How Freeze-Drying Protects Breast Milk Nutrients
Effect of Freeze-Drying Breast Milk on Nutrients
Study 1: Combination of High-Pressure Processing and Freeze-Drying as the Most Effective Techniques in Maintaining Biological Values and Microbiological Safety of Donor Milk (2021)
Aim: To compare high-pressure processing (HPP) ± freeze-drying with Holder pasteurisation for safeguarding both bio-activity and microbiological safety in donor human milk.
Summary: Using pooled donor milk, the authors applied six treatments: Raw, Holder pasteurisation (HoP 62.5°C for 30 min), HPP 600 MPa for 5 min, Freeze-drying (FD), HoP + FD and HPP + FD; and analysed insulin, leptin, adiponectin, pancreatic lipase activity and hepatocyte growth factor immediately and after storage to six months, alongside bacterial challenge tests. HoP sharply reduced every measured bio-active (lipase and HGF fell > 90%), whereas FD alone preserved nutrients, yet could not guarantee sterility. HPP kept most bio-actives near raw-milk levels and fully inactivated pathogens; when followed by FD, the powdered milk maintained these bio-active concentrations and remained free of bacterial growth throughout storage
Conclusion: High-pressure processing combined with freeze-drying delivered Holder-equivalent microbiological safety while best conserving key bio-active components over prolonged storage
Read the full study: Combination of High-Pressure Processing and Freeze-Drying as the Most Effective Techniques in Maintaining Biological Values and Microbiological Safety of Donor Milk (2021)
Study 2: Essential and Toxic Elements in Human Milk Concentrate with Human Milk Lyophilizate: A Preclinical Study (2020)
Aim: To compare baseline donor milk with its freeze-dried concentrate in terms of essential micronutrients and potentially toxic trace elements.
Summary: Forty-nine pooled donor-milk samples were analysed before (HM-baseline) and after direct lyophilisation (HM-concentrate). Using inductively coupled plasma-mass spectrometry and Bayesian mixed-effects modelling, the team quantified 12 elements (Al, As, Cd, Cr, Fe, Hg, Mn, Ni, Pb, Se, Sn, Tl). Concentrating the milk significantly raised only the essential nutrients manganese (+0.80 µg/L) and selenium (+6.74 µg/L) while reducing lead (-6.13 µg/L); all other elements showed no meaningful change, and toxic metals remained well below safety thresholds. Overall, the concentrate’s micronutrient profile was similar to or richer than that of milk from mothers of preterm infants, and no hazardous accumulation of contaminants was detected.
Conclusion: Freeze-drying donor milk can safely enhance key trace nutrients without increasing toxic-metal exposure, supporting its potential use as a nutritionally adequate human milk concentrate for vulnerable newborns.
Read the full study: Essential and toxic elements in human milk concentrate with human milk lyophilizate: A preclinical study (2020)
Study 3: Human Milk Powder an Alternative for Better Conservation and Healthier Use in the Banks of Human Milk (2019)
Aim: To determine whether transforming donor human milk into a freeze-dried powder can retain its nutritional quality while providing extended, microbiologically safe storage for use in milk banks.
Summary: In this pre-clinical investigation, pooled donor milk was freeze-dried to produce a shelf-stable powder and then tracked for macronutrients, key micronutrients, lipid oxidation and microbial growth across a range of storage temperatures (18°C, 25°C and 40°C). Protein and carbohydrate levels remained statistically unchanged during storage at 25–40°C, and no bacterial growth was detected; only lipid oxidation rose appreciably at the highest temperature, reaching about a one-third increase after prolonged exposure. Modelling projected that, when kept at 18°C, the powder would preserve its compositional integrity for roughly one year without significant deterioration.
Conclusion: Converting donor milk to a freeze-dried powder maintains essential nutrients and microbiological safety for at least a year under moderate conditions, supporting its practical adoption in human milk banks.
Read the full study: Human Milk Powder an Alternative for Better Conservation and Healthier Use in the Banks of Human Milk (2019)
Study 4: Evaluation of the Lipid Quality of Lyophilized Pasteurized Human Milk for Six Months by GC-FID and ESI-MS (2019)
Aim: To assess whether pasteurised human milk that is subsequently freeze-dried retains its lipid quality (acidity, total fat, fatty-acid profile and triacylglycerol composition) during six months of frozen storage.
Summary: Pasteurised pools of colostrum, transitional and mature donor milk were lyophilised, vacuum-packed and stored at –18°C; samples were reconstituted and analysed at 0, 30, 60, 90, 120, 150 and 180 days using gas chromatography-FID and electrospray ionisation mass spectrometry. Across all milk phases, Dornic acidity stayed within the safe 1–8° range, total lipid content (≈3.2 to 3.8%) showed no significant loss, and fatty-acid profiles; including key DHA, ARA and EPA; remained statistically unchanged, with only a small MUFA dip in transitional milk at day 180. The relative abundance of major triacylglycerols likewise showed no alteration, indicating preserved structural lipids; overall oxidative degradation was minimal under these conditions.
