-Stephenson et al. 10.3389/fnut.2024.1468886

antimicrobials in combination with alternative processing capacity of milk, remain unclear. A potential drawback of HTST is its
technologies should be further explored. logistical applicability to a milk bank setting.
In contrast, thermal processing by microwave heating has greater
accessibility and is less labor-intensive. However, data on this
4.4 Pulsed electric field treatment experimental technique remains scarce and an overall impact on
DHM components cannot yet be determined. Similarly, although
Pulsed electric field (PEF) treatment is a non-thermal novel findings on TUS processing are positive, the research is still in its
processing technique that results in bacterial inactivation whilst infancy and further studies are necessary to substantiate the results to
preserving the nutritional and sensory characteristics of food (195). date on novel technologies and to determine the optimal treatment
During PEF, the food is placed between two electrodes, and short parameters. Conversely, retort processing and pasteurization at high
high-voltage (approximately 20–80 kV/cm) pulses are applied (195). temperatures (>80°C) incur significant losses in various nutritional
Consequently, pore formation occurs in the microbial cell membrane, and bioactive factors, suggesting that the required conditions are too
this is known as electroporation (196). The structural changes to the harsh for DHM processing.
cell membrane as a result of electroporation cause an imbalance in cell Non-thermal methods have rightly generated much interest due to
homeostasis and induce cell death (197). To date, only two studies the protective capacity of such techniques on the heat-labile
have explored PEF processing of DHM. Sanchaya et al. (198) compounds of DHM. Of the non-thermal technologies, HPP stands
demonstrated the retention of the physiochemical properties and the out as the most promising and consequently most studied. Determining
lipid content of DHM following PEF. In addition, PEF at 35 kV for the optimal HPP parameters should therefore be a priority (201).
3,000 μs was sufficient to eliminate the naturally occurring microbes However, neglected areas of research remain, mainly, the impact on
of DHM. In contrast, bacterial growth persisted following processing micronutrient content of DHM, in particular mineral content and
at 25–30 kV for 3,000–4,500 μs. bioactive constituents including miRNA and milk hormones.
Separately, Zhang et al. performed nanosecond (ns)-PEF on DHM Furthermore, in comparison with HoP, further study of microbial
(199). The group used response surface methodology, a mathematical deactivation with pressurization is required. Although the potential of
modeling system, to determine two optimum processing conditions UV-C has been demonstrated, the limited existing data impedes a
for DHM. These were 15 kV voltage with 6,000 pulses at a frequency determination but does encourage further evaluation of this technique.
of 20 Hz or 50 Hz. Indeed, following nsPEF at 20 Hz, retention of Similarly, PEF and gamma IR are novel techniques with scarce findings
lysozyme, sIgA, and lactoferrin of 76, 108, and 74%, respectively, was requiring further evaluation. The applicability of these processing
observed. Moreover, nsPEF increased the levels of xanthine oxidase techniques in terms of feasibility, implementation, and consumer
and a greater than 60% retention of the enzyme lactoperoxidase acceptance should be considered. Unlike newer technologies, HoP has
was observed. been extensively examined; consequently, an unevenness in the volume
Of the sparse data available, the processing of DHM with PEF has and range of data exists. For a direct comparative analysis to be enabled,
encouraging results. However, it is evident that substantial research is further research must be carried out on these alternative processing
still required to fully establish its effect prior to its application in a milk technologies that remain relatively unexplored. In particular, the
bank setting. Nonetheless, the accomplished studies point to the capacity for bacterial spore inactivation should be a primary focus, as
potential processing parameters for future investigations. this is a major limitation of the currently favored HoP technique.
A core issue across all studies using various modes of
pasteurization is the variability in study designs. To date, studies have
5 Discussion differed vastly in terms of pre-processing collection, handling, and
storage of samples. Furthermore, the number of freeze–thaw cycles to
Human milk processing is an area of research that has undergone which a sample is exposed should be clearly presented where possible.
much investigation and progress in recent years. In particular, All of these factors may influence the milk constituents and their
alternative pasteurization technologies have emerged with the vulnerability to processing conditions. In addition, both the sample
potential for implementation by the milk bank sector. The emerging volume and number of donors per pool have differed significantly
technologies can be categorized into two groups: thermal and across studies. These factors can influence the total solids
non-thermal processing techniques. concentration and the required treatment time, potentially impacting
Heat treatment of food is a long-established method for food both the effectiveness of the technique, the subsequent analysis and
preservation. Indeed, the concept of heating milk before infant feeding the comparability of findings. Similarly, following processing,
was first suggested in 1824, and by 1889, heat-treated milk dispensaries variation in the analytical methods deployed is common. Collectively,
were conceived (200). Currently, HoP is the widely accepted milk these alternating approaches make it difficult to discern the true effect
processing technique established within milk banks. However, in of processing when contrasting results are reported. Recently, a review
recent years, the negative influence of HoP on valuable bioactive by Kontopodi et al. presents the most commonly used analytical
components of DHM has been substantiated. Consequently, the methods and suggests a template workflow for evaluating DHM
appraisal of substitute thermal processing techniques has been processing (83). Adherence to an accepted workflow and consideration
explored. Of these, HTST has been the most extensively studied. of the aforementioned factors would increase uniformity and may
Despite the potential superior retention of some DHM components, reduce the variability in findings. Evidently, a standardized approach
the effect on milk lipases and lactoferrin is degradative. Other when assessing milk processing techniques would facilitate future
components, such as cell signaling molecules and the bacteriostatic comparative analysis.

