Share this article on:

Bacteriological, Biochemical, and Immunological Modifications in Human Colostrum After Holder Pasteurisation

Espinosa-Martos, I.*; Montilla, A.; Segura, A. Gómez de*; Escuder, D.; Bustos, G.; Pallás, C.; Rodríguez, J.M.*; Corzo, N.; Fernández, L.*

Journal of Pediatric Gastroenterology and Nutrition: May 2013 - Volume 56 - Issue 5 - p 560–568
doi: 10.1097/MPG.0b013e31828393ed
Original Articles: Hepatology and Nutrition

Objective: The objective of this work was to evaluate the effect of Holder pasteurisation of human colostrum on a variety of microbiological, biochemical, and immunological parameters.

Methods: Colostrum samples from 10 donors, and 8 samples of mature milk used as controls, were heated at 62.5°C for 30 minutes. Bacterial counts and the concentration of furosine, lactose, myoinositol, glucose, lactulose, cytokines, and immunoglobulins were determined before and after the heat treatment.

Results: Mean bacterial counts in nonpasteurised colostrum samples oscillated between 2.72 and 4.13 log10 colony-forming units per millilitre in the agar media tested. Holder pasteurisation led to the destruction of the bacteria originally present in the samples. Furosine was detected in all samples before pasteurisation and increased significantly after the heat treatment (from 6.60 to 20.59 mg/100 g protein). Lactulose content was below the detection limit in nonpasteurised colostrum, but it was detected in all samples and quantified in 7 of them (from 10.68 to 38.02 mg/L) after Holder pasteurisation. Lactose, glucose, and myoinositol concentrations did not change after Holder pasteurisation. The concentrations of most cytokines and immunoglobulins were significantly higher in colostrum than in mature milk samples. Immunoglobulin content, both in colostrum and in milk samples, was reduced during pasteurisation, whereas, among cytokines, only macrophage inflammatory protein-1β, interleukin-7, and granulocyte-macrophage-colony-stimulating factor concentrations were affected by this heat treatment.

Conclusions: Lactulose and furosine content could be used as heat treatment indicators in colostrum samples. Holder pasteurisation modified the immunological profile of both colostrum and mature milk.

*Departamento de Nutrición, Bromatología y Tecnología de los Alimentos. Universidad Complutense de Madrid, Spain

Instituto de Investigación en Ciencias de la Alimentación (CIAL), CEI (CSIC+UAM), Madrid, Spain

Servicio de Neonatología. Hospital Universitario 12 de Octubre, Madrid, Spain.

Address correspondence and reprint requests to Dr. Leónides Fernández, Dpto. Nutrición, Bromatología y Tecnología de los Alimentos. Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n 28040 Madrid, Spain (e-mail:

Received 4 October, 2012

Accepted 15 December, 2012

This work was supported by the CYTED (IBERFUN 110AC0386), CSD2007-00063 (FUN-C-FOOD, Consolider-Ingenio 2010), and AGL2010-15420 projects from the Ministerio de Ciencia e Innovación (Spain), by projects FIS PS09/00040 (Instituto de Salud Carlos III) and S2009/AGR-1469 (Comunidad de Madrid, Spain), and by Red SAMID.

The authors report no conflicts of interest.

Colostrum is a yellowish species-specific fluid produced by the mammary glands in late pregnancy and in the first few days after birth. During this period, the tight junctions of the mammary epithelium are open, allowing paracellular transport of many substances from the maternal blood circulation. As a consequence, its composition is different from that of mature milk. Colostrum volume is usually low because it is adapted to the small size of the newborn stomach after birth; however, it contains a high concentration of nutrients, including carbohydrates and proteins, elements of the immune system, such as immunoglobulins, immunocompetent cells and cytokines, several other biologically active compounds, and commensal or potentially probiotic bacteria (1,2). Therefore, it is widely admitted that colostrum play a key role in the protection against infectious diseases and in the initial immunomodulation of the neonatal mucosa-associated lymphoid tissue (3–6). Colostrum also contains high amounts of sodium, potassium, chloride, and cholesterol, a combination that may lead to an optimal development of the infant's heart, brain, and central nervous system (7). In addition, the mild laxative qualities of colostrum encourage the passage of meconium. All these facts clearly indicate that the intake of human colostrum by neonates is highly desirable because of its unique properties.