Conclusion: Freeze-drying pasteurised human milk, followed by frozen storage, maintains lipid integrity for at least six months, supporting its suitability for long-term use in human milk banks.
Read the full study: Evaluation of the Lipid Quality of Lyophilized Pasteurized Human Milk for Six Months by GC-FID and ESI-MS (2019)
Study 5: Development of a human milk concentrate with human milk lyophilizate for feeding very low birth weight preterm infants (2019)
Aim: To create a freeze-dried human milk concentrate and assess its osmolality plus macro- and micronutrient composition immediately and after 3- and 6-month frozen storage.
Summary: Researchers lyophilised 50 donor-milk samples, reconstituted the powder with baseline milk to form an immediate concentrate (HMCI), and compared it with the original milk (HM baseline) and with concentrates stored frozen for 3 months (HMC3m) and 6 months (HMC6m). HMCI showed significant rises in energy, carbohydrates, total lipids, calcium and phosphorus versus baseline, while maintaining acceptable osmolality; after 3 and 6 months, most nutrients remained stable or increased, though small declines were noted in energy, lipids and copper, indicating partial but not complete stability over time. All samples passed pasteurisation and microbiological safety checks, and the simple single-step lyophilisation method minimised handling risks and costs.
Conclusion: Direct freeze-drying can yield a nutrient-dense, osmolality-safe human milk concentrate suitable for very-low-birth-weight infants; however, extended frozen storage may alter certain nutrients and warrants careful monitoring.
Read the full study: Development of a human milk concentrate with human milk lyophilizate for feeding very low birth weight preterm infants (2019)
Study 6: Human milk enriched with human milk lyophilisate for feeding very low birth weight preterm infants: A preclinical experimental study focusing on fatty acid profile (2018)
Aim: To determine how enriching donor milk with freeze-dried human milk powder affects total lipid content and fatty-acid composition immediately and after 3- and 6-month frozen storage.
Summary: Fifty donor samples were lyophilised, reconstituted with baseline milk to create an immediate concentrate (HMCI), and then stored at −18°C for 3 months (HMC3m) and 6 months (HMC6m). HMCI showed a higher total lipid concentration than the original milk. Across time points, the proportions of major fatty acids, palmitic, oleic, linoleic and α-linolenic, remained largely stable; arachidonic and docosahexaenoic acids declined modestly yet stayed present. Gas chromatography analysis detected no lipid peroxidation, indicating oxidative stability throughout storage.
Conclusion: Freeze-drying followed by enrichment yields a lipid-dense human milk concentrate whose fatty-acid profile remains largely intact over six months of frozen storage, supporting its potential for very-low-birth-weight infant nutrition.
Read the full study: Human milk enriched with human milk lyophilisate for feeding very low birth weight preterm infants: A preclinical experimental study focusing on fatty acid profile (2018)
Study 7: The freeze-drying does not influence the proteomic profiles of human milk (2018)
Aim: To determine whether the protein composition of human milk changes after freeze-drying.
Summary: Researchers collected nine fresh milk samples from three healthy mothers at 15 and 60 days postpartum. They analysed each sample before and after freeze-drying using shotgun proteomics and mass spectrometry, and compared 245 detected proteins using paired statistics. Neither freeze-drying nor the different lactation stages altered overall protein abundance or functional group distribution; all observed differences were statistically non-significant.
Conclusion: Freeze-drying preserved the full proteomic profile of human milk, suggesting the process does not compromise its protein-based bioactivity.
Read the full study: The freeze-drying does not influence the proteomic profiles of human milk (2018)
Study 8: Lipids and Fatty Acids in Human Milk: Benefits and Analysis (2018)
Aim: To review the lipid and fatty-acid composition of human milk, highlight their nutritional and physiological benefits for infants, and outline modern analytical methods used to characterise these lipids.
Summary: The research explains that fat supplies 50–60% of the energy in breast milk and is primarily stored as triacylglycerols enveloped by a phospholipid-protein membrane. Long-chain polyunsaturated fatty acids, such as DHA, EPA, and AA, are emphasised for supporting neural, immune, and tissue development, while the sn-2 positioning of palmitic acid is noted for enhancing calcium and fat absorption. The authors discuss how maternal diet, lactation stage, and processing can influence fatty acid profiles and describe key techniques, including gas chromatography, HPLC-ELSD, and mass spectrometry, for quantifying total lipids, positional distribution, and minor bioactive fractions. Together, these data provide a framework for monitoring milk quality and ensuring that preservation methods (including freeze-drying) do not compromise essential fatty acids.
Conclusion: Human milk fat is indispensable for infant growth and development, and precise analytical monitoring is critical to safeguard its complex fatty-acid profile during any form of storage or processing.
