Introduction

Human breast milk is widely accepted as the gold standard source for nutrition of the newborn and is particularly beneficial in preventing neonatal pathologies thanks to its complex composition.1 In this respect, breast milk contains numerous bioactive factors such as lactoferrin, lysozyme, secretory IgA and human milk oligosaccharides (HMOs)2 exhibiting antimicrobial and anti-inflammatory properties. Lactoferrin is an abundant milk protein known for its anti-inflammatory, immunomodulatory and prebiotic capacities.3,4 The bacteriostatic and bactericidal actions of lactoferrin are related to its ability to sequester iron, to interact with lipopolysaccharides of Gram-negative bacteria and lipoteichoic acid of Gram-positive bacteria and to inhibit the attachment of pathogens to host cells.5 Lysozyme is another bioactive protein detected at high concentrations in human breast milk which is able to degrade the bacterial cell wall.6 Moreover, lactoferrin’s antibacterial actions can be synergistic with lysozyme, particularly on Gram-negative bacteria.7 Altogether, the antimicrobial functions of human breast milk are paramount for infant health, and even more so with regard to vulnerable neonates.

Preterm birth is a major public health issue which can lead to short and/or long-term deleterious consequences.8 Premature infants are born with an immature immune system and gastrointestinal tract, making them highly susceptible to neonatal infections and severe intestinal pathologies such as necrotizing enterocolitis.9 Escherichia coli and group B Streptococcus (GBS) are two representative pathogens involved in early-onset sepsis occurring in both term and preterm infants. The incidence of E. coli and GBS infection is respectively 0.40 and 0.33 cases/1000 live births.10 These infections result from vertical transmission from mother to newborn infant before 72 h of life.10,11 Interestingly, higher concentrations of fecal E. coli were reported in cases of necrotizing enterocolitis, and are promoted by prematurity and feeding with preterm formula.12 In contrast with breast milk, bioactive factors are lacking in infant formula.13 Thus, when mother’s own milk is not available, the preferred feed for preterm infants is donor milk (DM), which is collected, treated and stored in human milk banks (HMBs). Inadequate hygiene during milk collection and processing in HMBs may result in contaminated maternal breast-milk through growth of E. coli or GBS,14,15,16 with potentially highly deleterious consequences for preterm babies.

To ensure safe long-term storage of DM, Holder Pasteurization (HoP) by heating to 62.5 °C for 30 min, is the reference method used extensively in HMBs. However, this treatment dramatically reduces the concentration of DM bioactive factors, particularly antimicrobial ones (e.g., immunoglobulins, lactoferrin, lysozyme).17,18 Recently, there has been increased interest in the century-old High Hydrostatic Pressure (HHP) processing to sterilize DM.19,20 It relies on various pressure concentrations (ranging from 100 to 600 MPa) at low temperature (from 19 to 38 °C) to sterilize the DM. Using different HHP protocols, several studies have already demonstrated the better preservation of milk bioactive factors, such as immunoglobulins and immunological factors,21,22,23 but only a few authors have investigated its impact on milk antimicrobial activity.24,25 Using a specific HHP protocol moderate 350 MPa pressure, 38 °C temperature, 1 MPa.s−1 application rate, with 4 cycles of 5 min each with a latency time of 5 min at normal pressure between each cycle, we recently showed a better preservation of DM metabolic hormones with HHP, contrary to HoP, making it an attractive surrogate for HoP processing.23 In this context, we aimed to assess the effects of HoP and our particular HHP processing on two major milk antimicrobial proteins, lactoferrin and lysozyme, together with their actions on DM antimicrobial activity against E. coli and GBS strains. We also prospectively evaluated the safety of HHP-treated DM storage conditions over a 6-month period.

Methods

Donor milk collection

The study protocol was approved by the ethics committee of the Groupe Francophone d’Hépatologie, Gastroentérologie et Nutrition Pédiatrique (2023–51). DM was collected from 11 volunteers for the antimicrobial factors assays and the bacterial cultures, and from 15 additional volunteers for the milk storage study, in the regional HMB (Lactarium Régional de Lille, Jeanne de Flandre Children’s Hospital, CHU Lille). All donors were fully informed about the research study and provided their written consent to participate.

Human breast milk preparation and treatments

Milk samples were divided into 8 different batches of DM with homogeneous composition as previously described.23,26 Each batch was composed of 3 aliquots of DM as follows: one untreated fraction (raw milk), one fraction treated by HoP using the standard HoP protocol (i.e., 62.5 °C for 30 min) and the last fraction was treated according to HHP processing as previously described.21,23 Following HoP and HHP treatments, the sterilization process efficacy was confirmed by microbiological analysis (Bacteriology Laboratory, Centre de Biologie-Pathologie, CHU de Lille). All aliquots were stored at −80 °C before analysis. For the DM storage study, DM samples were treated by HHP using the same protocol.

