Human Milk Processing: A Systematic Review of Innovative Techniques to Ensure the Safety and Quality of Donor Milk : Journal of Pediatric Gastroenterology and Nutrition

Secondary Logo

Journal Logo

Invited Reviews

Human Milk Processing: A Systematic Review of Innovative Techniques to Ensure the Safety and Quality of Donor Milk

Peila, Chiara; Emmerik, Nikki E.; Giribaldi, Marzia‡,§; Stahl, Bernd; Ruitenberg, Joost E.||; van Elburg, Ruurd M.†,¶; Moro, Guido E.#; Bertino, Enrico; Coscia, Alessandra; Cavallarin, Laura

Author Information
Journal of Pediatric Gastroenterology and Nutrition: March 2017 - Volume 64 - Issue 3 - p 353-361
doi: 10.1097/MPG.0000000000001435


What Is Known

  • Holder pasteurization is considered the best compromise to ensure microbiological safety and preservation of bioactive components of human milk.
  • Both thermal and nonthermal alternative processing techniques have been tested.
  • Processing technologies for human milk banks should be applicable to treat significant amounts of human milk on a daily basis.

What Is New

  • Data about microbiological safety are scarce for novel technologies, in particular for ultraviolet-C irradiation and (thermo-)ultrasonication.
  • The differences in the reported testing and processing conditions do not allow conclusions to be drawn on whether and to what extent alternative technologies are able to improve the nutritional quality and bioactivity of pasteurized human milk.

Breastfeeding and human milk (HM) are the normative standards for infant feeding and nutrition (1) and mother's own milk is considered to be the first choice for feeding all infants (2,3). When a mother's own milk is not available or cannot be given, donor HM represents the best alternative. The WHO/UNICEF Joint Statement clearly indicates: “human milk banks (HMBs) should be made available in appropriate situations” (2,3). In addition, the ESPGHAN Committee on Nutrition has recently advised: “future research should focus on the improvement of milk processing in HMBs, particularly of heat treatment” (4).

HM is a functional and dynamic biological system: it provides nutrients, bioactive components, such as immune factors, lipids, enzymes, cells, and beneficial bacteria, which promote the adequate and healthy growth of newborn infants. Milk delivered to HMBs, and mother's own milk in specific clinical situations, should be pasteurized to inactivate life-threatening viral and bacterial agents. A pasteurization process at 62.5°C for 30 minutes (holder pasteurization—HoP) is currently recommended in all international guidelines for the constitution of HMBs (5–8). The absence of bacterial growth after HoP is required and microbiological screening is usually performed after pasteurization. Milk pasteurized with HoP is known to retain many beneficial and protective effects of HM, when compared to preterm formula, such as a reduction in necrotizing enterocolitis and sepsis, although evidence on this topic mostly dates back to the 1980s (9–11). Also, some studies showed that donor HM is doing as well as fresh HM in reducing the risk on neonatal morbidities for preterm infants (12). Nevertheless, the reduction/disruption of important nutritional and non-nutritional biological factors, such as immunological factors, was reported in previous studies as being the main detrimental effect of HoP on HM (5). Therefore, HMBs and researchers are committed to developing novel or enhanced methods to process donor HM that can ensure microbial inactivation, while improving the preservation of its nutritional, immunological, and functional constituents (4).

The food industry, and the dairy industry in particular, has tested innovative alternatives to standard pasteurization over the past decades in an effort to maximize the retention of food taste and nutritional features (13). Alternative processing techniques that are currently being tested to investigate their effect on HM include High-Temperature–Short-Time (HTST), High Pressure Processing (HPP), and ultraviolet (UV) irradiation and (thermo-)ultrasonic processing.

HTST is a thermal pasteurization method that is well established in the dairy industry (“fresh” bovine milk is usually pasteurized by means of HTST). The method involves a thin-layered milk flow being heated rapidly to 72°C and being kept at this temperature for a few seconds (usually 15 seconds), and then immediately cooled down. This method preserves most of the sensory features and nutritional values of the milk, and ensures a lower degradation of proteins and vitamins (14).

HPP is considered a promising alternative to the thermal pasteurization of HM. HPP is a nonthermal processing method that can be applied to solid and liquid foods to provide microbiologically safe, nutritionally intact, and sensory high-quality products (15). This technology inactivates pathogenic microorganisms by applying hydrostatic high pressure (usually 400–800 MPa) during short-term treatments (<5–10 minutes) (16). The pressures applied are transmitted instantaneously to the food being processed, which is treated evenly throughout. Pressurization at room temperature is usually accompanied by a moderate temperature increase (5°C–15°C), termed adiabatic heating, which depends on the food composition; the food cools down during decompression, provided no heat is lost or gained during the pressure hold time. Pressurization induces several changes in the bacterial cells: inhibition of the key enzymes and proteins, and alterations in cell membranes, and in the genetic mechanisms of the microorganism, such as disruption of transcription and translation (15). In general, the chemical composition and sensory analysis of food processed by means of high pressure are similar to those of untreated products in color and aroma, and changes are less intense than those caused by a heat treatment (15).