Read the full study: Lipids and Fatty Acids in Human Milk: Benefits and Analysis (2018)
Study 9: The Effect of Lyophilization on Selected Biologically Active Components (Vitamin C, Catalase, Lysozyme), Total Antioxidant Capacity and Lipid Oxidation in Human Milk (2017)
Aim: To investigate how freeze-drying affects antioxidants, bactericidal proteins, total antioxidant capacity (TAC) and lipid-oxidation markers in mature human milk.
Summary: Milk from five healthy mothers was freeze-dried (removing 88% water) and then reconstituted for analysis of vitamin C, catalase activity, lysozyme, TAC, primary lipid peroxides (LP) and secondary oxidation products (TBARS). Vitamin C decreased by 31% and TAC by 17%, whereas catalase (-11%) and lysozyme (-9%) declined modestly and non-significantly. LP and TBARS remained unchanged, indicating no added lipid oxidation. The lyophilizate dissolved readily in water, suggesting practical usability.
Conclusion: Freeze-drying preserves lipid stability and most protein-based bioactivity in human milk, although it moderately reduces vitamin C and overall antioxidant capacity.
Read the full study: The Effect of Lyophilization on Selected Biologically Active Components (Vitamin C, Catalase, Lysozyme), Total Antioxidant Capacity and Lipid Oxidation in Human Milk (2017)
Study 10: Effect of Pasteurization, Freeze-drying and Spray Drying on the Fat Globule and Lipid Profile of Human Milk (2016)
Aim: To evaluate how pasteurisation, freeze-drying (lyophilisation) and spray-drying alter human milk fat content, fat-globule characteristics and fatty-acid composition.
Summary: Sixty mature milk samples from healthy Mexican donors were processed by pasteurisation (62–85°C), freeze-drying (−55°C, 10 Pa), or spray-drying (inlet temperature 150°C) and compared with raw milk. The fat content did not differ significantly across treatments, except in spray-dried milk, where a 23% loss was observed. Across all processes, the mean fat-globule diameter decreased markedly, and polydispersity increased, effects attributed to membrane rupture and mechanical disruption. The core fatty-acid profile remained stable after freeze-drying, whereas pasteurisation produced moderate shifts, lower palmitic and higher oleic/linoleic acids, likely due to selective adhesion of saturated fats to container walls; spray-drying showed similar trends. Overall, processing left macronutrient composition intact and reduced globule size, potentially enhancing lipid bioavailability.
Conclusion: Freeze-drying preserved total fat and fatty-acid composition while reducing globule size without the fat losses seen in spray-drying, indicating it is a nutritionally suitable preservation method for human milk.
Read the full study: Effect of Pasteurization, Freeze-drying and Spray Drying on the Fat Globule and Lipid Profile of Human Milk (2016)
Study 11: The Effect of Freeze-Drying on the Nutrient, Polyphenol, and Oxidant Levels of Breast Milk (2016)
Aim: To evaluate whether freeze-drying better conserves breast milk macronutrients, polyphenols and oxidative-stress markers than conventional freezing.
Summary: Fresh milk from 116 mothers was divided into three treatments: frozen at −80°C for six months (F), freeze-dried in 24 hours and then stored at 4°C for six months (FD), and freeze-dried and then stored at −80°C for six months (FD + F). Proteins, glucose, triglycerides, total polyphenols and multiple oxidant indicators (nitrites, superoxide anion, hydro- and lipoperoxides, γ-glutamyl transpeptidase) were measured before and after storage. Conventional freezing reduced glucose (p < 0.05) and tended toward higher oxidative products, whereas both freeze-dried groups preserved all macronutrients, polyphenols, and oxidant markers at baseline levels. Adding deep-freezing to the lyophilised samples (FD + F) conferred no extra benefit.
Conclusion: A single-day freeze-drying protocol maintained the nutritional, polyphenol and oxidative integrity of human milk for six months, outperforming standard deep freezing as a conservation method.
Read the full study: The Effect of Freeze-Drying on the Nutrient, Polyphenol, and Oxidant Levels of Breast Milk (2016)
Study 12: Vitamins, fatty acids, and antioxidant capacity stability during storage of freeze-dried human milk (2014)
Aim: To evaluate how three months of storage at 4°C or 40°C affects vitamin C, tocopherols, antioxidant capacity and fatty-acid composition in freeze-dried human milk.
Summary: Although freezing is the most common method used to preserve human milk, nutritional and immunological components may be lost during storage. Freeze-drying could increase the shelf life of human milk while preserving its original characteristics. Seventy-two samples of freeze-dried human milk were stored for varying periods, up to a maximum of 3 months, at either 4°C or 40°C. Vitamin C, tocopherols, antioxidant capacity, and fatty acids composition were analysed. A new HILIC–UHPLC method improving vitamin C determination was also validated. Ascorbic acid and total vitamin C concentrations significantly decreased at both temperatures, while antioxidant capacity only decreased at 40 °C. Fatty acid composition and both γ-tocopherol and δ-tocopherol contents remained unaltered. The stability after storage of freeze-dried milk was higher than that reported for frozen or fresh milk, indicating that freeze-drying is a promising option for improving the preservation of human milk in banks.
