Holder Pasteurization Research Articles

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.

Validating Tools to Detect and Inactivate Monkeypox Virus in Human Milk.

Objectives: Breastfeeding and human milk (HM) improve maternal and infant morbidities and mortality. Therefore, monitoring the safety of breastfeeding and access to HM is of critical importance. In this study, we assessed tools to monitor the presence of monkeypox virus (MPXV) in HM and whether standard Holder pasteurization inactivates MPXV. Materials and Methods: Heat-inactivated MPXV was added to HM or viral transport media (VTM) and analyzed using both research and clinical MPXV quantitative polymerase chain reaction (qPCR) tests. Infectious MPXV was added to HM and was exposed to 1 cycle of freeze-thaw, incubation for 1 hour at room temperature, or conditions of Holder pasteurization (62.5°C for 30 minutes) followed by infectious unit quantification by plaque assay. Results: Research and clinical nucleic acid tests detect MPXV that was added to HM but with reduced sensitivity compared with equivalent samples in VTM at low virus inoculum. MPXV added to HM to achieve a starting concentration of 225,000 plaque forming units (pfu)/mL remains infectious after freeze-thaw or 1 hour storage at room temperature. However, Holder pasteurization reduced infectious virus below the limit of detection, >2,000-fold reduction in viral titer. Conclusion: MPXV can be detected when added to HM using a clinical laboratory-developed qPCR test without modification, but the detection limit is reduced compared with equivalent samples in VTM. MPXV remains viable in HM should the virus ever gain access to HM, but Holder pasteurization reduces infectious MPXV to below detection limits and can be used to reduce the risk of MPXV transmission to infants who receive pasteurized (donor) HM.

The Sterilization of Human Milk by Holder Pasteurization or by High Hydrostatic Pressure Processing Leads to Differential Intestinal Effects in Mice.

Human milk banks (HMBs) provide sterilized donor milk (DM) for the feeding of preterm infants. Most HMBs use the standard method of Holder pasteurization (HoP) performed by heating DM at 62.5 °C for 30 min. High hydrostatic pressure (HHP) processing has been proposed as an alternative to HoP. This study aims to evaluate intestinal barrier integrity and microbiota composition in adult mice subjected to a chronic oral administration of HoP- or HHP-DM. Mice were treated by daily gavages with HoP- or HHP-DM over seven days. Intestinal barrier integrity was assessed through in vivo 4 kDa FITC-dextran permeability assay and mRNA expression of several tight junctions and mucins in ileum and colon. Cecal short chain fatty acids (SCFAs) and microbiota were analyzed. HHP-DM mice displayed decreased intestinal permeability to FITC-dextran and increased ileal mRNA expression levels of two tight junctions (Ocln and Cdh1) and Muc2. In the colon, mRNA expression levels of two tight junctions (Cdh1 and Tjp1) and of two mucins (Muc2 and Muc4) were decreased in HHP-DM mice. Cecal SCFAs and microbiota were not different between groups. HHP processing of DM reinforces intestinal barrier integrity in vivo without affecting gut microbiota and SCFAs production. This study reinforces previous findings showing that DM sterilization through HHP might be beneficial for the intestinal maturation of preterm infants compared with the use of HoP for the treatment of DM.

The Use of Saliva Samples to Test for Congenital Cytomegalovirus Infection in Newborns: Examination of False-Positive Samples Associated with Donor Milk Use.

A universal screening research study was conducted in six hospitals to identify the clinical sensitivity of polymerase chain reaction (PCR) testing on newborn dried blood spots (DBSs) versus saliva specimens for the diagnosis of congenital cytomegalovirus (cCMV). CMV DNA positive results from DBSs or saliva were confirmed with urine testing. Findings of several false-positive (FP) saliva PCR results prompted an examination of a possible association with donor milk. Documentation of the frequency of positive saliva results, including both true-positive (TP) and FP status from clinical confirmation, occurred. The frequency of donor milk use was compared for TP and FP cases. Of 22,079 participants tested between 2016 and 2022, 96 had positive saliva results, 15 were determined to be FP, 79 TP, and 2 were excluded for incomplete clinical evaluation. Newborn donor milk use was identified for 18 (19.14%) of all the positive saliva screens. Among the 15 FPs, 11 (73.33%) consumed donor milk compared to 7 of the 79 TPs (8.8%) (OR 28.29, 95% CI 7.10-112.73, p < 0.001). While milk bank Holder pasteurization inactivates CMV infectivity, CMV DNA may still be detectable. Due to this possible association, screening programs that undertake testing saliva for CMV DNA may benefit from documenting donor milk use as a potential increased risk for FP results.

