Raw Human Milk Research Articles

Protein-bound and free glycation compounds in human milk: A comparative study with minimally processed infant formula and pasteurized bovine milk

The role of the Maillard reaction and the accumulation of non-enzymatic glycation compounds in human milk have been scarcely considered. In this study, we investigated the proteins most susceptible to glycation, the identity of the corresponding modified residues and the quantitative relationship between protein-bound and free glycation compounds in raw human milk and, for comparison, in minimally processed infant formula and pasteurized bovine milk. In human milk, total protein-bound lysine modifications were up to 10% of the counterparts in infant formula, while Nε-carboxymethyllysine reached up to 27% of the concentration in the other two products. We demonstrated that the concentration of free pyrraline and methylglyoxal-hydroimidazolone were of the same order of magnitude in the three milk types. Our results delineate how the occurrence of some glycation compounds in human milk can be an unavoidable part of the breastfeeding and not an exclusive attribute of infant formulas and pasteurized bovine milk.

New alternatives to holder pasteurization in processing donor milk in human milk banks.

Infectious and toxicological risks are the main potential hazards that operators of Human Milk Banks (HMBs) encounter and must eliminate. HMBs are trying to implement procedures that allow to manage and sanitize human milk without altering significantly its nutritional and biologically protective components, obtaining a product characterized by a valid balance between safety and biological quality. The history of human milk processing is linked to the origins of HMBs themselves. And although other forms of sterilization were used originally, pasteurization soon became the recognized most effective means for sanitizing milk: all the milk that arrives at the HMB must be pasteurized. Holder pasteurization (HoP) is the most used methodology, and it is performed using low temperature and long time (+62.5°C for 30 min). With HoP some bioactive milk components are lost to varying degrees, but many other precious bioactive compounds are completely or partially preserved. To improve the quality of human milk processed by HMBs, maintaining in the meantime the same microbiological safety offered by HoP, new technologies are under evaluation. At present, High-Temperature Short-Time pasteurization (HTST) and High-Pressure Processing are the most studied methodologies. HTST is already utilized in some HMBs for daily practical activity and for research purposes. They seem to be superior to HoP for a better preservation of some nutritional and biologically protective components. Freeze-drying or lyophilization may have advantages for room temperature storage and transportation. The aim of this study is to evaluate the advancement regarding the processing of DHM with a literature search from 2019 to 2022. The effects of the new technologies on safety and quality of human milk are presented and discussed. The new technologies should assure microbiological safety of the final product at least at the same level as optimized HoP, with an improved preservation of the nutritional and bioactive components of raw human milk.

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

Handling of human milk to prevent acquired cytomegalovirus infection in Japanese neonatal intensive care units: The first nationwide survey.

Human milk (HM) has been proven to provide immunological and nutritional advantages to neonates; however, acquired cytomegalovirus (CMV) infection can be associated with raw HM. In Japan, there are no standardized guidelines concerning HM handling. This cross-sectional survey was performed to reveal specific trends in HM handling in neonatal intensive care units (NICUs) in Japan. A questionnaire was sent to 255 NICUs participating in the Japanese Neonatologist Association in May 2020. It involved HM handling practices, such as maternal screening, pasteurization, storage, and the workforce. Of 255 NICUs, 174 (67.8%) responded to the survey. Maternal CMV screening was carried out in 37 units (22.2%), and CMV inactivation in HM was performed in 44 units (26.5%). For CMV inactivation, a freeze-thawing method was employed in about 90% of units. In 70% of units providing CMV inactivation, CMV inactivation was conducted regardless of bodyweight and corrected gestational age of infants until the infants' discharge. Acquired CMV infection in preterm neonates was observed in 43 units (25.7%) in the survey period. A wide range of HM handling practices are used in Japanese NICUs. A national guideline for handling HM in NICUs should be created to promote the infection control of CMV.

Short-chain fatty acids levels in human milk are not affected by holder pasteurization and high hydrostatic pressure processing.