Conclusion: Freeze-drying followed by cool storage preserves human milk lipids and tocopherols, largely maintaining antioxidant capacity, although vitamin C is partially lost, supporting the method’s suitability for milk-bank preservation over at least three months.
Read the full study: Vitamins, fatty acids, and antioxidant capacity stability during storage of freeze-dried human milk (2014)
Study 13: Ultrasonication, Lyophilization, Freezing and Storage Effects on Fat Loss during Mechanical Infusion of Expressed Human Milk (1995)
Aim: To test whether ultrasonication continues to prevent fat losses when expressed human milk is stored frozen or lyophilised and later delivered by mechanical infusion.
Summary: Expressed milk was pasteurised, ultrasonicated, then stored either frozen (–18°C) or lyophilised for one or four months before being reconstituted and infused via gravity flow or pump. Fat recovery remained high, and protein losses were negligible in the ultrasonicated and frozen milk. In contrast, ultrasonicated milk that had been lyophilised showed significantly lower fat recovery during infusion. Applying ultrasonication after reconstitution largely restored these losses.
Conclusion: Ultrasonication safeguards milk fat during later tube feeding if the milk is stored frozen, but lyophilisation diminishes this benefit unless the milk is re-ultrasonicated after reconstitution.
Read the full study: Ultrasonication, Lyophilization, Freezing and Storage Effects on Fat Loss during Mechanical Infusion of Expressed Human Milk (1995)
Study 14: Evaluation of Freeze-Drying, Pasteurization, High-Temperature Heating and Storage on Selected Enzymes, B-Vitamins and Lipids of Mature Human Milk (1983)
Aim: To compare how freeze-drying, Holder pasteurisation, short high-temperature heating and subsequent storage affect key enzymes, B-vitamins and lipids in pooled mature human milk.
Summary: Researchers measured the effects of freeze-drying, pasteurisation, high-temperature heating, and storage on key enzymes, B vitamins, and lipids in pooled mature human milk. Freeze-drying significantly decreased (P<0.05) the activity of lactoperoxidase and lysozyme but had no effect on the lipase or protease of pooled human milk. Storage after freeze-drying destroyed lactoperoxidase activity but had no apparent effect on the other enzymes. Heating at 62.5°C for 30 min or 75°C for 15 min significantly decreased (P<0.05) the activities of lactoperoxidase, lipase and protease. Lysozyme was inactivated significantly only by heating at 75°C. Storage at −25°C following pasteurisation had no significant effect on enzyme activity. Biotin, niacin and pantothenic acid appeared to be quite stable and were not significantly altered by freeze-drying, heating and/or storage. Similarly, there were no significant differences in lipid components following processing and storage.
Conclusion: Freeze-drying preserves human milk's B-vitamins and lipids, but partially inactivates certain protective enzymes, effects that are broadly similar or milder than those caused by common heat treatments.
Read the full study: Evaluation of Freeze-Drying, Pasteurization, High-Temperature Heating and Storage on Selected Enzymes, B-Vitamins and Lipids of Mature Human Milk (1983)
Study 15: Manufacture, quality control and use of freeze-dried human milk (1960)
Aim: To describe a pilot system for producing lyophilised (freeze-dried) human milk and to evaluate its microbiological safety and nutritional suitability for infant feeding.
Summary: After reviewing the literature on freeze-dried human milk, the authors describe the development of their own technique. Data are presented on the effects of storage of freeze-dried milk for up to 6 yr. on the amino acid composition and acidity and on the effects of freeze drying on the bacterial content. Clinical trials, using 1-6 months-old freeze-dried milk for feeding premature infants, indicated that freeze-dried human milk could satisfactorily replace fresh human milk for infant feeding. E.J.M.
Conclusion: Early clinical experience indicated that properly manufactured freeze-dried human milk could be stored safely and reconstituted for infant use with minimal loss of nutritional value.
Read the full study: Manufacture, quality control and use of freeze-dried human milk (1960)
Study 16: Preservation of human milk (1960)
Aim: To test practical methods, refrigeration, freezing and the then-new technique of lyophilisation, for keeping expressed human milk nutritionally intact and bacteriologically safe over extended periods.
Summary: From 13,000 to 15,000 litres of human milk are now collected yearly at the Vienna milk bank, which dates back to 1909. Some of it is now freeze-dried after pasteurisation. The product is a loose whitish-yellow powder, almost without odour. Its moisture content is 2 to 3%. The proteins, especially β-lactoglobulin, are not denatured and minerals, enzymes and vitamins are little altered. It is packed in hermetically sealed aluminium cartons. The powder is easily reconstituted with boiled water at 40°C. The present cost is 15 sohillings for 1/4 litre carton. Attempts are being made to produce dried milk from unpasteurised breast milk.