Unfortunately, the mother's colostrum is not always available for her infant. In the last years, access to donor breast milk has increased through the creation of a rapidly growing network of human milk banks; however, availability of colostrum is still low and, in fact, scientific literature dealing with pasteurisation effects on such biological fluid is scarce, in contrast with the variety of studies focused on human milk (8–10). Protein concentration is significantly higher in colostrum than in human milk, whereas lactose content is lower (11,12). Therefore, the known effects of Holder pasteurisation (or other thermal treatments) on milk components should not be extrapolated directly to colostrum.

In this context, the objectives of this work were to evaluate the effect of Holder pasteurisation on the colostrum microbiota and the potential nutritional damage of this thermal treatment using the furosine and lactulose indices. Both indices, furosine and lactulose, are useful markers for evaluating the extent of heat damage in milk and infant formulas (13,14). Finally, changes in the concentration of immunoglobulins and a wide range of cytokines present in human colostrum and mature milk after this treatment were also evaluated.

Back to Top | Article Outline


Colostrum and Mature Milk Samples

Ten colostrum and 8 mature milk samples (10 mL) were obtained from the human milk bank located at the Hospital Universitario 12 de Octubre (Madrid, Spain). Colostrum collection was performed following a specific protocol for donor mothers approved by the local ethics committee. Samples were donated by women that fulfilled the milk bank requirements and informed consent was signed by each donor. An aliquot from each colostrum or mature milk sample was separated before pasteurisation, whereas the rest of the sample was pasteurised by heating it at 62.5°C for 30 minutes; then, it was cooled in a shaking water bath (Jeiotech BS-21, Lab Companion, Seoul, Korea) filled with ice-cold water and provided with temperature control. Once the sample temperature reached 4°C (always within the first 15 minutes of cooling), it was stored at −20°C until analysis. A thermometer coupled to an external temperature sensor (DT132, Fourier Systems, Fairfield, CT) was introduced in a control bottle (containing the same volume of cow's colostrums or mature milk) to monitor the temperature during the whole heating/cooling process.

Back to Top | Article Outline

Bacterial Cultures and Identification of Isolates

Ten pairs of colostrum samples (10 before and 10 after pasteurisation) were plated onto Brain Heart Infusion (BHI, Oxoid, Basingstoke, UK), Columbia Nadilixic Acid Agar (CNA, BioMerieux, Marcy l’Etoile, France), Baird Parker (BP, BioMerieux), MacConkey (MCK, BioMerieux), Polymyxin-Pyruvate-Egg Yolk-Mannitol with bromothymol blue (PEMBA, Oxoid), de Man, Rogosa, and Sharpe (MRS, Oxoid), and Wilkins Chalgren (WCh, Oxoid) agar plates, after proper dilution in peptone water, as described previously by Gómez de Segura et al (15). Representative colonies from plates where bacterial growth was detected were isolated and stored at −20°C in the presence of glycerol (20%, vol/vol).

Identification of the isolates was performed by polymerase chain reaction sequencing of a 470-bp fragment of the 16S rRNA gene as described previously (16). The amplicons were purified using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and sequenced at the Genomics Unit of the Universidad Complutense de Madrid, Spain. The resulting sequences were used to search sequences deposited in the EMBL database using BLAST algorithm, and the identity of the isolates was determined on the basis of the highest scores (>98%).

Back to Top | Article Outline

Determination of Furosine

Furosine analysis was carried out by ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) following the method of Resmini et al (17) as described by Gómez de Segura et al (15) but adapted to smaller volumes. Colostrum volume for hydrolysis was reduced to 1 mL and, accordingly, mixed with 3 mL of 10.6 N HCl under inert conditions at 110°C for 24 hours in a Pyrex screw-cap vial with polytetrafluoroethylene-faced septa. The hydrolysate was filtered through Whatman No. 40 filter paper, and 0.5 mL of filtrate was applied to a previously activated Sep-Pak C18 cartridge (Millipore, Milford, MA). Furosine was eluted with 3 mL of 3N HCl and 50 μL was injected into the chromatograph.

RP-HPLC analysis of furosine was carried out in a C8 column (250 mm × 4.6 mm, 5 μm) (Alltech furosine dedicated; Alltech Associates, Laarne, Belgium) maintained at 35°C using a linear binary gradient at a flow rate of 1.2 mL/min. Mobile phase was constituted by solvent A, 0.4% acetic acid, and solvent B, 0.3% KCl in phase A. The elution program was as follows: 100% A from 0 to 12 minutes, 50% A from 20 to 22.5 minutes, and 100% A from 24.5 to 30 minutes. Detection was performed using a variable-wavelength UV detector at 280 nm (Beckman System 166, Fullerton, CA). Acquisition and processing of data were achieved with Karat 8.0 Software (Beckman Coulter Inc, Brea, CA). Calibration was performed by external standard method using commercial standard of pure furosine (Neosystem Laboratories, Strasbourg, France). Protein concentrations were determined by the bicinchoninic acid (BCA) assay using the BCA Protein Assay Reagent (Thermo Fisher Scientific Inc, Rockford, IL) and bovine serum albumin as standard. All analyses were done in duplicate, and the values were expressed as milligrams of furosine per 100 g of protein.