UV irradiation (200–280 nm wavelength) is classified as a nonthermal disinfection method. UV-C has a high germicidal effect, between 250 and 270 nm, and is capable of destroying bacteria, viruses, protozoa, yeasts, moulds, and algae, although it has a low penetration capacity that limits its use to liquid foods and flat surfaces (17). UV-C irradiation induces chemical reactions that damage the DNA, which in turn leads to photoproducts such as pyrimidine dimers, and cross-linking with proteins. On rare occasions, damage of DNA strands occurs (18).

Ultrasonic processing (20–100 kHz) is an emerging technology for the preservation of foods through the induction of inertial cavitation. Inertial cavitation results in the formation of microscopic bubbles, which rapidly collapse and produce shock waves and localized heating (19). During the collapse of these bubbles, localized hotspots occur, with temperatures of roughly 5000°C, pressures of approximately 50 MPa, and a lifetime of a few microseconds. The pressure changes resulting from these implosions create shock waves that disrupt the cellular membranes of bacteria, and this results in cell lysis and homogenization of lipid vesicles, such as the fat globules of milk (20). When mild heating is combined with ultrasonic processing, the treatment is referred to as thermoultrasonication.

The aim of the present study was to systematically review the published evidence on the efficacy of non-HoP techniques as a means of ensuring the safety of donor HM, and to compare the results on the effects of the microbiological, biochemical, and nutritive components of HM for its potential use in HMBs. Both thermal (HTST) and nonthermal processing techniques (HPP; UV-C; ultrasonic and (thermo-)ultrasonic processing) were reviewed.


Identification of Articles

A systematic search of the literature was conducted to find studies published in any language (provided that at least an abstract in English was available) that examined the effects of HTST, HPP, UV, and (thermo-)ultrasonic processing on the microbiological content and/or on the biochemical/bioactive/nutritional components of HM. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (21) have been followed in the present systematic review.

Literature searches of the Medline (via PubMed and PubMed Central), Embase, Scopus, and CAB Abstracts databases were performed on several occasions. The last database search was performed on April 20, 2016. The search strategy involved a 4-step process using “MeSH” and “Title/Abstract” terms. The first 3 searches were performed separately, and these were followed by the fourth search, which combined the results from the first 3 searches in each database to obtain the articles that were to be screened for relevance and subsequent review. The first search was on the following words: human milk OR breast milk OR breastmilk OR breast fed OR breastfed OR breastfeed OR donor milk OR banked milk; the second was on HTST OR Flash OR High Pressure OR UV OR ultrasonic OR non-thermal; the third was process OR pasteuris OR pasteuriz. This procedure was followed for all the databases, except for variation in the search terms specific to a single database: Scopus was searched for “Title/Abstract/Keywords,” CAB Abstracts for “All fields.”

The investigators determined eligible studies by screening the titles and available abstracts of all of the studies compiled from the final electronic database search. Bibliographies from studies included in the systematic review were also examined for additional applicable studies.

Screening of the Articles

To minimize the bias of this systematic review, 4 of the authors (C.P., N.E., M.G., and B.S.) evaluated the list of entries separately, according to predetermined inclusion/exclusion parameters, and finally consensus was reached by these authors. The inclusion criteria were primary (original) research published in a peer-reviewed journal; studies including only HM (either preterm or term HM and colostrum, transitional, or mature HM); and studies including, or excluding, a comparison with holder method pasteurization of the same milk. The exclusion criteria were the study design was a review, letter to the editor, or conference paper; the article included animal milk; the article did not clearly define the technique that had been applied; the article used a method that was not attributable to 1 specific technique; the technique was not intended for HMB use. The latter case led to the elimination of studies on Flash pasteurization, a low technology pasteurization method that was developed for home use to mimic industrial HTST pasteurization.

Data extracted from each relevant article included number of donor mothers included in the experimentation (when available); processing of pooled or individual samples; type of HM (when available); parameters of processing technique (including treatment, duration, intensity, equipment); comparison with either raw and/or holder pasteurized HM; type of outcome (microbiological and/or biochemical/nutritional/bioactive components).

All outcomes other than those requiring direct clinical trials (eg, growth measures) were considered in the systematic review.


Study Characteristics

The Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow chart of the database search is reported in Supplemental Digital Content 1, Figure 1 ( A total of 99 entries were found in the 4 databases, 12 of which were duplicate entries. Seventeen entries were excluded, because they were review or conference papers. Another 36 were excluded after checking the abstract because they did not deal with HM pasteurization, and 12 because only HoP was tested. Finally, 7 articles were eliminated because the Flash heating used in those articles was intended for home use (fifth exclusion criteria). All 15 remaining articles were read carefully, and the references were checked, and this led to the addition of 11 articles, for a total of 26 articles in the systematic review. The studies included in the present report are summarized in Supplemental Digital Content 2, Table 1 (, and are listed according to author and publication year, type of HM and number of mothers, and investigated pasteurization method. Of the 26 articles, 2 examined both HPP and HTST, 10 only examined HPP, 10 only examined HTST, 2 articles examined UV irradiation, and 2 articles examined (thermo-)ultrasonic processing. Seven articles did not include direct comparison with same samples pasteurized with holder method.