Conclusion: Early clinical experience showed that carefully manufactured freeze-dried human milk could be stored safely at room temperature and reconstituted on demand with minimal nutrient loss, offering a viable preservation alternative when refrigeration or freezing capacity is limited.
Read the full study: Preservation of human milk (1960)
Study 17: Freeze-drying for the preservation of breast milk (1955)
Aim: To investigate whether freeze-drying alters the protein integrity of human milk.
Summary: The authors freeze-dried expressed breast milk, reconstituted it, and compared its electrophoretic protein pattern with that of fresh milk. The two patterns were virtually identical, indicating that the major milk proteins were not denatured by the lyophilisation process; no additional structural changes were reported.
Conclusion: Early experimental evidence showed that freeze-drying preserved the native protein profile of human milk, supporting its feasibility as a gentle preservation method.
Read the full study: Freeze-drying for the preservation of breast milk (1955)
Study 18: A new method of preserving raw breast milk (1953)
Aim: To evaluate freeze-drying as a means of conserving raw human milk without destroying its biologically active components.
Summary: Clinicians produced a white, porous powder by freeze-drying expressed milk directly in sterile infant bottles, then reconstituted it with sterile water for feeding premature or sick infants. Analytical testing revealed that key bioactive factors, collectively referred to as inhibins, vitamins, and enzymes, remained unchanged by lyophilisation, with the sole exception of vitamin A, which decreased by 17-38%. Because the powder could be sealed and shipped at room temperature with preserved “raw” properties, the authors argued that freeze-drying solved both the transport and safety challenges faced by existing milk-bank systems that relied on heat treatment.
Conclusion: Early clinical experiments demonstrated that freeze-drying can preserve nearly all biological activity in raw breast milk while producing a shelf-stable powder suitable for safe transport and later reconstitution.
Read the full study: A new method of preserving raw breast milk (1953)
How Freeze-Drying Safeguards Breast Milk Immune Defenders
Effect of Freeze-Drying Breast Milk on Immune Factors
Study 1: Impact of pasteurization/freeze-drying on available immunoglobulin content of the mature human milk: Use in human milk banking of hospitals (2017)
Aim: To determine how three standard pasteurisation temperatures followed by freeze-drying affect the concentrations of immunoglobulins (A, G, M) and complement C3 in mature human milk intended for hospital milk banks.
Summary: Aliquots of mature donor milk were pasteurised at 62.5°C for 30 min, 72°C for 15 min, or 85°C for 5 min, rapidly cooled, and then freeze-dried for 36 hours. Total protein and each immune factor were quantified nephelometrically. Pasteurisation at 62.5°C preserved the highest overall protein and immunoglobulin levels, yet the 72°C treatment retained the greatest proportion of immunoglobulins once the samples had been freeze-dried. Regardless of heat step, all freeze-dried powders met microbiological standards and showed clinically relevant amounts of IgA, IgG, IgM and C3.
Conclusion: Combining pasteurisation with freeze-drying provides a practical way for milk banks to extend shelf life while conserving key immune factors in mature human milk.
Read the full study: Impact of pasteurization/freeze-drying on available immunoglobulin content of the mature human milk: Use in human milk banking of hospitals (2017)
Study 2: Spray and freeze drying of human milk on the retention of immunoglobulins (IgA, IgG, IgM) (2016)
Aim: To compare how spray-drying versus freeze-drying at different plate temperatures affects the preservation of immunoglobulins A, G and M in human milk powder.
Summary: Mature donor milk was converted to powder either by spray-drying (inlet 160°C or 180°C) or by freeze-drying with plate temperatures of 20°C, 30°C or 40°C, and the resulting powders were analysed for moisture and residual IgA, IgG and IgM. Spray-drying produced a low-moisture powder (≈2%) with high retention of IgG (>88%) and moderate retention of IgM (~70%), but IgA fell sharply to 38%. Freeze-drying retained a higher proportion of all three immunoglobulins at 20°C and 30°C (≈75% IgA and 80% IgG/IgM); only at 40°C did IgA retention drop to 55%, albeit still above the spray-dried level. The authors conclude that IgA is the most heat-sensitive antibody and that careful freeze-drying (30°C) provides the best overall immunoglobulin preservation.
Conclusion: Freeze-drying at ≤ 30°C better conserves all major immunoglobulins in human milk than spray-drying, making it the preferred drying method when immune-factor retention is a priority
Read the full study: Spray and freeze drying of human milk on the retention of immunoglobulins (IgA, IgG, IgM) (2016)
Study 3: Effect of Microwave Radiation, Pasteurization and Lyophilization on the Ability of Human Milk to Inhibit Escherichia coli Adherence to HEp-2 Cells (1996)
Aim: To assess how microwave heating, pasteurisation, and freeze-drying influence mature milk and colostrum’s ability to block enteropathogenic E. coli (EPEC) from adhering to human epithelial (HEp-2) cells.