Back to Top | Article Outline

Determination of Carbohydrates

Lactose, myoinositol, glucose, and lactulose were determined by gas chromatography with flame ionisation detector in the 10 pairs of colostrum samples (before and after pasteurisation), following the method described by Montilla et al (18) with minor modifications. For this purpose, 0.2 mL of sample was made up to 2 mL with methanol in a volumetric flask to remove proteins and fat. Mixtures were gently stirred, followed by standing for at least 1 hour at room temperature until the supernatant became transparent. The clear supernatant was used for carbohydrate analysis and a solution of 0.04% (wt:vol) phenyl-β-D-glucoside in ethanol/water (70:30, vol/vol) was added as internal standard.

Before derivatisation, equal volumes (0.5 mL) of supernatant and internal standard solution were mixed and dried at 38 to 40°C in a rotary evaporator. For derivatisation 100 μL of N,N-dimethylformamide was added to the dried mixtures and held at 70°C for 1 hour to obtain a constant anomeric composition. Then, 100 μL of N-trimethylsilylimidazole was added to silylate the carbohydrates, and the reaction was completed in 30 minutes at 70°C. Silylated carbohydrates were extracted with 0.1 mL of hexane and 0.2 mL of water. Volumes in the range of 1 to 2 μL of the organic phase containing silyl derivatives were injected into the column.

The trimethylsilyl ethers of carbohydrates were analysed in an Agilent Technologies 7890A gas chromatograph equipped with a commercial 30 m × 0.32 mm inside diameter, 0.5-μm film fused silica capillary column SPB–17, bonded, cross-linked phase (50% diphenyl/50% dimethylsiloxane) (Supelco, Bellefonte, PA). Separation was performed at 235°C for 9 minutes, followed by an increase up to 270°C at a rate of 15°C/min and maintenance of this temperature for 15 minutes. Temperatures of the injector and flame ionisation detector were 280° and 290°C, respectively. Injections were carried out in split mode 1:30, using 1 mL/min of nitrogen as carrier gas. Data acquisition and integration were performed using Agilent Chem-Station Rev. B.03.01 software (Wilmington, DE). To study the response factor relative to the internal standard, solutions containing glucose, myoinositol, lactose, and lactulose were prepared over the expected concentration range in colostrum samples. The identity of carbohydrates present in colostrum samples was confirmed by comparison with relative retention times of standard samples. Quantitative analysis was carried out by the internal standard method. Response factors were calculated after triplicate analysis of 4 standard solutions (glucose, myoinositol, lactose, and lactulose) at concentrations ranging from 1 to 6 g/L for lactose and from 1 to 50 mg/L for glucose, myoinositol, and lactulose. The limit of detection and the limit of quantification of the gas chromatography method for lactulose were 4 and 10 mg/L, respectively.

Back to Top | Article Outline

Analysis of Cytokines

The concentration of 17 cytokines, chemokines, and growth factors (interleukin [IL]-1β, IL-6, IL-12[p70], interferon-γ, tumour necrosis factor-α, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-8, macrophage-monocyte chemoattractant protein [MCP]-1(MCAF), macrophage inflammatory protein [MIP]-1β, granulocyte-colony-stimulating factor [G-CSF], IL-7, and granulocyte-macrophage-colony-stimulating factor [GM-CSF]) in 10 colostrum and 8 mature milk samples was determined in duplicate using a Bioplex system instrument (Bio-Rad, Hercules, CA) and the TH1/TH2 Human Cytokine 17-Plex assay kit (Bio-Rad). Previously, and to avoid interferences in the immunoassays, the fatty layer and the somatic cells were removed from the samples. Briefly, sample aliquots (1 mL) were centrifuged at 800 g for 15 minutes at 4°C; the intermediate aqueous phase was collected and stored at −20°C until analysis. In parallel, levels of active transforming growth factor (TGF)-β2 were measured 2 hours after acid incubation by ELISA with the RayBio Human TGF-β2 ELISA kit (RayBiotech, Norcross, GA). All the determinations were carried out following the manufacturer's protocol, and standard curves were performed for each analyte.