Novel Technologies

The results of the microbiological validation studies for both thermal (HTST) and nonthermal (HPP, UV-C and (thermo-)ultrasonic processing) technologies are listed in Table 1. The effects of non-HoP pasteurization on HM composition are listed in Table 2 for HTST, in Table 3 for HPP, and in Table 4 for both UV-C and (thermo-)ultrasonic processing. In most of the published studies, the authors also performed HoP, to offer a direct comparison of the results obtained with standard processing technology (see Supplementary Table 1).

Microbiological validation of innovative human milk processing technologies
Effects of High-Temperature–Short Time pasteurization on human milk components
Effects of High Pressure Processing on human milk components
Effects of (thermo-)ultrasonic processing or ultraviolet-C irradiation on human milk components

A considerable variability can be observed between the studies in terms of HM type (preterm vs term milk and colostrum vs transitional vs mature milk). The study populations also varied in number of donor mothers (1–60 donors per study). Moreover, the pasteurization parameters are also variable, even for HTST, which is considered a “standard” pasteurization technique in the food industry.


Main Findings

Thermal Alternatives to Holder Pasteurization: High-Temperature–Short-Time Pasteurization

HTST was the first non-HoP technique tested to improve the nutritional and immunological quality of milk, because it has been established in the dairy industry since the 1930s (14). HTST is usually performed by heating thin-layered milk in continuous flow systems at 72°C for 15 seconds, but in the past batch processes were sometimes performed to simulate HTST on HM, and/or to have shorter heating times, or higher temperatures than standard practice (5 seconds instead of 15; 75°C instead of 72°C).

Immunological components, and in particular immunoglobulins (Igs), are known to be affected by HoP (22–25), and were often targeted as qualitative/functional parameters in studies on alternative HM pasteurization technologies. Goldsmith et al (26) tested HTST pasteurization on HM for the first time using a stainless steel laboratory capillary heat exchanger. They reported comparable degradation after HTST and HoP for Igs and lactoferrin. One year later, using a plate-type industrial heat exchanger and injecting HM into a sterile water stream, the eradication of inoculated cytomegalovirus and endogenous bacteria by means of HTST was firstly reported (27). Moreover, no significant change was found in the contents of either the total and secretory IgAs (sIgAs—the dimeric form of IgAs), or lactoferrin. Hamprecht et al (28) reported that 5 seconds at 72°C were sufficient to destroy cytomegalovirus infectivity, although a comparable loss in sIgAs content with that of HoP was found. In that study, HTST was obtained by rotating 20 mL of HM to simulate thin layering. The data on the efficacy of HTST pasteurization were confirmed by challenge tests on HM samples inoculated with Escherichia coli and Staphylococcus aureus, using a small-scale device for continuous flow HTST pasteurization (29). The retention of HM Igs was found to decrease as the temperature and holding time were increased, thus indicating that the optimal compromise between microbiological safety and biological quality was to be searched for considering the pasteurization equipment and conditions. Continuous flow HTST pasteurization was also highly effective against inoculated lipid-enveloped viruses (HIV and marker viruses for hepatitis B and C) and bacteria (E. coli, S. aureus, and Streptococcus agalactiae) (30). On the contrary, a limited effect was found for nonlipid-enveloped viruses, including hepatitis A; thus, this system represents a main concern for its use in HMBs when in the presence of donor mothers infected by such viruses (30).

The concentration of lactoferrin, a protein with known antibacterial activity, was reported to be modified following HTST (26,31,32), although less so than by HoP. The decrease was due to protein aggregation by disulfide bonds (32), and the lactoferrin concentration after HTST was almost twice that of HoP. Because these data were measured on small volumes (40 μL) of skimmed HM, by submerging glass capillary tubes into a heated water bath, which is a significantly different way from the common HMB practice, they should be considered with some caution and need to be confirmed, as well as the high reduction of IgAs measured after both HTST and HoP (33).

The data on the effect of HTST on the concentration and activity of lysozyme, an enzyme with an important bactericidal activity, are divergent: it was reported to increase (27), following release from sequestered complexes, to decrease (28), and to be unaffected (33). It should, however, be pointed out that a different method was used for HTST in each study, which partially contributed to these differences.

Silvestre et al (34) evaluated the effect of HoP and HTST, performed as bulk processes at a higher temperature (75°C rather than 72°C), on HM bactericidal capacity (the capacity of HM to prevent the growth of inoculated test microorganisms) against E. coli. Although each pasteurization process induced a significant reduction in bactericidal capacity, the reduction was more pronounced when higher heating temperatures were used, thus confirming that heating affects the concentration of important immunological factors.