Summary: Pooled colostrum and mature milk were treated with microwave radiation, Holder pasteurisation (62.5°C for 30 min), or lyophilisation, then challenged in vitro with EPEC O111:H-. All treatments preserved the samples’ capacity to inhibit bacterial adherence; total protein remained unchanged, while pasteurisation, but not freeze-drying, lowered IgA and anti-EPEC IgA titres, though residual IgA still sufficed for protection. Western blot analyses confirmed that treated and untreated milks recognised the 94 kDa EPEC adhesin intimin, indicating maintenance of antigen-specific activity.
Conclusion: These results suggest that the protection of colostrum and milk to infantile diarrhoea due to EPEC remains unalterable after the physical treatments studied.
Read the full study: Effect of Microwave Radiation, Pasteurization and Lyophilization on the Ability of Human Milk to Inhibit Escherichia coli Adherence to HEp-2 Cells (1996)
Study 4: Effect of Ultrasonication, Lyophilization, Freezing and Storage on Lipids and Immune Components of Human Milk (1989)
Aim: To examine how ultrasonication and subsequent frozen or freeze-dried storage alter milk lipids, free-fatty-acid (FFA) and peroxide levels, fatty-acid profile, IgA concentration, and fat/protein delivery during tube infusion.
Summary: Expressed human milk was pasteurised, ultrasonicated, or ultrasonicated and pasteurised. Then stored for one or four months, either frozen or lyophilised, analyses included FFA, peroxides, detailed fatty acid profiles, IgA, and recovery of fat/protein after mechanical infusion. Non-pasteurised ultrasonicated samples showed larger FFA rises yet smaller peroxide increases than pasteurised controls, while lyophilised storage accentuated both effects. Ultrasonication plus storage reduced certain saturated and long-chain PUFA (C12:0, C18:0, C20:4 n-6, C22:4 n-6) and caused a modest 6% loss of IgA, far less than the 33% loss reported for pasteurisation alone. During tube feeding, frozen ultrasonicated milk delivered virtually all fat and protein, whereas lyophilisation reversed this benefit by enlarging fat globules, a second ultrasonication just before infusion restored efficient nutrient delivery.
Conclusion: Ultrasonication followed by frozen storage best preserves milk fat delivery and most immune and lipid qualities, while lyophilisation introduces only minor IgA and fatty acid losses and necessitates re-ultrasonication to ensure full nutrient delivery.
Read the full study: Effect of Ultrasonication, Lyophilization, Freezing and Storage on Lipids and Immune Components of Human Milk (1989)
How Freeze-Drying Preserves Breast Milk Probiotic Power
Effect of Freeze-Drying Breast Milk on Probiotics
Study 1: The impact of freeze-drying on the glycoproteomic profiles of human milk (2020)
Aim: To determine whether freeze-drying alters the abundance of glycoproteins in mature human milk.
Summary: Fresh milk samples (15 and 60 days postpartum) were split into two aliquots: one was frozen, and the other was freeze-dried, and then analysed by liquid chromatography mass spectrometry. Across 203 identified glycoproteins, statistical testing showed no significant differences between frozen versus freeze-dried milk (p > 0.05). Partial-correlation analysis confirmed strong concordance in glycoprotein levels across lactation stages (r = 0.897) and processing status (r = 0.887), indicating structural stability of these immune-active proteins after lyophilisation.
Conclusion: Freeze-drying preserves the full glycoproteomic profile of human milk, supporting its suitability for milk-bank storage when maintenance of complex protein functions is crucial.
Read the full study: The impact of freeze-drying on the glycoproteomic profiles of human milk (2020)
Study 2: The human milk oligosaccharides are not affected by pasteurization and freeze-drying (2017)
Aim: To determine whether Holder pasteurisation and subsequent freeze-drying alter the composition of human milk oligosaccharides (HMOs).
Summary: Nine mature-milk samples from three mothers (collected in weeks 1 to 3 postpartum) were analysed before and after Holder pasteurisation (62.5°C for 30 min) followed by 36 hours of freeze-drying. Using MALDI-TOF/TOF mass spectrometry, the researchers profiled 203 HMOs and found strong correlations between pre- and post-treatment samples (r² = 0.989–0.999, p < 0.001), indicating that neither processing step changed individual or overall HMO patterns; variation was attributable only to differences between mothers, not to lactation stage or treatment.
Conclusion: Holder pasteurisation plus freeze-drying preserves the full HMO profile of human milk, supporting the method’s suitability for milk-bank storage when oligosaccharide integrity is essential.
Read the full study: The human milk oligosaccharides are not affected by pasteurization and freeze-drying (2017)
Study 3: Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice (2017)
Aim: To determine whether spray-drying compromises the viability, immunomodulatory profile, or anti-inflammatory efficacy of a breast-milk-derived Bifidobacterium animalis subsp. lactis strain (INL1) compared with the commercial strain BB12.