Effect of High Hydrostatic Pressure Processing and Holder Pasteurization of Human Milk on Inactivation of Human Coronavirus 229E and Hepatitis E Virus.

In preterm infants, sterilized donor milk (DM) is frequently used for feeding when breast milk is lacking. Most human milk banks use the Holder pasteurization method (HoP) to ensure the microbiological safety of DM. However, this method degrades many bioactive factors and hormones. Recently, high hydrostatic pressure (HHP) processing, which preserves bioactive factors in human milk, has been proposed as an alternative method to ensure the safety of DM. Although HHP treatment has been shown to be effective for viral inactivation, the effect of HHP on viruses that may be present in the complex nutritional matrix of human milk has not yet been defined. In the present study, we compared the efficacy of two HHP protocols (4 cycles at 350 MPa at 38 °C designated as 4xHP350 treatment, and 1 cycle at 600 MPa at 20 °C designated as 1xHP600 treatment) with the HoP method on artificially virus-infected DM. For this purpose, we used human coronavirus 229E (HCoV-229E) and hepatitis E virus (HEV) as surrogate models for enveloped and non-enveloped viruses. Our results showed that HCoV-229E is inactivated by HHP and HoP treatment. In particular, the 4xHP350 protocol is highly effective in inactivating HCoV-229E. However, our results demonstrated a matrix effect of human milk on HCoV-229E inactivation. Furthermore, we demonstrated that HEV is stable to moderate pressure HHP treatment, but the milk matrix does not protect it from inactivation by the high-pressure HHP treatment of 600 MPa. Importantly, the complex nutritional matrix of human milk protects HEV from inactivation by HoP treatment. In conclusion, we demonstrated that HHP and HoP treatments do not lead to complete inactivation of both surrogate virus models, indicating that these treatments cannot guarantee total viral safety of donor milk.

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.

Survival of lactic acid bacteria species in Filipino donor milk subjected to different heating temperatures

This study aimed to characterize the behaviors of a cocktail of lactic acid bacteria (LAB) isolated from human donor milk after subjecting to processing at different temperatures. The isolates (n = 12) were identified using 16S rRNA sequencing prior to the propagation and challenge studies. BLAST comparisons revealed that the isolates were Streptococcus spp., Lactobacillus spp., and Enterococcus spp. (% Identity 91.20–98.67%). At an initial inoculation level (8.0 log CFU/ml), the isolates exhibited a non-linear inactivation curve in all temperatures. In the initial logarithmic-linear phase, a decreasing D-value that ranged from 1.70 to 0.20 min was observed with increasing heating temperatures (55 °C, 57 °C, 60 °C, and 63 °C). Microbial load reduction in this initial phase (LR1) also increased (3.31–5.42 log CFU/ml). Moreover, the time necessary to reach LR1 (tLR1), prior to the inactivation curve inflection to tailing, generally decreased. During tailing, minimal to no additional microbial load changes were observed. Total reduction (TLR) increased (3.32–5.42 log CFU/ml), the time it took to reach TLR (tTLR) decreased (60.00–40 min), and the survivor population decreased (4.83–2.63 log CFU/ml) with increasing temperature. The study was able to establish the thermal challenging behaviors and kinetic parameters of a cocktail of lactic acid bacteria (LAB) in human donor milk with known composition and physicochemical properties. The inactivation kinetic parameters were determined from the curves and significantly varied with increasing heating temperature. This study provides a basis for evaluation and possible improvement of the Holder pasteurization (HoP) protocols for human donor milk, specifically temperatures below HoP that allow for keeping the milk biologically active without compromising quality.

The Effects of Refrigerated Storage of Donor Human Milk with Limited Bacterial Presence After Holder Pasteurization on Nutrient Concentration and Bacterial Growth.