The activities of important biologically active enzymes (alkaline phosphatase [ALP], lipases) can be suitable markers for an effective HM pasteurization, because they are completely destroyed by both HoP and HTST (27–29). ALP is already employed in the dairy industry, because it is inactivated by a proper pasteurization. A higher bile salt–stimulated lipase concentration and total lipase activity following HTST, with respect to HoP, was also reported (31).

Although it was reported that HTST, and HoP, did not affect the total protein content of HM (28,31,35), it was reported to affect protein quality to some extent, by causing a differential occurrence of carbonylation (31), and a highly significant loss of available lysine (35), although this result has not been confirmed in a more recent study (31). Available lysine, an important nutritional marker, is also a common target of analysis to estimate protein damage and digestibility, as lysine can be modified and blocked by the Maillard reaction (31). The discrepancies between the 2 studies (31,35) could be due to the different processes (continuous flow vs bulk heating in water bath), and to the different temperatures (72°C vs 75°C), used for HTST. Other important nutritional and functional HM constituents, including folic acid and group B and C vitamins, were not significantly altered after HTST (27,28), and neither were several HM cytokines (36).

Silvestre et al (37) investigated oxidative stress markers (reduced glutathione, glutathione peroxidase activity, malondialdehyde, and total antioxidant capacity), and showed that the pasteurization of HM implies a decrease in its antioxidant properties, especially in the glutathione balance, but HTST caused a smaller loss in antioxidant potential than HoP.

From published evidence, it has emerged that HTST is at least equivalent to HoP in ensuring HM microbiological safety, but is better at preserving the HM antioxidant potential, lactoferrin content and structure, B and C vitamins, and some cytokines.

Nonthermal Alternatives to Holder Pasteurization: High Pressure Processing

Pressurization as a tool for HM pasteurization has been applied in several researches. A review on the topic, which includes 4 articles, has recently been published (25). In the present systematic review, 8 additional studies have been reviewed. The published studies tested a wide range of parameters: pressure, ranging between 200 and 900 MPa; duration, between 1 to 120 minutes; and temperature, ranging between 8°C and 80°C. In many cases, the variability in the studied conditions makes a comparison of the results from the different authors complicated.

As observed for thermal technologies, different microorganisms react with different degrees of resistance, even to HPP treatment: there can be vast difference in HPP sensitivity among bacterial species and even among strains, and between vegetative cells and spores (15). This variability was confirmed by Viazis et al (38), who assessed the efficacy of HPP (400 MPa) in reducing the pathogenic bacteria contamination of inoculated HM during challenge tests. Treatments of 2 and 4 minutes were sufficient for the complete inactivation of Listeria monocytogenes and S. agalactiae, respectively, whereas after 30 minutes of HPP, E. coli and 1 strain of S. aureus were only reduced by 6-log. HoP achieved higher levels of bacterial reduction than HPP in these conditions. In another report (39), HPP (400, 500, and 600 MPa for 5 minutes at 12°C) was as effective as HoP in reducing native total bacterial and Enterobacteriaceae counts to undetectable levels. Nevertheless, only 1 sample containing Enterobacteriaceae was analyzed, and the authors specified that samples collected from volunteer donor mothers for the study were likely less contaminated than those obtained from routine milk bank donors. Thus, it is not possible to assure the effectiveness of HPP in these conditions on routine banked milk with a higher bacterial content.

Immune components have also been targeted in most studies as qualitative parameters in the case of HPP. Viazis et al (40) found that HPP at 400 MPa preserved IgA immunoactivity more than HoP. The suitability of HPP for a better conservation of immunological components was confirmed (39,41), although a higher pressure than 600 MPa resulted in a comparable Igs level to that of HoP (39,41,42), also in colostrum (43). Mayayo et al (33) have recently reported that the degradation of IgAs increased as the HPP processing time and pressure increased, with pressures >500 MPa causing a comparable reduction to that induced by HoP. In their experiment, a significant variability, with respect to HMB practice, was noticed, because all the treatments were performed on skimmed HM. On the contrary, lysozyme activity was unaffected by HPP at any condition in either the colostrum or HM (33,40,43).

The effects of HPP on lactoferrin denaturation were also investigated: HPP in the 300 to 600 MPa range at 20°C for 30 minutes resulted in a lactoferrin decrease much smaller than following HoP (32). The authors also pointed out that lactoferrin aggregation does not take place after HPP, whereas it does occur with thermal technologies. The other immunological components that were profiled included the leukocyte content, which was almost destroyed by both HPP and HoP (41,42).

HPP at 300 to 600 MPa for 1 minute had a minimal effect on the levels of most cytokines, but harsher conditions (60°C–80°C and a pressure of 900 MPa) caused a significant decrease in some cytokines (41). In milder conditions, the effect was generally lower, especially in comparison to HoP (44).