Sterilized donor milk (DM) is frequently used for feeding preterm infants. To date, the effect of different modes of DM sterilization on short-chain fatty acids (SCFAs) remains unknown. We aimed to quantify SCFAs in DM samples after two types of milk sterilization: the Holder pasteurization (HoP) and a high hydrostatic pressure (HP) processing. Eight pooled DM samples were sterilized by HoP (62.5°C for 30 min) or processed by HP (350 MPa at 38°C). Raw DM was used as control. Six SCFAs were quantified by gas chromatography/mass spectrometry. Compared to raw milk, both HoP and HP treatment did not significantly modulate the concentration of acetate, butyrate, propionate and isovalerate in DM. Valerate and isobutyrate were undetectable in DM samples. In conclusion, both HoP and HP processing preserved milk SCFAs at their initial levels in raw human milk.

Is glycogen an important constituent of human milk?

Human milk stands as the optimal natural source of nutrition for infants, harboring a plethora of complex nutrients including water, carbohydrates, lipids, minerals, proteins, and vitamins, as well as a diverse array of bioactive molecules such as oligosaccharides and growth factors, etc. These components facilitate and safeguard the growth and development of infants. Furthermore, the composition and concentration of human milk subtly evolve across different stages of breastfeeding, adapting to the specific requirements of infants. Therefore, attaining a more precise and comprehensive understanding of human milk composition holds significant value. In this study, a succinct overview of the primary constituents of human milk and their fluctuations during various lactation periods was provided. Then, existing research is critically examined concerning the existence of free glycogen in raw human milk because previous studies indicate the presence of glycogen in human milk but do not determine whether glycogen exists in free form. Glycogen within human milk may exert influence on the growth, development, and establishment of the immune system in infants, but precise mechanisms must be determined. Elucidation of the existing form of glycogen particles in human milk will not only update our understanding of human milk composition, but will also strengthen the roles of glycogen in infant development. Through our exploration, the presence of free glycogen in human milk appears plausible. However, further experiments are needed to confirm the claim via solid evidence.

High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation

BackgroundDonor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. ObjectivesWe aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. MethodsPooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300–500 MPa at 16–19°C (due to adiabatic heating) for 1–9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. ResultsTreatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. ConclusionCompared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Temperature curve of raw human milk heated by different methods: experimental study.

To analyze the temperature curve of raw or pasteurized human milk exposed to different heating methods. Experiments with volumes of 5 ml to 100 ml of human milk were carried out between 2016 and 2021 and analyzed according to the exposure time by different heating methods. Descriptive statistics included the calculation of means, medians, minimum and maximum values, measures of dispersion and standard deviation. The thermal curve made it possible to identify the heating of human milk close to body temperature when subjected to a water bath and microwaves. Milk exposed to room temperature (21°C) was unable to reach this temperature. When heated in a water bath at 40°C, smaller volumes reached body temperature between 3 and 5 minutes, while in a microwave at 50% power, practically all volumes reached temperature. The temperature curves of raw or pasteurized human milk were constructed, and it was possible to verify its behavior using different heating methods for administering the food in a neonatal intensive care unit, considering the volume, type and time of heating and temperature.

Temperature curve of raw human milk heated by different methods: experimental study

RESUMO Objetivo: Analisar a curva de temperatura do leite humano cru ou pasteurizado exposto a diferentes métodos de aquecimento. Método: Experimentos com volumes de 5 ml a 100 ml de leite humano foram realizados entre 2016 e 2021 e analisados segundo o tempo de exposição por diferentes métodos de aquecimento. A estatística descritiva incluiu o cálculo das médias, medianas, valores mínimos e máximos, medidas de dispersão e desvio padrão. Resultados: A curva térmica permitiu identificar o aquecimento do leite humano próximo da temperatura corporal quando submetidos a banho-maria e micro-ondas. O leite exposto à temperatura ambiente (21°C) não foi capaz de atingir tal temperatura. No aquecimento em banho-maria a 40°C, volumes menores alcançaram a temperatura corporal entre 3 e 5 minutos, enquanto em micro-ondas na potência de 50%, praticamente todos os volumes alcançaram essa temperatura. Conclusão: As curvas de temperatura do leite humano cru ou pasteurizado foram construídas, sendo possível verificar o seu comportamento mediante diferentes métodos de aquecimento para administração do alimento em unidade de terapia intensiva neonatal, considerando o volume, tipo e tempo de aquecimento e temperatura.