Back to Top | Article Outline

Analysis of Immunoglobulins

The concentration of immunoglobulin (Ig)G1, IgG2, IgG3, IgG4, IgM, IgA, and IgE in the colostrum and milk samples was determined in duplicate using the Bio-Plex Pro Human Isotyping Assay kit (Bio-Rad) in the Bioplex system instrument (Bio-Rad). For this purpose, the samples were conditioned as described above for cytokine analysis. Analyses were carried out following the manufacturer's protocol and standard curves were performed for each analyte.

Back to Top | Article Outline

Statistical Analysis

Microbiological data, recorded as colony-forming units (CFU) per millilitre of colostrum, were transformed to logarithmic values before statistical analysis. Biochemical data (furosine, sugars, and bioactive compounds) were tested for normality of distribution by Shapiro-Wilk tests. Furosine and sugar concentrations were expressed as the mean and 95% confidence interval (CI) of the mean, whereas immunological data were expressed as the median and interquartile range (IQR) because they were not normally distributed.

The effect of Holder pasteurisation on furosine and sugar concentration was analysed by paired Student t tests. Differences in the cytokines and immunoglobulins concentration in colostrum and mature milk were evaluated using Mann-Whitney U tests. Wilcoxon signed-rank tests were used to determine the effect of the pasteurisation on the cytokine and immunoglobulin concentration present in colostrum and mature milk samples. Differences were considered significant at P < 0.05. Statgraphics Centurion XVI version 16.1.15 (Statpoint Technologies Inc, Warrenton, VA) and Bio-Plex Data Pro 1.0 (Bio-Rad) softwares were used to perform these analyses.

Back to Top | Article Outline


Bacterial Counts in the Colostrum Samples and Identification of the Isolates

Viability of bacteria using both general and selective growth media was evaluated in 10 samples of donor colostrum before and after heating at 62.5°C for 30 minutes. Bacterial growth was observed in all nonpasteurised samples when they were cultured on BHI, BP, CNA, MRS, PEMBA, and WCh agar plates (Table 1). Globally, the bacterial counts in nonpasteurised samples oscillated between 2.84 and 5.27 log10 CFU/mL in BHI medium, with a mean (95% CI) value of 4.03 (3.43–4.63) log10 CFU/mL. The lowest mean bacterial count of donor milk samples was found in MRS medium, and was 0.51 log units lower than in BHI agar plates (Table 1). Most of the bacteria isolated from the raw colostrum samples, both qualitatively and quantitatively, belonged to the genera Staphylococcus (BHI, CNA, MRS, WCh agar plates), Streptococcus (CNA, MRS, WCh agar plates), and to the Lactobacillus group (MRS).



In contrast, bacteria could only be isolated from 3 (30%) nonpasteurised samples when inoculated on MCK plates (Table 1). The bacterial counts in these samples oscillated between 2.00 and 3.40 log10 CFU/mL; these isolates belonged to the coliform group and, most of them, to the species Escherichia coli or to the genera Klebsiella and Enterobacter.

Pasteurisation treatment was effective in destroying the bacterial population present in colostrum samples (Table 1); in fact, bacterial growth could not be detected from most pasteurised samples after culturing onto BHI, BP, CNA, MCK, MRS, PEMBA, or WCh agar plates. Bacterial survival after pasteurisation was only observed in 2 samples when cultured on WCh (2.49 log10 CFU/mL) agar plates (Table 1). In both cases, the microorganisms isolated were staphylococci that did not survive Holder pasteurisation when they were inoculated afterwards in sterile colostrum at an initial concentration of ∼4.0 log10 CFU/mL, indicating that their presence was most likely because of postprocessing contamination (results not shown).

Back to Top | Article Outline

Evaluation of the Maillard Reaction in Pasteurised Colostrum By Furosine Determination

Furosine is used as an indirect measurement of Amadori compounds because it is formed during the Maillard reaction, which takes place during heat treatment of milk. The ion-pair RP-HPLC method used in this work to determine this compound allowed to detect furosine in all colostrum samples analysed. Furosine levels in nonpasteurised colostrum ranged from 3.50 to 11.60 mg/100 g protein, with a mean value of 6.60 mg/100 g protein (Fig. 1). After Holder pasteurisation, the furosine content in the acid hydrolysates increased significantly (paired Student t test, P < 0.001), and the observed values in pasteurised colostrum ranged from 13.0 to 28.6 mg/100 g protein, with a mean value of 20.59 mg/100 g protein (Fig. 1). These results indicate the progression of the Maillard reaction during the heat treatment of colostrum samples.