Lactoferrin and lysozyme assays

Lactoferrin and lysozyme concentrations were determined in the three fractions of DM (i.e., raw, HoP- and HHP-treated DM) including 8 DM batches per fraction and using ELISA kits ab200015 and ab108877 assays respectively (Abcam, Paris, France). As recommended by the manufacturer, defatted milk was used and assays were optimized for linear-range detection of each protein before protein concentrations assessment.

Bacterial culture and colony counting

Raw milk samples were contaminated with high concentration of endogenous bacteria and multiplicity of species (103−104 CFU/mL) (data not shown). To avoid confounding factors, such as growth competition between endogenous and seeded bacteria,27 only HoP and HHP-treated milk were further studied for the antimicrobial activity experiment. The impact of both treatments (i.e., HoP and HHP) on DM antimicrobial properties of 4 DM batches was assessed on two bacterial strains: E. coli ATCC 25922 and S. agalactiae ATCC 131813 as a Group B Streptococcus representative (GBS). Briefly, bacteria were pre-cultured overnight at 37 °C on Luria Bertani (LB) (Sigma-Aldrich) or of tryptic soy (TS) (Difco, Fisher) plates respectively for E. coli and GBS. Colonies were resuspended in 10 mL of LB or TS broth respectively and incubated at 37 °C under agitation (150 rpm) to reach exponential growth phase. Then, LB or TS broth (i.e., controls for E coli and GBS), HoP- or HHP-treated milks, were inoculated 1:10 volume with bacterial suspension (mean OD 600 nm = 0.97 and 0.35 for E. coli and GBS, respectively) corresponding to a final concentration of 106 CFU/mL. Bacterial inoculum was then quantified by plating adequate dilutions (from 10−1 to 10−6 for E. coli and from pure to 10−4 for GBS) at several timepoints: every 30 min for 4 h, then every hour for 2 h, and at 24 h (i.e., endpoint). All plates were incubated for 24 h at 37 °C. The limit of detection was 100 CFU/mL. Antimicrobial effect was expressed as Log reduction between Log of bacterial counts in control media comparatively to Log of bacterial counts in treated-milk samples. Following this procedure, all HoP and HHP samples (n = 4) were analysed in duplicate.

Donor milk storage study

Aliquots of the 15 HHP-treated DM samples provided by 15 additional donor mothers were stored at +4 °C and −20 °C under appropriate conditions for a six month period. The microbiological contamination of each refrigerated and frozen DM sample was assessed prospectively by plating 0.5 mL of DM on Columbia horse blood agar plates incubated for 48 h at 37 °C. The limit of detection was 2 CFU /mL. In case of positive samples, bacterial/microbial identification was performed directly on the colonies by MALDI-TOF mass spectrometry according to the manufacturer recommendations (Brüker, Wissembourg, France).

Statistical analysis

All data are expressed as mean ± SEM. Statistical analyses were performed with GraphPad Prism 8.0. Software (San Diego, CA). Outliers were excluded using Grubb’s test. Variables normality was assessed by a D’Agostino-Pearson test. If necessary, a log transformation was applied to reach normality. For the ELISAs, statistical differences were tested by One-way Anova (Tukey’s post-test). For the bacterial growth study, Kruskal-Wallis (followed by Dunn’s post-test) was used for group comparisons. Correlation between the values of area under the curve (AUC) and lactoferrin or lysozyme concentrations for each pool of DM, treated by HoP or HHP, was tested by Spearman’s correlation; p < 0.05 was considered significant.

Results

Donor milk antimicrobial factors storage

The effects of HoP and HHP treatments on lactoferrin and lysozyme concentrations are reported in Fig. 1. Lactoferrin concentration was similar between raw DM and HHP-treated DM (p = 0.99) and thus not affected by the treatment (Fig. 1a). Conversely, it was decreased by 30% with HoP treatment compared to raw milk (16.6 g/l ± 2.3 versus 23.6 g/l ± 1.3, p = 0.019). In contrast, lysozyme levels were not statistically different between the three groups (Fig. 1b).

Fig. 1: Lactoferrin and Lysozyme concentrations after donor milk treatments.
figure 1

Concentrations of Lactoferrin (a) and Lysozyme (b) in raw donor milks or after Holder Pasteurization or High Hydrostatic Pressure processing. Values are expressed in mean ± SEM. n = 8 per group. HoP Holder Pasteurization. HHP High Hydrostatic Pressure. Statistically differences for unpaired comparisons: *p < 0.05. ns not significant.