Conformidade no manuseio do leite materno cru doado aos bancos de leite humano em relação à qualidade microbiológica

Abstract Objectives: microbiological quality of raw human milk (HM) from donors at the HMB (Human Milk Bank) of Santa Casa de Misericórdia de Ouro Preto was evaluated. Methods: the microbiological quality of milk from each of 10 donors in triplicate (30 samples) was evaluated by enumeration of Staphylococcus aureus and total aerobic mesophiles (surface plating) and total and thermotolerant coliforms (Most Probable Number - MPN). A questionnaire and a checklist were applied after the end of sample collection to verify the compliance of milk extraction and storage procedures by the donors. Results: inadequacies in the procedures for expression and storage of donated HM were observed. The presence of all microorganisms studied were verified in the milk of all donors, and in some cases exceeded the safety limits. The checklist indicated that some necessary hygienic-sanitary procedures were not carefully performed, making it important to properly guide the donors. Conclusions: this study points to an unfavorable scenario for the safety and quality of donated HM, and it is important to encourage further studies to be conducted so that the Brazilian network of HMB adopts stricter criteria for instructions on procedures and support for HM donors.

Nanosecond pulsed electric field treatment of human milk: Effects on microbiological inactivation, whey proteome and bioactive protein

In the current study, nanosecond pulsed electric field (nsPEF) was investigated at lab-scale to optimise processing conditions of donor human milk to reduce bacterial counts, and to evaluate its effect on the bioactive proteins in human milk. Response surface methodology was utilized to optimise critical processing parameters. Two optimal nsPEF processing conditions were validated: 15 kV voltage, 6000 pulses at 20 Hz frequency, and 15 kV voltage, 6000 pulses at 50 Hz frequency. Compared to raw human milk, nsPEF processed milk had over 60 % retention of lysozyme, lactoperoxidase and lactoferrin, and 100 % retention of xanthine oxidase and immunoglobulin A. The contents of the five proteins were significantly higher after nsPEF processing when compared with Holder pasteurization. Liquid chromatography-mass spectrometry analysis showed that loss of milk proteins was smaller for samples treated with nsPEF than Holder pasteurization. These results indicated that nsPEF is a promising novel pasteurization method.

Lipid Profile of Human Milk in Different Lactation Stages Submitted to Pasteurization, Lyophilization and Spray-Drying Processes

The objective of this study was to evaluate the effect of pasteurization, lyophilization and spray-drying on the lipid profile of donated human milk (DHM) at different lactation stages. Nine frozen samples of colostrum, transitional and raw mature human milk (HM) were collected from the Human Milk Bank (HMB) of the Hospital Universitário de Maringá (HUM). Samples were thawed and pooled according to lactation stage. Thereafter, it was submitted to pasteurization, lyophilization and spray-drying processes and later the fatty acid (FA) composition and the lipid profile analyses were performed. Gas chromatograph (GC) with flame ionization detector (FID) and electrospray ionization (ESI) mass spectrometer (MS) were used, respectively. In the FA composition analysis, it was observed that the same classes of saturated FA (SFA), monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) were preserved throughout the process and in all lactation stages. The lipid profiles were also preserved after processing. Therefore, the lyophilization and spray-drying processes are promising techniques to preserve the DHM in the HMB, once the components evaluated were preserved, and both techniques facilitate the transport and storage, as the techniques reduce the sample volume.

Comparing the effects of hydrostatic high-pressure processing vs holder pasteurisation on the microbial, biochemical and digestion properties of donor human milk

In this study, hydrostatic high-pressure processing (HHP), a non-thermal pasteurisation method, was used to achieve the microbiological safety of donor human milk. After HHP, no bacteria were detected in human milk processed at 400 MPa for 5 min. Activities of a selection of bioactive components, including lysozyme, xanthine oxidase, lactoperoxidase, immunoglobulin A, lactoferrin, lipoprotein lipase and bile salt-stimulated lipase, did not decrease significantly. This study further investigated the gastrointestinal digestion kinetics of HoP and HHP milk compared with raw human milk, using an in vitro static infant digestion model. After 60 min of ‘gastric digestion’, the microstructure and protein profile of HHP milk samples were more similar to raw milk samples than HoP milk samples. Overall, HPP showed a better retention in milk nutrients and closer digestion behavior than that of HoP.