The effects of HPP were tested on the HM fatty acids (FAs) profile, and on vitamins C and E (45). Compared to HoP, HPP led to a better retention of vitamin C, and to the same levels of FAs and vitamin E. When HM was treated at more severe conditions (65°C or 80°C at 300, 600, and 900 MPa, for 1 minute), vitamin E was also reduced, and the most extreme treatment (900 MPa at 80°C) caused intense changes in the FAs profile (41). HPP at 600 MPa at room temperature significantly decreased the level of vitamin E, and a reduction in proportions of some key FAs (eg, α-linolenic and docosahexaenoic acids) (44).

Contador et al (46) have recently evaluated the volatile profile of pressurized HM. The volatile profile (particularly odorous volatiles) reflects the image of the odor and aroma of milk, and its analysis could therefore be used to assess global changes that occur after processing, especially adverse reactions (eg, lipid oxidation). In general, the effect of HPP on the volatile profile was less intense than that caused by HoP. The volatile compounds were similar to those of raw HM, when treated at 400 and 600 MPa for 3 minutes. HPP at 600 MPa for 6 minutes, however, changed the original volatile profile, by increasing the formation of unwanted aldehydes, furans, and pyrans. All these compounds were associated with an increase in lipid oxidation and with the degradation of sugars and amino acids via the Maillard reaction, thus supporting previous findings on a potential detrimental effect of higher operating pressure on nutritional and functional HM components.

Mateos-Vivas et al (47) studied the effects of HoP and HPP on the nucleotide contents in HM samples. The content of nucleotides in the HPP samples was equal to those of the untreated samples, whereas the nucleotide content in the samples treated by means of HoP increased, thus indicating that free nucleotides could be generated from polymeric nucleotides and/or nucleotide adducts by increasing the temperature during the pasteurization step.

In short, the data so far indicate that HPP, at least at room temperature or below, and with applied pressures not exceeding 400 MPa, may increase the retention of important immunological components (Igs, lysozyme, some cytokines), and also of some vitamins and FAs, with respect to HoP.

Nonthermal Alternatives to Holder Pasteurization: Ultraviolet-C Irradiation

HM is difficult to treat with UV-C, due to its high absorption coefficient, which increases as the total solids concentration increases, and thus limiting the penetration depth of the photons. This limitation can be overcome through the application of a vertical flow of HM around a UV-C source (48,49). To the best of the authors’ knowledge, only 1 study has assessed UV-C effects on HM microbiology so far (48). The authors placed a germicidal UV-C lamp diagonally into a glass beaker containing HM, and the milk was stirred during irradiation to generate a low velocity, laminar flow vortex. HM samples were tested at increasing concentrations of soluble solids (by fat adjustment), inoculated with S. epidermidis, Enterobacter cloacae, Bacillus cereus, and E. coli K12; a close correlation between the bactericidal efficacy and the total solids concentration of the samples was observed, and with increasing irradiation times, and thus indicating that UV-C treatment should be adjusted, according to the sample to be pasteurized, to ensure complete removal of the bacteria. UV-C irradiation was also shown to preserve the bioactivity of lipase, ALP, and the FA profile of HM (with the exception of FA 8:0), and thus retaining factors essential to the health of (pre-)term infants.

In another study (49), the authors found that the retention of the tested immunological proteins, including lactoferrin, lysozyme, and sIgAs, was dependent on the exposure dosages during UV-C irradiation. A lower dosage resulted in a higher retention rate, and vice versa. Nevertheless, UV-C irradiation preserved significantly higher levels of immunological proteins than HoP, and thus contributed to a slower bacterial growth and higher bactericidal capacity, for UV-C treated HM, than HoP.

There is currently a lack of data on the effects of UV-C irradiation on the microbiological and viral contamination of HM. Fundamental knowledge is still lacking, and extensive research is therefore required to obtain a reliable alternative to the conventional thermal pasteurization.

Nonthermal Alternatives to Holder Pasteurization: Ultrasonic and Thermoultrasonic Processing

Some studies have used ultrasonic processing to effectively eliminate various food-borne pathogens, including L. monocytogenes, Salmonella spp, E. coli, S. aureus, and B. subtilis from bovine milk and fruit juices. The microbial inactivation rates that can be achieved using ultrasounds can be improved through a combination with mild heating (thermoultrasonic processing) (19).

Czank et al (50) evaluated the effect of ultrasonic and thermoultrasonic processing on artificially contaminated HM samples (challenge tests). The authors used an ultrasonic cell disruptor that produced acoustic waves of 150 W peak power, and, for thermoultrasonic processing, the unit was mounted onto a precision water bath, which was heated at 45°C and 50°C. The thermoultrasonic processing was considerably more effective than ultrasounds alone against inoculated S. epidermidis and E. coli, and in achieving homogenization, which is a desirable side effect for milk. Lysozyme retention after the ultrasonic processing was approximately 65%, whereas it was lower after thermoultrasonic processing at 45°C and 50°C. Lipase activity was sensitive to both treatments, with a 30% activity being retained in the mildest applied conditions. On the contrary, the sIgA loss rate was found to be similar for the ultrasonic and thermoultrasonic processing at both temperatures.