Background: The Human Milk Banking Association of North America (HMBANA) sets the purity and quality standards for donor human milk (DHM) in the United States, which includes zero bacterial presence after Holder pasteurization. This study aimed to determine if nutrient and bacterial composition of DHM with limited bacterial presence after pasteurization change over 4 days of refrigerated storage. Methods: Twenty-five unique samples of DHM with limited bacterial growth postpasteurization were collected from two HMBANA milk banks. Infant formula was used as a comparison. Samples were stored in the refrigerator and a portion of milk was removed at 24-hour intervals beginning at hour 0 to 96 for analysis. Aerobic bacteria, protein, lactose, and immunoglobulin A (IgA) content were measured. Longitudinal changes between 0 and 96 hours were analyzed using repeated measures analysis of variance and mixed models test. p < 0.05 was deemed significant. Results: There was no significant difference in lactose, protein, bacteria, or IgA content over storage duration (p = 0.649, p = 0.690, and p = 0.385, p = 0.805, respectively). Total aerobic bacteria were less than 102 colony-forming units (CFUs) in 81% of the time points tested for DHM samples. Total aerobic bacteria were too many to count (>300 CFUs) in the infant formula sample at all time points. Conclusion: In periods of high demand for DHM, DHM with low bacteria growth postpasteurization may be an option as a supplemental food for the growing number of healthy infants who receive DHM. Future studies should investigate the bacterial strains in this milk.

High-Temperature Pasteurization Used at Donor Breast Milk Banks Reduces Melatonin Levels in Breast Milk.

Background and Objective: Donor human milk banks are used when breast milk directly from mothers is unavailable or insufficient. Breast milk contains melatonin, which exhibits a 24-hour pattern. Melatonin promotes sleep onset and is barely detected in daytime milk but rises in the evening and peaks early in the morning. Melatonin supports the development of an infant's own circadian rhythm and is important for neurodevelopment. Currently, donor banks pasteurize breast milk using a Holder Pasteurization (HoP) technique where breast milk is treated at a high temperature (+62°) for 30 minutes before cooling to eliminate any pathogens before it is given to infants. It is not known how the pasteurization process affects the melatonin levels in breast milk. The aim of this study was to investigate whether the pasteurization process reduces melatonin levels in breast milk. Materials and Methods: Ten night-time breast milk samples were collected and each divided into two groups; one group remained unpasteurized and the other group was pasteurized using the HoP technique. Results: Melatonin levels between the unpasteurized and pasteurized groups were compared. Results showed that there was a significant reduction after pasteurization (mean ± standard deviation = 51.92 pg/mL ± 19.54 versus 39.66 pg/mL ± 13.05, p = 0.01). Conclusions: It is important to understand that pasteurization can reduce melatonin levels in breast milk because this hormone is considered important to support the neurodevelopment of infants, especially those born preterm. Further focus on the effect of pasteurization techniques on melatonin in donor breast milk is warranted.

Prevention of human milk-acquired cytomegalovirus infection in very-low-birth-weight infants

BackgroundThe aim of the study was to determine the rate of cytomegalovirus virolactia in the human milk (HM) of mothers of VLBW infants, compare the CMV infection rates and the changes in CMV DNA viral load and nutrient profile among different HM preparation methods.MethodsA prospective randomized controlled study was performed in infants with gestational age < 32 weeks or birth-weight < 1500 g admitted to neonatal intensive care unit of Asan Medical Center and Haeundae Paik Hospital who were given mother’s own milk. Enrolled infants were randomized into three groups according to the HM preparation methods: freezing-thawing (FT), FT + low-temperature Holder pasteurization (FT + LP), and FT + high-temperature short-term pasteurization (FT + HP). Urine CMV culture and PCR were obtained at birth and at 4, 8, and 12 weeks. HM CMV culture and PCR were obtained at birth and at 3, 6, 9, and 12 weeks. Changes in macronutrients in HM was obtained at 4 ~ 6 weeks.ResultsOf 564 infants, 217 mothers (38.5%) produced CMV PCR positive milk. After exclusion, a total of 125 infants were randomized into the FT (n = 41), FT + LP (n = 42), and FT + HP (n = 42) groups, whose rate of HM-acquired CMV infection was 4.9% (n = 2), 9.5% (n = 4), and 2.4% (n = 1), respectively. Out of seven CMV infected infants, two infants fed with FT + LP HM developed CMV infection- associated symptoms. Ages at diagnoses were earlier (28.5 days after birth) and at younger post conceptional age (< 32 weeks) in comparison to infants with asymptomatic CMV infection. CMV DNA viral load significantly decreased after pasturizations, especially in FT + HP group.ConclusionsHM-acquired symptomatic CMV infection rate is low and its impact on clinical course was not serious in our VLBW infants. However, evidences showing poor neurodevelopmental outcome in later life, we need to generate a guideline to protect VLBW infant form HM transmitted CMV infection. Based on our small sized study, we did not find any superiority in pasteurizing HM with frequently used LP in comparison to frozen or HP HM. More research is needed to determine the method and duration of pasteurization to reduce the HM-acquired CMV infection.