High Hydrostatic Pressure Treatment Ensures the Microbiological Safety of Human Milk Including Bacillus cereus and Preservation of Bioactive Proteins Including Lipase and Immuno-Proteins: A Narrative Review.

Breast milk is the nutritional reference for the child and especially for the preterm infant. Breast milk is better than donated breast milk (DHM), but if breast milk is not available, DHM is distributed by the Human Milk Bank (HMB). Raw Human Milk is better than HMB milk, but it may contain dangerous germs, so it is usually milk pasteurized by a Holder treatment (62.5 °C 30 min). However, Holder does not destroy all germs, and in particular, in 7% to 14%, the spores of Bacillus cereus are found, and it also destroys the microbiota, lipase BSSL and immune proteins. Another technique, High-Temperature Short Time (HTST 72 °C, 5–15 s), has been tried, which is imperfect, does not destroy Bacillus cereus, but degrades the lipase and partially the immune proteins. Therefore, techniques that do not treat by temperature have been proposed. For more than 25 years, high hydrostatic pressure has been tried with pressures from 100 to 800 MPa. Pressures above 400 MPa can alter the immune proteins without destroying the Bacillus cereus. We propose a High Hydrostatic Pressure (HHP) with four pressure cycles ranging from 50–150 MPa to promote Bacillus cereus germination and a 350 MPa Pressure that destroys 106 Bacillus cereus and retains 80–100% of lipase, lysozyme, lactoferrin and 64% of IgAs. Other HHP techniques are being tested. We propose a literature review of these techniques.

Fluid-structure interaction modeling of lactating breast: Newtonian vs. non-Newtonian milk

Breastfeeding is a highly dynamic and complex mechanism. The suckling process by the infant involves compression and intra-oral vacuum pressure, leading to milk expression from breast. The accumulated milk from the nipple varies depending on the milk properties and transient flow rate during the suckling cycle. Rheological studies on raw human milk indicate that milk has a non-Newtonian shear-thinning flow behavior. This study aims to investigate the effect of non-Newtonian milk on flow behavior through the breast ductal system using fluid–structure interaction (FSI) simulation. The results of the non-Newtonian effects on flow velocity and the volumetric flow rate of expressed milk are presented. The results show that non-Newtonian Carreau model is promising for the simulation of human milk flow through the breast ductal systems. Also, the results show that the non-Newtonian effects on the milk flow behavior appear for 30–35% of the suckling cycle. Therefore, the Newtonian model is acceptable for the purpose of numerical simulation.

Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages

In milk and milk products, small membrane-enclosed vesicles can be found, commonly termed extracellular vesicles (EVs). Milk-derived EVs have previously been suggested to have immunoregulatory properties, especially important for infants without a fully functioning immune system. In the present study, EV fractions were isolated from human milk, mature and colostrum bovine milk, and two dairy fractions, and successively surveyed for their immunomodulating effects on lipopolysaccharide (LPS)-stimulated macrophages (RAW264.7). RAW264.7 cell material and supernatant were evaluated by monitoring degradation of IκBα in the NF-κB pathway, and IL-6 and IL-1β cytokine production, using Western blotting and enzyme-linked immunosorbent assaying, respectively. The results revealed that preincubation with EVs derived from raw human and bovine milk lowered the LPS-activated response of the NF-κB pathway. Additionally, it was found that preincubation with EVs, from human and bovine milk as well as dairy whey or skim milk-derived fractions, decreased secretion of proinflammatory cytokines from LPS-activated RAW264.7 cells. The findings that milk-derived EVs can change the inflammatory response in macrophages support the notion that milk EVs have an important role in mother-to-infant communication and protection of a newborn.