Christen et al (51) performed ultrasonic processing at constant power for different times, observing that inoculated E. coli viability decreased exponentially over treatment time. A stable ultrasound energy (1000 J) was then tested with different time-power ratios: the combination of the longest treatment time and the lowest power was the most effective in reducing the viability of inoculated E. coli, but it was also responsible for an increase in HM temperature, confirming that temperature is a critical parameter for the efficacy of ultrasounds. In their study, the viability of E. coli was reduced by 5-log, with minimal heat damage, by applying 13.8 kJ of energy, using high ultrasound power over a short exposure time to ensure that the temperature remained below the critical level for protein denaturation. Lipase activity was only reduced significantly when the longest treatment time was applied at the lowest power, and thus demonstrating that the longer exposure time, rather than the high ultrasound power, was responsible for the increase in temperature, and the subsequent denaturation of the enzyme.

As previously observed for UV-C, few microbiological data are available concerning the safety of (thermo-)ultrasonic processing for use in HMBs. Moreover, the heating of HM the process is critical to ensure positive qualitative advantages, with respect to HoP, and should be kept at a minimum.

Strengths and Limitations

Several studies have been conducted, some of them recently, describing the effects of novel techniques on the nutritional, immunological, and microbiological contents of HM. Depending on the technology exploited, specific immunological and/or nutritional features of HM are better preserved than following HoP, in particular when the increase in temperature is limited.

The most extensively studied methods are HTST and HPP, but both still need further confirmation of the safety and benefits that can be derived from their exploitation in HMBs. The efficacy of HTST in eliminating bacterial and viral loads, although pointed out in several reports, should be confirmed with appropriate commercial devices, because the studies published to date were performed under different laboratory conditions and using different equipment (plate type heat exchangers, capillary tubes, rotary equipment; bulk, continuous treatments; variable holding times and temperatures).

As far as nonthermal technologies are concerned, few microbiological data are available to date to ensure their efficacy for use with donor milk, especially considering the strict HMB requirements (no bacterial growth after pasteurization). The efficacy of these methods on viruses, fungi, and spore-forming bacteria should also be investigated more thoroughly. For example, only 2 reports studied HPP efficacy as a means of ensuring HM microbiological safety, and no article has been found that describes its effect on viruses. A further complication for HPP is represented by the differences in the 3 (instead of 2) parameters that need to be controlled (pressure, temperature, time). It would be difficult to draw final conclusions on this technique until consensus on the applied parameters is reached.

Another main limitation of all the studied novel techniques is that, to date, no other commercial device for HM pasteurization, apart from the HoP device, is available on the market, so that there is a significant variability in the tested conditions. Moreover, in almost all the reviewed studies, with the exception of (28,29,36), no monitoring of goal parameters inside the tested HM samples, including HM temperature control, is reported. This represents a further complication for the generalization of conclusions about the safety and quality of the resulting pasteurized HM. During the 2015 congress of European Milk Banks Association, 2 commercial prototypes of alternative pasteurizers for HMBs, one based on HTST (52) and the other on HPP (53), were presented, thus representing, when they will be available on the market, a possible solution to fill the gap between research and routine use of these technologies. The HTST prototype was also described more extensively in a recently published paper (54).

Finally, a limitation for the evaluation of benefits of each technique with respect to HMB standard pasteurization method is represented by the difficult quantification of qualitative advantages on bioactive components, because HoP is simulated on small HM aliquots in most of the studies, and not performed in real HMB conditions (24).

Agreement and Disagreement With Other Reviews

To our knowledge, only 1 critical review on HPP (25) recently described the evidences to date for its use in HMBs. The authors agreed about the lack of sufficient data to ensure the adequacy and advantages of HPP when compared to HoP for treating HM, at present. In particular, the absence of studies on the effect of HPP on spore-forming bacteria, viruses, fungi, and prions, was considered an important limitation, as in our systematic review.


In conclusion, fundamental knowledge on novel HM treatments and their effects on safety and composition are still lacking, and extensive research is still required. Moreover, other newly available techniques should be tested, such as pulsed electric fields, or the combination of different novel approaches. A thorough analysis of the implementation potential of these techniques should also be made to ensure their readiness of use and reduced operating costs, in particular in the HMB operating environment, characterized by variability of volumes of processed donor milk. Finally, the technical and safety measurements of new methods for the treatment of HM samples should be accompanied by new, validated bioanalytical techniques and assays, to determine the most relevant functional ingredients for the nutrition and health benefits of those infants who receive donor milk.