Impact of time-temperature combinations on the anti-Cytomegalovirus activity and biological components of human milk.

There is extensive evidence that Holder pasteurization (HoP) (30 min at 62.5 °C) has harmful effects on the bioactivities of human milk (HM). We previously demonstrated that lowering HoP temperature is sufficient to inactivate Cytomegalovirus (HCMV). Here, we analyzed the effect of lowering time/temperature on the antiviral activity against HCMV and IgA levels of HM. Eighty HM samples from five mothers were pasteurized in a range of temperature (62.5-56 °C) and time (40-10 min) in a conventional setting of Human Milk Bank. Unpasteurized HM from each mother was used as control. The samples were assayed against HCMV-AD169 strain in cell cultures and IgA levels were determined by ELISA. All HM samples exhibited anti-HCMV activity, to a different extent. An improvement of antiviral activity was observed in samples treated at 60, 58 and 56 °C compared to those at 62.5 °C, with ID50 values near those of unpasteurized milk. Similarly, better retention in IgA levels was observed by reducing the temperature of treatment. We demonstrated that a 2.5 °C reduction of heat treatment significantly preserved the IgA content and fully restored the anti-HCMV activity of HM, supporting this variant of HoP as a valid alternative to preserve HM bioactivities. This work questions the standard HoP and opens the debate on whether the pasteurization temperature commonly used in Human Milk Banks should be lowered to better preserve the biological components of the milk. A reduction of HoP temperature at 60 °C determined a significant preservation of anti-HCMV activity and IgA content of donor HM, compared to standard HoP. This alternative HoP is highly feasible compared to other substitute pasteurization techniques, since it would employ the same pasteurizer equipment found in most Human Milk Banks.

Comparative analyses of holder pasteurization vs. HTST pasteurization for donor milk: a cost-minimization study applicable to human milk banks

BackgroundHigh-temperature short-time (HTST) pasteurization (72–75 °C, 15 s) is an alternative treatment to traditional Holder pasteurization (HoP) (62ºC, 30 min) for donor milk. HTST pasteurization guarantees the milk’s microbiological safety and retains more of its biologically and nutritionally active compounds, but the cost of implementing this technology for a human milk bank is unknown.MethodsA cost-minimization study was carried out on the facilities of a regional human milk bank in a public hospital. Total production costs (fixed plus variables) were quantified using HTST pasteurization and HoP in three hypothetical scenarios: (1) costs of the first 10 L of pasteurized milk in a newly opened milk bank; (2) costs of the first 10 L of pasteurized milk in an active milk bank; and (3) costs using the maximum production capacity of both technologies in the first two years of operation. The following costs were analyzed: health care professionals, equipment and software, external services, and consumables.ResultsIn scenario 1, the total production costs were € 228,097.00 for the HTST method versus € 154,064.00 for the HoP method. In scenario 2, these costs were similar (€ 6,594.00 for HTST pasteurization versus € 5,912.00 for HoP). The cost of healthcare professionals was reduced by more than half when pasteurization was carried out by the HTST method versus the Holder method (€ 84.00 and € 191.00, respectively). In scenario 3, the unit cost of milk pasteurized by the HTST method decreased from the first to the second year by 43.5%, while for the HoP method, it decreased by 30%.ConclusionsHTST pasteurization requires a high initial investment in equipment; however, it provides a significant minimization of production costs in the long term, pasteurizes large quantities of donor milk per working day and achieves a more efficient management of the time of the health care professionals in charge of the bank’s operation compared to HoP.