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Growth kinetics of E. coli and GBS in HoP and HHP-treated milks

We further analysed DM antibacterial activity after HHP treatment on E. coli and group B Streptococcus (GBS). Results showed a significant bacteriostatic effect of HHP DM pools on E. coli growth at early timepoints (i.e., 2.5 h, 3 h, 4 h and 5 h), reaching a maximum of 1.36 Log difference (p < 0.05) between HHP and control media after 4 h of incubation (Fig. 2a). Likewise, an early bacteriostatic effect starting at 2 h post-infection was observed on GBS (Fig. 2b). This continuous activity was extended by a clear bactericidal effect from 5 to 24 h post-inoculation (up to five Log), dropping under the limit of detection with no regrowth detected at endpoint (i.e., 24 h). When considering bacterial growth in HoP DM pools, E. coli loads were not statistically different between HoP and control conditions at all time-points (Fig. 2a). Conversely, GBS loads in HoP had similar kinetic features as in HHP DM pools with a significant decrease starting at 2.5 h post-inoculation to endpoint (Fig. 2b). However, regardless of the bacteria, no significant difference was noticed between HoP and HHP treated DM pools or among individual fractions (Supplementary Figs. 1–2). Finally, there were no significant correlations between the bacterial AUC and the concentrations of lactoferrin or lysozyme for each DM pool (data not shown).

Fig. 2: Bacterial growths after donor milk treatments.
figure 2

Effects of donor milk treated by Holder Pasteurization or High Hydrostatic Pressure on Escherichia coli (a) and Group B Streptococcus (b) growth over time. Values expressed in mean ± SEM. n = 4 per group. CFU colony forming unit. HoP Holder Pasteurization. HHP High Hydrostatic Pressure. LOD Limit of Detection. Statistically significant differences using Kruskal-Wallis with Dunn’s post-test between HHP and control: *p < 0.05, **p < 0.01, and HoP vs control: #p < 0.05.

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Donor milk storage over 6 months

Of note, no further contamination of the 15 HHP treated DM fractions was detected when assessed monthly in both storage conditions (i.e., −20 °C and +4 °C). These results confirm the safety of HHP treatment on a long-term DM storage.

Discussion

While Holder pasteurization and HHP have been developed for more than a century, these two methods still provide efficacy and safety when applied to food. Moreover, avoiding the alteration of essential food composition such as maternal milk makes the HHP process attractive for DM treatment. Our HHP protocol consists of 4 cycles of moderate pressures (350 MPa, application rate: 1 MPa/s i.e., 350 s to reach 350 MPa before each cycle, latency time with normal pressure between each cycle) at a temperature (38 °C) close to human milk when secreted by the mammary glands. Interestingly, this protocol has been reported in two studies to be effective in destroying the vegetative forms of bacteria colonizing donor milk and Bacillus cereus spores inoculated in donor and infant milk, which are often difficult to eliminate.21,28,29 However, the discrepancies in efficacy of bacterial spores’ elimination observed between these studies performed at moderate pressure and temperature reinforce the need to further confirm and better characterize the effects of this protocol. These distinctive features contrast with the wide range of pressure (100–600 MPa) and temperature (10–70 °C) previously described, highlighting the lack of consensus regarding a standardized protocol.22,24,25,29,30,31,32

In this study, we showed a better preservation of DM lactoferrin after HHP compared with HoP, and no impact of these treatments on DM lysozyme concentrations. Lactoferrin and lysozyme are bioactive factors of human milk involved in infant’s defences against intestinal infections by inhibiting bacterial growth.33 We confirmed the significant decrease of lactoferrin in DM following HoP but not after HHP treatment as described in most studies.21,22,30,32,34,35 Indeed, comparing DM treated with HoP and HHP, Pitino et al. (2019) and Demazeau et al. (2018), using specific HHP protocols (500 MPa, 8 min, 4 °C and the same protocol as ours, respectively), showed a better preservation of lactoferrin concentrations and almost intact lactoferrin retention rates (93–97%) after HHP compared with HoP.18,21 Intriguingly, our concentrations of lactoferrin in raw (23.6 ± 1.3 g/l) and treated DM (HHP: 24.0 ± 0.7 g/l, HoP: 16.6 ± 2.3 g/l) were far higher than previously reported. The correlation between lactation stage and lactoferrin concentrations has been well described.36,37 Lactoferrin concentrations in DM display a considerable variability between donors, ranging from 2 g/L to 40–45 g/L.37 Thus, the establishment of homogeneous pools from DM samples with a high lactoferrin content could explain the elevated lactoferrin concentrations found in our study. In addition, the use of different methods to estimate lactoferrin concentration in human milk (ELISA, liquid chromatography and mass spectrophotometry) could also explain, at least in part, some differences observed between studies37,38 Here, we showed the absence of negative effects of both techniques (HoP and HHP) on DM lysozyme concentration. Overall, HHP processing displays a good retention of DM lysozyme activity, preferentially assessed by most authors.35,39,40,41 These results, pooled together with our study, corroborate the absence of adverse effects of HHP on DM lysozyme. Indeed, following the same HHP protocol as ours (350 MPa), Demazeau et al. (2018) reported almost intact retention rates of lysozyme concentrations with HHP (>95%), which were much lower after HoP (52%).21 Moreover, a similar lysozyme activity retention between the two techniques and raw milk has also been reported.18 Finally, we confirmed that our HHP protocol was protective against HoP-associated degradation of lactoferrin with no impact on lysozyme concentrations. However, although several potential mechanisms have been suggested to explain the passage of milk bioactive factors into the infant’s circulation (e.g., high gastric pH, immaturity of pancreatic enzymatic activity, paracellular diffusion),42 the detailed activity of these molecules on newborns’ intestines and other organs after milk treatment has not been reported. Further studies, such as the use of ex vivo intestinal explants or in vitro receptor-binding assays in cellular models would be relevant to answer this question.