Back to Top | Article Outline

Effect of Pasteurisation on the Carbohydrate Fraction of the Colostrum

Table 2 shows the mean, minimum, and maximum values of lactose, myo-inositol, glucose, and lactulose concentrations found in analysed colostrum samples, both before and after pasteurisation. Mean (95% CI) concentration value of lactose in nonpasteurised colostrum samples was 50.70 (48.54–52.86) g/L. Other minor carbohydrates such as myoinositol and glucose were also found and quantified, and their concentrations were 273.12 (243.28–302.95) and 142.95 (110.91–175.00) mg/L, respectively. These compounds showed a considerable sample-to-sample variation, which was particularly relevant in the case of glucose (coefficient of variation equal to 50%). Changes in lactose, glucose, and myoinositol concentration after Holder pasteurisation of colostrum samples were not statistically significant (paired Student t test, P > 0.05).



Lactulose, a disaccharide formed by isomerisation of lactose during heat treatment of milk and used as chemical indicator to differentiate heated milk, was not found in nonpasteurised colostrum samples; however, this neoformed disaccharide was quantified in 7 of 10 pasteurised samples and its mean (95% CI) concentration was 22.62 (12.25–32.99) mg/L. Lactulose was also detected in the other 3 pasteurised colostrum samples, although the amounts were below the quantification limit of the analytical method (10 mg/L) (Table 2).

Back to Top | Article Outline

Concentration of Cytokines in Colostrum and Milk Samples Before and After Pasteurisation

The concentration of a variety of cytokines, chemokines, and growth factors in 10 colostrum and 8 mature milk samples was measured before and after Holder pasteurisation (Table 3). Globally, the values obtained for all these immune factors showed a high degree of variability depending on the donor. Only IL-6, TGF-β2, IL-8, MCP-1, MIP-1β, and IL-7 were detected in all the samples, whereas G-CSF could not be detected in any sample. The concentration of all cytokines tested was higher in colostrum than in mature milk samples, with the exception of IL-7.



In relation to the cytokines involved in innate immunity (IL-1β, IL-6, IL-12(p70), interferon-γ, and tumour necrosis factor-α), the levels of IL-1β, IL-6, and IL-12 were significantly higher in colostrum than in milk samples (Mann-Whitney U test, P = 0.019, P = 0.001, and P = 0.003, respectively) and pasteurisation did not modify significantly their levels (Table 3). Regarding those cytokines involved in acquired immunity (IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and TGF-β2), the median concentrations were also higher in colostrum than in mature milk samples, although this difference was statistically significant only for IL-17 and TGF-β2 (Mann-Whitney U test, P = 0.003 and P = 0.029, respectively). Heat treatment did not change significantly the levels of this group of cytokines related with acquired immunity (Table 3). Similar results were obtained for the chemokines included in the study (IL-8, MCP-1, and MIP-1β), which showed a higher concentration in colostrum than in mature milk samples (Mann-Whitney U test, P = 0.003 for MCP-1 and P = 0.002 for MIP-1β). When comparing the concentration of these chemokines before and after heat treatment, a significant reduction in the levels of MIP-1β in milk was observed after pasteurisation (Wilcoxon signed-rank test, P = 0.002) (Table 3). Finally, only 2 haematopoietic-stimulant compounds were detected in the analysed samples (IL-7 and GM-CSF). It is noteworthy to mention that their concentrations were higher after pasteurisation and that this increase was significant for IL-7 levels in colostrum and for both IL-7 and GM-CSF in mature milk samples (Wilcoxon signed-rank test, P = 0.002, P = 0.030 and P = 0.007, respectively). Regarding GM-CSF, the number of colostrum samples where it could be detected increased from 3 to 9 as a consequence of the heat treatment, although its concentration did not vary significantly (Wilcoxon signed-rank test, P = 0.575) (Table 3).