Insulin, Testosterone, and Albumin in Term and Preterm Breast Milk, Donor Milk, and Infant Formula

Infants have three options for feeding: their own mother's breast milk, donor milk, or infant formula. Insulin, testosterone, total protein, and albumin levels were measured in breast milk samples from the first 6 months of lactation, in donor milk samples, and in different infant formulas. Mothers who gave birth to term (n = 19) or preterm (n = 19) infants were recruited to collect breast milk samples during the first 6 months of lactation. The Breast Milk Collection Center (Unified Health Institution, Pécs, Hungary) provided 96 donor milk (DM) samples for analysis in our study. Insulin, testosterone, total protein, and albumin levels were measured in breast milk, donor milk, and infant formulas. During the first 2 months of lactation, the concentration of insulin was lower (-27.4%) while the testosterone concentration was higher (+20.8%) compared to the period between the 3rd and 6th months only in the preterm breast milk samples. The infant formulas examined did not contain insulin or testosterone. Holder pasteurization (HoP) did not influence the level of testosterone in human milk, although HoP decreased the insulin (-53.6%) and albumin (-38.6%) concentrations. Diet impacts the hormone intake of infants, underlining the importance of breastfeeding and the possible supplementation of formula-fed infants.

The metabolome of human milk is altered differentially by Holder pasteurization and high hydrostatic pressure processing.

The milk metabolome is composed of hundreds of molecules that can impact infant development. In preterm infants, sterilized donor milk (DM) is frequently used for their feeding. We aimed to identify differences in the metabolome of DM after two types of milk sterilization: the Holder pasteurization (HoP) and a high hydrostatic pressure (HP) processing. DM samples were sterilized by HoP (62.5°C for 30 min) or processed by HP (350 MPa at 38°C). 595 milk metabolites were analyzed using an untargeted metabolomic analysis. Both treatments differentially altered several classes of compounds. The major changes noted included decreased levels of free fatty acids, phospholipid metabolites, and sphingomyelins. Decreases were more strongly noted in HP samples rather than in HoP ones. Both HoP and HP treatments increased the levels of ceramides and nucleotide compounds. The sterilization of human milk altered its metabolome especially for lipids.

Effect of Holder pasteurisation and UV-C irradiation on bacteriophage titres in human milk.

Human milk is the optimal nutrition source for infants and contains a complex mix of bioactive compounds and microorganisms. When unavailable, pasteurised donor milk may be provided, particularly to preterm infants. Holder pasteurisation (HP) is typically implemented in human milk banks to prevent pathogen transmission. Given the impact of heat on milk bioactives, ultraviolet-C irradiation (UV-C) is an alternative being explored and has demonstrated effective bactericidal activity. In addition to bacteria, milk contains viruses including primarily bacteriophages (phages) and which likely influence the developing bacterial microbiome of infants. However, the effect of pasteurisation on human milk phages is unknown. This study assessed the effect of HP and UV-C on titres of exogenous bacteriophages inoculated into human milk. Ten donor human milk samples were tested in parallel with water controls. Milk samples or water controls were inoculated to a final concentration of 1× 104 PFU/mL (±1 log) each of a thermotolerant Escherichia coli phage (T4) and a thermosensitive Staphylococcus aureus phage (BYJ20) and subjected to HP and UV-C treatments. UV-C inactivated both phages within milk and water controls, however, HP was ineffective against the thermotolerant T4 phages. Initial data suggests that UV-C treatment may eliminate phage with potential to affect preterm infant gut colonization. Further studies should extend this to other phages.

Digestion of human milk processed by high pressure processing and Holder pasteurization using a dynamic in vitro model of the preterm infant

Holder pasteurization (HoP) (62.5 °C, 30 min) of donor human milk is widely used to inactivate potential pathogens but may lead to denaturation and aggregation of bioactive proteins, reducing their functionality. In contrast, high pressure processing (HPP) is a non-thermal technique that minimally affects assessed bioactive components; however, it is unclear how HPP affects protein digestion, and retention of functional bioactive proteins. Raw or processed (HoP; HPP[500 MPa,10 min]) pools of milk (N = 3, from 9 donors) were subjected in triplicate to in vitro digestion simulating the preterm infant gastrointestinal tract. Compared to raw or HPP, HoP increased intestinal proteolysis of lactoferrin and bioactive milk fat globule membrane proteins. Lysozyme activity was impacted by digestion following HoP (72 % to 7 %)—significantly more than HPP (75 % to 34 %) or raw (100 % to 39 %), which did not differ. Proteins in HPP-treated donor milk are digested no different than raw milk, while preserved bioactivity remains functional upon digestion.