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Conboy-Stephenson et al. 10.3389/fnut.2024.1468886

Despite the wide range of technologies examined to date, some of milk banking pasteurization. Most importantly, any future
areas remain relatively unexplored, for instance, biopreservation. technology should mitigate the risk of bacterial contamination whilst
The potential use of antimicrobials in food preservation is gaining promoting the optimal retention of bioactive components, ensuring
momentum in dairy and other foodstuffs (202–204). Future studies DHM that confers the highest quality nutrition to the
should endeavor to assess their potential in DHM, in particular, as newborn recipient.
part of hybrid processing with other technologies. Indeed, hybrid
processing, as demonstrated in a small number of studies, may
reduce the severity of processing conditions required. In addition, Author contributions
the production of DHM in powdered form may provide greater
transport and storage options—in addition to prolonged shelf life. RC-S: Writing – original draft, Writing – review & editing. RPR:
These factors are particularly important in under-resourced areas, Writing – review & editing. AK: Writing – review & editing. CS:
where refrigeration storage may not be possible and milk supply is Writing – review & editing.
low. Another technique under investigation by both the dairy and
infant formula industries is the use of microfiltration as a method
of milk pasteurization. This technique is yet to be studied in DHM Funding
and should be evaluated in the future. Furthermore, although
beyond the scope of this review, DHM may be enhanced by The author(s) declare that financial support was received for the
fortification to replace lost nutrients—or indeed, inoculated to research, authorship, and/or publication of this article. This
allow for the re-introduction of mothers’ own milk microbiota publication has been supported by grants from Science Foundation
(205–210). The combination of optimal processing techniques with Ireland to APC Microbiome Ireland [grant number SFI/12/
the potential for personalized nutrition may produce the highest RC/2273-P2] and VistaMilk [grant number 16/RC/3835].
value DHM.
Human milk is a highly valuable food that influences and shapes
the infant’s gut, physiological development, and immune system Conflict of interest
maturation. Breast milk promotes the survival and development of the
infant while ensuring tailored nutrition with an ample supply of The authors declare that the research was conducted in the
immune proteins. The impact of alternatively processed DHM on the absence of any commercial or financial relationships that could
clinical value for the newborn recipient should be further assessed, be construed as a potential conflict of interest.
and any processing technique under consideration should first
be assessed for its efficacy in microbial inactivation. Bacterial spores
present a persistent challenge that future technologies should Publisher’s note
be equipped to overcome.
In conclusion, the heterogeneity in sample history, study design, All claims expressed in this article are solely those of the authors
and analytical methods used have contributed to divergence in the and do not necessarily represent those of their affiliated
available information for optimal processing of DHM and prevented organizations, or those of the publisher, the editors and the
a complete understanding of the emerging processing techniques. A reviewers. Any product that may be evaluated in this article, or
uniform approach to future studies and comprehensive information claim that may be made by its manufacturer, is not guaranteed or
regarding sample history will allow further insight and optimization endorsed by the publisher.

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