1. European Milk Bank Association (EMBA). The sharing of breastmilk. EMBA Web site Published December 2011. Accessed December 28, 2015.
2. American Academy of Pediatrics (AAP)Breastfeeding and use of human milk. Pediatrics 2012; 129:e827.
3. World Health Organization (WHO)/United Nations Children's Fund (UNICEF)Meeting on infant and young child feeding. J Nurs Midw 1980; 25:31–38.
4. Arslanoglu S, Corpeleijn W, Moro G, et al. European Society for Paediatric Gastroenterology Hepatology, Nutrition (ESPGHAN) Committee on NutritionDonor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr 2013; 57:535–542.
5. Arslanoglu S, Bertino E, Tonetto P, et al. Italian Association of Human Milk Banks (AIBLUD)Guidelines for the establishment and operation of a donor human milk bank. J Matern Fetal Neonatal Med 2010; 23:1–20.
6. Human Milk Banking Association of North America (HMBANA)Guidelines for the Establishment and Operation of a Donor Human Milk Bank. 9th EdRaleigh, NC: Human Milk Banking Association of North America; 2015.
7. Rede Brazileira do Bancos de Leite Humano. Banco de leite humano: funcionamento, prevenção e controle de riscos. Brazilia: Ed. Agência Nacional de Vigilância Sanitária; 2008.
8. Excellence NIoHaC. Donor milk banks: the operation of donor milk bank services. NICE 2010. Published February 2010. Accessed August 5, 2016.
9. McGuire W, Anthony MY. Donor human milk versus formula for preventing necrotising enterocolitis in preterm infants: systematic review. Arch Dis Child Fetal Neonatal Ed 2003; 88:F11–F14.
10. Boyd CA, Quigley MA, Brocklehurst P. Donor breast milk versus infant formula for preterm infants: systematic review and meta-analysis. Arch Dis Child Fetal Neonatal Ed 2007; 92:F169–F175.
11. Quigley M, McGuire W. Formula milk versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database Syst Rev 2014; 4:CD002971.
12. Cossey V, Vanhole C, Eerdekens A, et al. Pasteurization of mother's own milk for preterm infants does not reduce the incidence of late-onset sepsis. Neonatology 2013; 103:170–176.
13. Ortega-Rivas E, Salmerón-Ochoa I. Nonthermal food processing alternatives and their effects on taste and flavor compounds of beverages. Crit Rev Food Sci Nutr 2014; 54:190–207.
14. Holsinger VH, Rajkowski KT, Stabel JR. Milk pasteurisation and safety: a brief history and update. Rev Sci Tech 1997; 16:441–451.
15. Considine KM, Kelly AL, Fitzgerald GF, et al. High-pressure processing-effects on microbial food safety and food quality. FEMS Microbiol Lett 2008; 281:1–9.
16. Huppertz T, Smiddy MA, Upadhyay VK, et al. High-pressure-induced changes in bovine milk: a review. Int J Dairy Tech 2006; 59:58–66.
17. Jay JM. Modern Food Microbiology. 6th ed.Gaithersburg, MD: Aspen Publishers Inc; 2000.
18. Shama G. Robinson RK, Batt C, Patel P. Ultraviolet light. Encyclopedia of Food Microbiology 3rd ed.London: Academic Press; 1999. 2208–2214.
19. Piyasena P, Mohareb E, McKellar RC. Inactivation of microbes using ultrasound: a review. Int J Food Microbiol 2003; 87:207–216.
20. Cameron M, McMaster LD, Britz TJ. Electron microscopic analysis of dairy microbes inactivated by ultrasound. Ultrason Sonochem 2008; 15:960–964.
21. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 2009; 6:e1000097.
22. Akinbi H, Meinzen-Derr J, Auer C, et al. Alterations in the host defense properties of human milk following prolonged storage or pasteurization. J Pediatr Gastroenterol Nutr 2010; 51:347–352.
23. Chang JC, Chen CH, Fang LJ, et al. Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins. Pediatr Neonatol 2013; 54:360–366.
24. Peila C, Moro GE, Bertino E, et al. The effect of Holder pasteurization on nutrients and biologically-active components in donor human milk: a review. Nutrients 2016; 8:477.
25. Sousa SG, Delgadillo I, Saraiva JA. Human milk composition and preservation: evaluation of high-pressure processing as a nonthermal pasteurization technology. Crit Rev Food Sci Nutr 2016; 56:1043–1060.
26. Goldsmith SJ, Dickson JS, Barnhart HM, et al. IgA, IgG, IgM and lactoferrin contents of human milk during early lactation and the effect of processing and storage. J Food Protection 1983; 1:4–7.
27. Goldblum RM, Dill CW, Albrecht TB, et al. Rapid high-temperature treatment of human milk. J Pediatr 1984; 104:380–385.
28. Hamprecht K, Maschmann J, Müller D, et al. Cytomegalovirus (CMV) inactivation in breast milk: reassessment of pasteurization and freeze-thawing. Pediatr Res 2004; 56:529–535.
29. Dhar J, Fichtali J, Skura BJ, et al. Pasteurization efficiency of a HTST system for human milk. J Food Sci 1996; 61:569–572.
30. Terpstra FG, Rechtman DJ, Lee ML, et al. Antimicrobial and antiviral effect of high-temperature short-time (HTST) pasteurization applied to human milk. Breastfeed Med 2007; 2:27–33.