Variation in the level of the biocomponents immunoglobulin A and lactoferrin in human milk after Holder pasteurization

Background: breast milk is the ideal food for newborns and infants, but there are factors that can prevent the practice of breastfeeding. Human milk banks (BLH) are a strategy to increase breastfeeding coverage; the donated milk is subjected to Holder pasteurization to guarantee its innocuousness, undergoing large changes in temperature and a decrease in the concentrations of biocomponents such as Immunoglobulin A (IgA) and lactoferrin (LF). This article describes the results of recent studies on the impact of Holder pasteurization on IgA and LF in human milk. Material and methods: a search for research articles related to the topic of interest was carried out in various databases and in accordance with inclusion criteria that considered the type of study, date of publication and quality of the journal. Results: the impact of Holder pasteurization on IgA and LF concentrations is not clear, given that the literature reports a variety of protocols and different results; however, the percentage reductions of both biocomponents are significant and consistent in the studies reviewed, suggesting the importance of establishing a standard protocol for their quantification. Conclusions: Holder pasteurization guarantees the microbiological quality of the milk distributed in HMB, but affects the amount of beneficial biocomponents for the final recipient. Government entities that regulate HMBs should evaluate the possibility of using other techniques that reduce the impact on biocomponents while preserving the microbiological quality of the product.

Characterization of D-amino acids in colostral, transitional, and mature preterm human milk.

D-Amino acids are regulatory molecules that affect biological processes. Therefore, being able to accurately detect and quantify these compounds is important for understanding their impact on nutrition and health. There is a paucity of information regarding D-amino acids in human milk. We developed a fast method for simultaneous analysis of amino acid enantiomers in human milk using liquid chromatography with tandem mass spectrometry. The method enables the separation of 41 amino acids without chemical derivatization. Our results revealed that human milk from mothers of preterm infants contains concentrations of D-amino acids that range from 0.5 to 45% that of their L-counterparts and that levels of most D-amino acids decrease as the milk production matures. Moreover, we found that Holder pasteurization of milk does not cause racemization of L-amino acids. To our knowledge, this is the first study to describe percentages of D-amino acid levels in human milk; changes in D-amino acid concentration as the milk matures; and the effect of Holder pasteurization on D- and L-amino acid concentrations in human milk.

Development of a human milk protein concentrate from donor milk: Impact of the pasteurization method on static in vitro digestion in a preterm newborn model

The impact of high temperature short time (HTST, 72 °C, 15 s), Holder pasteurization- (63 °C, 30 min) and high hydrostatic pressure (HHP, 600 MPa-10 min) was evaluated on the digestibility of human milk protein concentrate (HMPC) by using a static in vitro gastrointestinal digestion system. The results showed that the processing steps used to produce the HMPC induced a decrease in readily available nitrogen (non-protein nitrogen and peptides). Overall, digestibility was similar between pasteurized and raw HMPC (degree of hydrolysis ranged from 26 to 34 %). Lactoferrin was more susceptible to gastric and intestinal digestion after thermal pasteurization. Additionally, the resistance of β-casein to digestion increased after HHP and Holder pasteurization due to aggregation and changes in protein structure. During intestinal digestion, Holder pasteurization induced a higher release of arginine, phenylalanine and tyrosine from HMPC compared to raw and HHP-treated HMPC. Overall, protein structural changes induced by human milk (HM) processing (freeze–thawing and filtration) and pasteurization treatments affected HMPC proteolysis during in vitro digestion. However, protein digestion behaviors were quite similar for raw and HHP-treated HMPC compared to the thermal-treated HMPC, with no effect on lactoferrin digestion. Consequently, pasteurization of HMPC by HHP represents an interesting non-thermal process that preserves the HM bioactive proteins during digestion.

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.