Because HHP better preserved DM lactoferrin, we decided to assess DM antimicrobial activity on the growth kinetics of two main bacteria responsible for serious neonatal diseases and which effects have been mostly described in relation to human milk, E. coli and group B Streptococcus.9,14,24,25,43,44,45,46,47 Indeed, several studies have now reported a clear link between the maintenance of the native form of lactoferrin and its bio-functionalities, such as its antibacterial activity.48,49,50 We aimed to determine milk antibacterial activity in a systematic manner against both bacteria over a long time period (from 0 to 24 h), when other authors have chosen shorter timescales (e.g., 2 h for E. coli)24,43 or 8 h period (e.g., Staphylococcus aureus).9 In the present study, after 2 h of incubation, E. coli loads were similar in HoP-, HHP-treated DM and control, while GBS growth in HHP-milk was already significantly reduced when compared to control. Conversely, Silvestre et al. reported a significant reduction of near 70% of E. coli counts in milk treated by HoP,43 while Barbaska et al. presented a significant but limited 12% decrease of E. coli growth by HoP but non-significant reduction with HHP 450 MPa and HHP 200 + 400 MPa.24 From 2.5 h to 6 h, HHP revealed a transient bacteriostatic activity against E. coli and a gradually increasing bactericidal property against GBS. These effects were less dramatic with HoP, which led to a non-significant bacteriostatic effect against E. coli and a significant bactericidal activity at only two timepoints (2.5 h and at 24 h) against GBS. Nonetheless, HHP- and HoP-treated DM antimicrobial activities were not significantly different from each other against both bacteria. Moreover, previous studies showed that HoP-treated milk also presented a lower growth inhibition for E. coli and S. aureus compared to untreated DM.9,24 Consequently, our findings reveal similar preservation of milk antimicrobial effects by HHP processing compared to HoP, although some differences remain subtle. We hypothesized that these small changes on antibacterial activity between the two treatments could have been due to slight variations in the composition of a few bioactive factors in DM. This assumption is supported by the absence of direct correlations between DM lactoferrin concentrations and bacterial growth curves, suggesting the involvement of other milk bioactive factors such as the HAMLET protein, lactoperoxidase, secretory IgA or HMOs,51,52 that act as major microbial growth regulators. Finally, we observed specific and inherent trends in each DM pool, with specific growth kinetics for E. coli and GBS irrespective of the treatment procedure. This reflects the variability in human milk composition between donors, depending on a multitude of human and technical factors (e.g., stage of lactation, DM composition in bioactive factors, milk sterilization protocols and storage) and involves taking these parameters into account before administration to vulnerable newborns. Last, the roles of HMBs are to ensure long term storage and an optimal preservation of DM content. Since HoP remains the intuitional gold standard,17 we confirm that HHP is a valuable alternative to promote a suitable preservation of breast milk.53 Interestingly, a recent study demonstrated that the beneficial effects of the HHP technique on the preservation of bioactive compound concentrations and the microbiological safety of milk were maintained when it was combined with freeze-drying.53 Although the sterilization conditions were not clearly detailed and this type of treatment would need to be further characterized, this report emphasizes the relevance of associating HHP with freeze-drying treatment for long-term storage of DM in a context of low levels of both milk stocks and donation.54 At this point, the combination of the freeze-drying technique following our HHP protocol may be envisaged and further supported by long-term storage studies.

To conclude, our HHP protocol allows a higher preservation of donor milk lactoferrin and a similar antimicrobial activity against E. coli and GBS compared to HoP. These effects could be particularly valuable for preterm infants dependent on donor milk from HMBs in early life. HHP-treated milk may improve gut health and the establishment of microbiota in these vulnerable infants.