31. Baro C, Giribaldi M, Arslanoglu S, et al. Effect of two pasteurization methods on the protein content of human milk. Front Biosci Elite 2011; 3:818–829.
32. Mayayo C, Montserrat M, Ramos SJ, et al. Kinetic parameters for high-pressure-induced denaturation of lactoferrin in human milk. Int Dairy J 2014; 39:246–252.
33. Mayayo C, Montserrat M, Ramos SJ, et al. Effect of high pressure and heat treatments on IgA immunoreactivity and lysozyme activity in human milk. Eur Food Res Technol 2016; 242:891–898.
34. Silvestre D, Ruiz P, Martínez-Costa C, et al. Effect of pasteurization on the bactericidal capacity of human milk. J Hum Lact 2008; 24:371–376.
35. Silvestre D, Ferrer E, Gayá J, et al. Available lysine content in human milk: stability during manipulation prior to ingestion. Biofactors 2006; 26:71–79.
36. Goelz R, Hihn E, Hamprecht K, et al. Effects of different CMV-heat-inactivation-methods on growth factors in human breast milk. Pediatr Res 2009; 65:458–461.
37. Silvestre D, Miranda M, Muriach M, et al. Antioxidant capacity of human milk: effect of thermal conditions for the pasteurization. Acta Paediatr 2008; 97:1070–1074.
38. Viazis S, Farkas BE, Jaykus LA. Inactivation of bacterial pathogens in human milk by high-pressure processing. J Food Protection 2008; 71:109–118.
39. Permanyer M, Castellote C, Ramírez-Santana C, et al. Maintenance of breast milk immunoglobulin A after high-pressure processing. J Dairy Sci 2010; 93:877–883.
40. Viazis S, Farkas BE, Allen JC. Effects of high-pressure processing on immunoglobulin A and lysozyme activity in human milk. J Human Lactation 2007; 23:253–261.
41. Delgado FJ, Contador R, Álvarez-Barrientos A, et al. Effect of high pressure thermal processing on some essential nutrients and immunological components present in breast milk. Innovative Food Sci Emerg Technol 2013; 19:50–56.
42. Contador R, Delgado-Adámez J, Delgado FJ, et al. Effect of thermal pasteurisation or high pressure processing on immunoglobulin and leukocyte contents of human milk. Int Dairy J 2013; 32:1–5.
43. Sousa SG, Santos MD, Fidalgo LG, et al. Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum. Food Chem 2014; 151:79–85.
44. Delgado FJ, Cava R, Delgado J, et al. Tocopherols, fatty acids and cytokines content of Holder pasteurised and high-pressure processed human milk. Dairy Sci Technol 2014; 94:145–156.
45. Moltó-Puigmartí C, Permanyer M, Castellote AI, et al. Effects of pasteurisation and high-pressure processing on vitamin C, tocopherols and fatty acids in mature human milk. Food Chem 2011; 124:697–702.
46. Contador R, Delgado FJ, García-Parra J, et al. Volatile profile of breast milk subjected to high-pressure processing or thermal treatment. Food Chem 2015; 180:17–24.
47. Mateos-Vivas M, Rodríguez-Gonzalo E, Domínguez-Álvarez J, et al. Analysis of free nucleotide monophosphates in human milk and effect of pasteurisation or high-pressure processing on their contents by capillary electrophoresis coupled to mass spectrometry. Food Chem 2015; 174:348–355.
48. Christen L, Lai CT, Hartmann B, et al. Ultraviolet-C irradiation: a novel pasteurization method for donor human milk. PLoS One 2013; 8:e68120.
49. Christen L, Lai CT, Hartmann B, et al. The effect of UV-C pasteurization on bacteriostatic properties and immunological proteins of donor human milk. PLoS One 2013; 8:e85867.
50. Czank C, Simmer K, Hartmann PE. Simultaneous pasteurization and homogenization of human milk by combining heat and ultrasound: effect on milk quality. J Diary Res 2010; 77:183–189.
51. Christen L, Lai CT, Hartmann PE. Ultrasonication and the quality of human milk: variation of power and time of exposure. J Diary Res 2012; 79:361–366.
52. Bertino E, Di Nicola P, Tonetto P, et al. HTST treatment for human milk. In: 3rd International Congress of the European Milk Bank Association (EMBA) Abstract book. Lyon, France. Published October 8–9, 2015. Accessed August 5, 2016.
53. Demazeau G. A new high hydrostatic pressure treatment of human milk leading both to microbial safety and preservation of the activity of the main components. In: 3rd International Congress of the European Milk Bank Association (EMBA) Abstract book. Lyon, France. Published October 8–9, 2015. Accessed August 5, 2016.
54. Giribaldi M, Coscia A, Peila C, et al. Pasteurization of human milk by a benchtop high-temperature short-time device. Innov Food Sci Emerg Technol 2016; 36:228–233.

High Pressure Processing; High-Temperature–Short-Time; human milk banks; ultrasounds; ultraviolet-C

Supplemental Digital Content

Copyright 2016 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.