Evaluation of the lipid composition of the three lactation phases of raw, pasteurized and lyophilized pasteurized human milk

The aim of this study was to analyze the acidity, fatty acid (FA) composition and the triacylglycerol (TAGs) profile of human milk (HM) from three lactation phases (colostrum, transitional and mature) submitted to different treatments (raw milk, pasteurized, pasteurized in conjunction with lyophilization), in order to verify whether these processes applied to the samples can influence the characteristics of the analyzed components. To carry out the analyzes, the project was approved by the ethics committee and the HM was acquired at the Human Milk Bank (HMB) of the University Hospital of Maringá - HUM (Paraná, Brazil). The acidity analysis was performed using the titratable acidity method in Dornic degrees (ºD), the composition in FAs from Gas Chromatography with Flame Ionization Detector (GC-FID), and the TAGs profile by Mass Spectrometry with Electrospray Ionization source (ESI-MS). From the results obtained, it was possible to observe that the Dornic acidity and the composition in AGs did not undergo significant changes by the Tukey test (p&lt;0.05) and the TAG profile remained similar after the application of the processing, when compared samples of raw HM from their respective phase. Therefore, the pasteurization technique in conjunction with freeze drying can be a promising alternative for HM storage and conservation in HMBs, as it guarantees the preservation of the evaluated components, in addition to reducing the storage volume and facilitating the transport of this HM.

Gut response to pasteurized donor human milk in a porcine model of the premature infant.

This study investigated the tolerance and safety of pasteurized donor human milk (PDHM) given either alone or together with commercially-used supplements in a porcine model of premature infants. A porcine model, mimicking human neonates at 30-32 weeks of gestational age, was used. The 7-day experiment was performed on 20 piglets. After birth, the piglets were infused with porcine immunoglobulins via the umbilical artery and surgically fitted with a stomach port. The piglets were then randomized into five groups and fed either PDHM, different variants of fortified PDHM or 'raw' human milk (RHM). Preterm piglets fed PDHM showed signs of gastrointestinal intolerance. Four piglets across the various PDHM-fed groups died, none of them were from the group fed PDHM supplemented with long-chain polyunsaturated fatty acids (LC PUFA). In all groups fed PDHM, macroscopic features of enterocolitis were observed, however, these pathological gut changes were less manifested in piglets receiving PDHM supplemented with LC PUFA. The piglets fed RHM had no specific signs of gut damage. The poor tolerance to PDHM suggests changes in milk composition caused by the Holder pasteurization. The supplementation with LC PUFA probably improves tolerance to PDHM.

A human milk-mimicking fluid for PIV experiments

It is essential to formulate a refractive index (RI)-matched fluid to use in silicone phantom models for particle image velocimetry (PIV) experiments. In this work, an extensive effort to develop a non-Newtonian human milk-mimicking fluid (HMMF) with different concentrations of sodium iodide (NaI), glycerol (Gly), xanthan gum (XG), and distilled water (DW) is conducted. Measurements of RI and density are fitted onto a linear empirical expression relating to the NaI and Gly concentration. The Cross model was utilized to find the nonlinear viscosity model of raw human milk. HMMF solution is generated from a multi-constrained optimization. The matched HMMF, suitable for use in the lactating human breast phantom with bifurcated ductal structures, is achieved with composition of 15.69% NaI, 30.27% Gly, 54.02% water and 0.02% XG by weight percentage resulting in a non-Newtonian viscosity matched with that of human milk.

Molecular characterization of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from human milk samples in Brazil.

Human milk is the best nutrient for infants. The donor human milk is stored in a milk bank before pasteurization. However, the human milk is not sterile and could be colonized with different types of bacteria. Many studies have shown S. aureus to be the most prevalent potential pathogen detected in human milk. This study characterized 22 methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from raw human milk for the presence of virulence genes and agr type. Moreover, the genotypic as identified characterization was realized. The presence of virulence genes sei, seg, sec, seh, and etb was identified in resistant and sensitive strains. We observed the predominance of agr type II. The presence of SCCmec IV (67%, 4/6) and V (33%, 2/6) characterized resistant strains as CA-MRSA. Endemic lineages detected (ST1635/CC5-t002, ST5/CC5-t002, ST72/CC5-t126, ST1/CC1-t127, ST45/CC45-t065, and ST398/t1451) could be related to epidemic clones, such as USA800/ST5, USA700/ST72, USA400/ST1, USA600/ST45, and ST398. This study made it possible to understand the characteristics of virulence and clonality of some strains that circulate in breast milk in our region. The discovery of human milk colonization by MSSA and MRSA strains with molecular characteristics similar to infectious clones spread globally demonstrates the importance of monitoring strains that can spread and cause serious infections.