Human milk (HM) is the recommended type of enteral nutrition for premature infants but it is frequently pasteurized to render inactive viruses and reduce bacterial counts (1, 2). Holder pasteurization (HoP) represents the current gold standard for pasteurization whereby HM is heated to a plateau temperature of 62.5 ± 0.5°C with a holding time of 30 min. However, all heat treatments degrade a wide range of bioactive HM components, thus affecting HM quality and may therefore ultimately affect the clinical outcome of premature infants (3–5). Treatments that limit heat exposure, such as high-temperature short-time treatment (HTST), i.e., heating HM at 62–72°C for mere seconds, is therefore beneficial in retaining those bioactive components, and is currently being tested for treating human milk (6).

Recently, because of hygienic concerns and feasibility issues, solid bodies such as aluminum alloy thermostats (dry-tempering, DT) were introduced into clinical use. Those thermostats consist of electrically heated aluminum alloy blocks with customized individual boreholes for feeding bottles. Automatic processes guided by a sensor fitted reference bottle perform a Holder pasteurization whereas the milk is heated by the alloy block, foregoing any need of water as heat conductor. However, the effects of such thermostats on HM quality compared to water-bath pasteurizers have not been assessed.

We hypothesized that heat conduction while dry-tempering may be less efficient, potentially resulting in HM's longer heat exposure and an even further drop in its beneficial protein content compared to water-bath based pasteurization.

We aimed to assess the impact of water-bath based pasteurization (WB-HoP) compared to dry-tempering pasteurization (DT-HoP) on the retention rates of lactoferrin (LF), secretory immunoglobulin A concentration (sIgA), and alkaline phosphatase activity (ALP) contrasting those results to the latest HTST technology.

The time-temperature curves of the three different pasteurization processes are shown in Figure 2.

Mean (±SD) time to reach 62°C was 2.02 ± 0.2 min for HTST, to reach 62.5°C was 23.7 ± 1 min for WB-HoP and 38 ± 2 min for DT-HoP (WB-HoP vs. DT-HoP, p > 0.001). Time from 62.5°C to 10°C was 15.7 ± 1.2 min for WB-HoP and 43.1 ± 8 min for DT-HoP (WB-HoP vs. DT-HoP, p > 0.001) and time to 4°C was 39.3 ± 4 min for WB-HoP and 69 ± 14 min for DT-HoP (WB-HoP vs. DT-HoP, p > 0.001). Mean (SD) peak temperature was 62.7 ± 0.1°C for WB-HoP, and 64.6 ± 0.1°C for DT-HoP (WB-HoP vs. DT-HoP, p > 0.001).

In the unpasteurized samples, mean (±SD) values were 0.68 ± 0.6 g/L for sIgA and 15.2 ± 8.6 g/L for LF concentration, ALP activity was 0.84 ± 0.3 mU/mL. Protein retention rates of ALP, sIgA and LF are illustrated in Figure 3.

In general, HTST treatment revealed higher retention rates than Holder methods. ALP activity was below the detection limit after any HoP. Otherwise, the two Holder methods' retained protein concentrations differed. sIgA concentrations were 0.47 ± 0.4 g/L after WB-HoP and 0.38 ± 0.3 g/L after DT-HoP (p = 0.001). LF concentrations measured 7.2 ± 6.9 g/L after WB-HoP and 3.8 ± 2.6 g/L after DT-HoP (p = 0.07).

The generally adverse effects of treating HM thermally, i.e., protein inactivation due to different degrading mechanisms, are well known (3). In this study, we demonstrated that selecting the technical application for Holder pasteurization further influences the retention rate of immunoactive HM proteins.

HoP desktop appliances that avoid traditional water-bath devices for heating milk are potentially more appealing in a clinical milk bank setting for hygienic and feasibility reasons. However, they can potentially deteriorate HM quality further, as our study shows. Both heating and cooling times differed in the tested HoP devices, as the heat-transfer performance appears to be superior in the water-bath compared to dry-tempering devices. The resulting lag times due to the diverse heat conduction displayed by the systems appear to be responsible for the differences in protein retention we observed. Furthermore, thermic inertia may also be the cause of higher peak temperatures in the dry-tempering than the water-bath device.

In line with our findings, Buffin et al. similarly compared water-bath pasteurization to air pasteurization, and demonstrated significant differences in milk's heat exposure between these two pasteurization methods, suggesting that this might significantly compromise HM's bioactive components (8).

The generally adverse effect of thermally treating HM are well described (3). Increasing temperatures resulted in higher rates of alkaline phosphatase enzyme inactivation whereas bovine milk immunoglobulins could maintain their structure resisting temperatures up to 75°C for 15 s albeit losing their antigen-binding efficacy (9, 10). Lactoferrin concentration has been consistently shown to be reduced after milk heat treatments but appears to retain its antibacterial activity even after heat treatments of 85°C for up to 10 min (11).

However, in the absence of alternative feasible treatment options, heat treatment in form of HoP is the best compromise currently available. There have been various attempts to optimize HoP. Buffin et al. demonstrated increased retention rates of IgA and LF when comparing an updated and optimized Holder pasteurizer to the same manufacturer's previous model, despite both models adhering to standard HoP criteria (12). Another modification of HoP included using a variation in the time-temperature curve as tested by Capriati et al. They applied a shortened pasteurization cycle lasting about 65 min and a peak temperature close to 72°C immediately followed by the cooling process, which revealed a trend toward better retention of triglycerides compared to standard HoP (13). By shortening the ramp time and thus heat exposure of HM proteins, Kontopodi at al. observed a tendency toward improved protein retention in milk that was rapidly preheated with an experimental HTST device before being Holder-pasteurized (14).

To preserve HM's immunologic and nutritional profile for premature infants, methods other than Holder pasteurization, such as HTST, have been evaluated. These methods were developed with the aim to achieve antiviral or antibacterial efficacy approximating the gold standard HoP, while at least weakening its detrimental impact on HM (15). Reports of increased protein retention by comparing HTST devices at 62–87°C plateau temperatures and 1–18.5 s holding times to HoP are in line with our findings (14, 16–19). Unlike those experimental continuous flow devices, we utilized a commercially available device originally designed for cytomegalovirus inactivation (7). However, this batch device appears to be less efficient in reducing bacterial counts than HoP or continuous flow HTST devices (20).

Given the available body of evidence on HTST treatment of HM, efforts should concentrate on developing non-HoP pasteurizers for actual routine use in a clinical setting (6, 15). HoP remains the best compromise currently available.

The dry-tempering of human milk impairs the quality of HM for premature infants more than water-bath Holder pasteurization does. Universal technical standards and specifications for HM pasteurizers are lacking. Therefore, vigorous quality control measures must be carried out before adopting new appliances.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

This study was approved by the Ethics Committee of Albert-Ludwigs-University of Freiburg, Germany (332/19, 8th October 2019). The patients/participants provided their written informed consent to participate in this study.

KM participated in the acquisition of the HM samples and performed the pasteurization process, analyzed the data, and wrote the first draft of the manuscript. DK conceived and designed the study and helped acquire the HM samples, analyzed and interpreted the data, and contributed to drafting the manuscript. OM and JB contributed to drafting the manuscript. FW, LT, and EN designed the study, conducted the biochemical analysis, and contributed to drafting the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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