Summary: Fresh cultures of INL1 and BB12 were spray-dried in 20% skim-milk carrier (inlet ~138°C, outlet ~83°C), yielding powders with ≈ 97–98% survival. In vitro, spray-dried cells retained a high IL-10/IL-12 induction ratio in human PBMCs, mirroring fresh cultures. In TNBS-induced acute colitis, both fresh and spray-dried forms of each strain reduced macroscopic and histological inflammation scores by ≥ 54% and downregulated pro-inflammatory cytokine genes equivalently. Similar protection was confirmed in a chronic DSS-colitis model, and bacterial survivability plus functionality remained intact throughout processing.
Conclusion: Spray-drying at food-grade conditions maintains the viability and anti-colitic efficacy of breast-milk-derived B. lactis INL1, supporting this low-cost drying method for probiotic formulation without loss of functional benefits.
Read the full study: Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice (2017)
Study 4: Effects of Extended Freezer Storage on the Integrity of Human Milk (2016)
Aim: To investigate how storing freshly expressed or briefly refrigerated human milk at −20°C for up to nine months affects pH, bacterial counts, macronutrients, immune components and osmolality.
Summary: Mothers’ milk samples were divided into two groups, frozen immediately (“fresh-frozen”) or refrigerated for 72 hours, then frozen (“refrigerated-frozen”); and analysed at 1, 3, 6 and 9 months. In both groups, pH and total/Gram-positive bacterial counts declined significantly over time, whereas Gram-negative counts and overall bacterial load also decreased, but more gradually. Non-esterified fatty acids rose with storage duration. Crucially, total protein, fat, lactoferrin, secretory IgA and osmolality remained unchanged throughout the nine-month period.
Conclusion: Freezer storage at −20°C for up to nine months preserves key nutritional and immunological constituents of human milk despite minor shifts in pH, microbial counts and free fatty acids, supporting current milk-storage guidelines.
Read the full study: Effects of Extended Freezer Storage on the Integrity of Human Milk (2016)
Study 5: Human Milk Bactericidal Properties: Effect of Lyophilization and Relation to Maternal Factors and Milk Components (2015)
Aim: To compare lyophilisation with freezer storage at −20°C and −80°C for their impact on microbial load and bactericidal activity of human milk, and to explore links between bactericidal capacity, maternal variables and milk components.
Summary: In 125 mature-milk samples from 65 donors, researchers measured mesophilic aerobic bacteria, Staphylococcus epidermidis, and the milk’s ability to kill pathogens, alongside protein, fat, lactose, gangliosides and sialic acid. Lyophilisation and storage at −80°C each cut microbial counts more effectively than storage at −20°C, yet neither treatment altered overall bactericidal activity. Protective activity correlated positively with ganglioside content but not with macronutrients, and was higher in term-delivery and later-lactation milk than in pre-term or early-lactation samples.
Conclusion: Freeze-drying maintains human milk bactericidal power while further lowering microbial load, offering a safe preservation option that keeps key antibacterial functions intact.
Read the full study: Human Milk Bactericidal Properties: Effect of Lyophilization and Relation to Maternal Factors and Milk Components (2015)
Additional links: Human Milk Bactericidal Properties
Study 6: Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions (2012)
Aim: To test whether microencapsulating a breast-milk-derived B. animalis strain improves its survival through freeze-drying, four-month storage, and simulated infant digestion.
Summary: The probiotic cells were coated in a prebiotic–calcium-alginate matrix (optimised at 2.1% alginate, 2.9% prebiotic and 21.7% glycerol) before lyophilisation; response-surface modelling predicted 81% post-drying survival, which matched experimental results. Encapsulated powders kept significantly higher viability than free cells during 120 days at 4°C or 25°C and withstood sequential gastric (pH 3–4, 90 min) and intestinal (pH 7.5, 5 h) challenges, confirming a stable synbiotic formulation.
Conclusion: Prebiotic–alginate microencapsulation effectively shields B. animalis during freeze-drying and subsequent storage, maintaining robust probiotic viability through conditions that mimic the infant gut.
Read the full study: Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions (2012)
Study 7: Viability of Bifidobacterium Pseudocatenulatum G4 after Spray-Drying and Freeze-Drying (2010)
Aim: To compare the survival of a breast-milk-derived B. pseudocatenulatum G4 strain after spray-drying versus freeze-drying in skim-milk carriers.
Summary: The probiotic was dried either by spray-drying (air-outlet at 75°C or 85°C, with or without prior heat adaptation) or by lyophilisation with various skim milk/sugar protectants. Spray-drying caused > 99% loss of viability irrespective of outlet temperature or pre-heating, demonstrating that heat adaptation did not improve thermotolerance. In contrast, freeze-drying preserved 72 – 82% of cells, yielding 9.3 – 9.5 log 10 cfu g⁻¹. Adding sucrose or lactose offered no additional benefit over 10% skim milk alone, and the resulting powder had low residual moisture (~4.4%).
Conclusion: Freeze-drying in 10% skim milk is far superior to spray-drying for maintaining the viability of B. pseudocatenulatum G4, making it the preferred method for formulating stable breast-milk-derived probiotic powders.
Read the full study: Viability of Bifidobacterium Pseudocatenulatum G4 after Spray-Drying and Freeze-Drying (2010)
Freeze-Drying Breast Milk Making Headlines
Freeze-Drying Breast Milk In The News
New kiosk at River Oaks' Motherhood Center helps working moms keep breastfeeding (2020)
Working mums in Houston can now drop off pumped milk at a new kiosk in River Oaks’ Motherhood Center, where local start-up Milkify collects, freeze-dries, and mails it back as shelf-stable powder, offering up to three years’ storage and fewer nutrient losses than conventional freezing.
Read the full article: New kiosk at River Oaks' Motherhood Center helps working moms keep breastfeeding (2020)
Breastmilk available to every newborn thanks to new technology (2020)
ABC News reports that scientists have perfected a gentle freeze-drying process that lets the new Australian Breast Milk Bank stockpile donor milk as shelf-stable powder, aiming for 33,000 litres, enough to rush life-saving feeds to any infant nationwide, even during disasters or remote births.
Read the full article: Breastmilk available to every newborn thanks to new technology (2020)
Recommendations for the Establishment and Operation of Human Milk Banks in Europe: A Consensus Statement From the European Milk Bank Association (EMBA) (2019)
The European Milk Bank Association synthesised existing national rules into 108 evidence-based recommendations covering every step: donor screening, milk collection, pasteurisation, storage, and distribution; to harmonise safety and quality standards for human milk banks across Europe and guide new facilities.
Read the full article: Recommendations for the Establishment and Operation of Human Milk Banks in Europe: A Consensus Statement From the European Milk Bank Association (EMBA) (2019)
Mother's Milk: From Handsfree Pumps to Spray Dried Powder, Redesigning Breastfeeding for Mothers and Infants (2019)
This MOLD feature surveys emerging tools that ease modern mums’ feeding challenges, hands-free pumps, milk-shipping services, and low-cost spray-drying that retains ~90% of breast milk nutrients while extending shelf life, positioning these design advances as keys to broader, equitable access to human milk.
Read the full article: Mother's Milk: From Handsfree Pumps to Spray Dried Powder, Redesigning Breastfeeding for Mothers and Infants (2019)
Freeze-Drying of Breast Milk: A new-old way to make breast-milk available and long-lasting for at-risk premature infants (2018)
This ELACTA (European Lactation Consultants Alliance) paper outlines a nationwide plan to collect surplus donor milk and convert it into freeze-dried powder, arguing that the gentler process preserves antibodies and other bio-actives better than pasteurisation, offers an 18-month shelf life, and could meet the annual shortfall for 260,000 litres of milk needed by at-risk newborns.
Read the full article: Freeze-Drying of Breast Milk: A new-old way to make breast-milk available and long-lasting for at-risk premature infants (2018)
Powdered Breast Milk: Coming Soon (2017)
Spray-drying specialists at the University of Guadalajara in Mexico converted donor milk into a shelf-stable powder that keeps about 90% of its nutrients and can be stored for six months without freezing, laying groundwork for Mexico’s first powdered-milk bank and easier distribution to remote infants.
Read the full article: Powdered Breast Milk: Coming Soon (2017)
Here’s How Powdered Breast Milk Works (2016)
Texas NPR affiliate KUT explains that researchers at the University of Guadalajara are testing spray-drying technology to turn donor milk into shelf-stable powder, allowing storage at room temperature, slashing banking costs, and retaining most key nutrients and immunoglobulins. These advances, milk-bank leaders say, could make human milk cheaper and easier to distribute.
Read the full article: Here’s How Powdered Breast Milk Works (2016)
Freeze-Drying of Breastmilk Retains More of its Healthy Properties (2015)
A Spanish study of 125 donor-milk samples showed that lyophilisation caused no microbiological contamination and preserved the milk’s antibacterial activity and nutrients as well as −80°C storage, while outperforming standard −20°C freezing.
Read the full article: Freeze-Drying of Breastmilk Retains More of its Healthy Properties (2015)
The Breast Milk Products of the Future (2015)
Milk Genomics’ SPLASH! Article surveys emerging innovations, from spray- or freeze-dried donor milk powders to breast milk-derived probiotics and targeted nutrient fortifiers, that aim to make human milk nutrition more shelf-stable, customisable, and widely available beyond the limits of frozen storage.
Read the full article: The Breast Milk Products of the Future (2015)
Elaboration of a Lyophilized Human Milk Concentrate To Be Used To Feed Very Low Birth Weight Preterm Newborns (2013)
A project at the Universidade Federal de São Paulo is testing a low-cost evaporation centrifugation freeze-drying workflow to produce nutrient-dense human milk concentrate in Brazilian milk banks, aiming to replace expensive cow's milk fortifiers and better nourish very low-birth-weight newborns.
Read the full article: Elaboration of a Lyophilized Human Milk Concentrate To Be Used To Feed Very Low Birth Weight Preterm Newborns (2013)