Back to Top | Article Outline

Concentration of Immunoglobulins in Colostrum and Milk Samples Before and After Pasteurisation

The concentration of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE in colostrum and milk samples before and after pasteurisation, expressed as median and IQR values, is shown in Table 4. The most abundant immunoglobulin, both in colostrum and in milk samples, was IgA, its median (IQR) concentration being similar in both types of samples: 7.18 (6.53–7.64) and 5.96 (2.81–6.79) g/L, respectively. IgA levels in both colostrum and mature milk samples were reduced significantly after pasteurisation treatment (55 and 50% reduction, respectively; Wilcoxon signed-rank test, P = 0.003 and P = 0.000, respectively). IgM was detected in all samples, although its level was significantly higher in colostrum than in milk (Mann-Whitney U test, P = 0.000); however, Holder pasteurisation reduced its concentration significantly in both colostrum and milk (37% and 34%, respectively; Wilcoxon signed-rank test, P = 0.014 and P = 0.006, respectively).



Regarding IgGs, the subclass IgG4 was found in practically all colostrum and milk samples, and its level in milk samples was reduced significantly after pasteurisation; IgG1 and IgG3 were detected only in a few colostrum and milk samples; and IgG2 was not present in any of the samples analysed. Finally, the less abundant immunoglobulin in human colostrum and milk was IgE, although it was detected in most samples.

Back to Top | Article Outline


Presence of viable bacteria in nonpasteurised fresh human colostrum supports results obtained in previous reports (2). Both colostrum and breast milk provide commensal and potentially probiotic bacteria to the infant gut, such as staphylococci, streptococci, bifidobacteria, and lactic acid bacteria (19,20), that play an important role in the colonization of the healthy infant gut (21). These commensal bacteria are killed by the pasteurisation process but spore-forming bacteria, such as Bacillus sp, may survive the heating process and can grow faster than in raw milk because of the heat damage to the milk bacteriostatic systems (15,22,23). Although any milk bottle with a culture-positive result after pasteurisation is rejected by most milk banks, a previous study revealed that all B cereus strains isolated from pasteurised milk did not possess a high virulence potential (15). Anyway, in the present study Bacillus sp could not be detected in any colostrum sample after pasteurisation. The use of milk pumps to collect the samples by donors participating in the present study may explain high bacterial counts in many milk samples, and in particular enterobacteria, which are not usual components of the colostrum and milk microbiota (15,24,25).

In this work, a modified RP-HPLC method, which included the use of a different detector, allowed to detect furosine in all nonpasteurised colostrum samples at levels ranging from 3.50 to 11.60 mg/100 g protein, and this value increased from 2 to 3 times after Holder pasteurisation. In a previous work to study some biochemical modifications in human donor milk after this heat treatment, furosine could not be detected in any of the 21 human milk samples analysed, neither before nor after pasteurisation (15). This fact can be attributed to a higher sensitivity of the new detector used in the HPLC analytical equipment used to quantify furosine. The reprocessing and analysis of the above-mentioned 21 pairs of human milk samples using the methodology described here allowed to detect furosine in all nonpasteurised samples (from 1.80 to 7.80 mg/100 mg protein) and the furosine concentration also increased after the heat treatment (from 6.60 to 19.60 mg/100 g protein). Higher protein content in colostrum compared with human milk may explain lower furosine formation in pasteurised human milk samples.

Holder pasteurisation did not significantly modify the concentration of lactose, glucose, and myoinositol in human colostrum; however, lactulose, which could not be detected in any of the nonpasteurised samples, was formed during pasteurisation and its concentration (mean value of 22.62 mg/L) was similar to what was found in Holder-pasteurised human milk (mean value of 18.96 mg/L) in a previous work (15). Lactulose is used as a thermal index to differentiate heat-treated cow's milk depending on the intensity of the heat treatment applied (26,27). The lactulose levels in pasteurised colostrum and human milk were slightly higher than those found in commercial pasteurised cow's milk (26), which could be explained by higher intensity of the Holder pasteurisation compared with the regular heat treatments applied in the dairy industry, together with the higher lactose content of human milk and the high amounts of sodium and potassium present in human colostrum (28).

Furosine formation also depends on the intensity of heat treatment and storage conditions, and a correlation between lactulose and furosine has been described in bovine milk subjected to more severe heat treatment, such as ultrahigh temperature and in-container sterilised milk (26,29). The lactulose:furosine ratio in human colostrum ranged from 0.5 to 1.8, which was similar to those reported by Villamiel et al (30) for commercial bovine pasteurised milks (0.3–1.1). Therefore, it can be concluded that human colostrum experienced few changes in their nutritional value as the indices used to evaluate damage by heat treatment (lactulose and furosine) were in the same range found in commercial pasteurised milk.

The mean lactose and glucose concentrations in donor colostrum reported in this work were lower than those in milk samples (15). The lactose content of human milk at different stages of lactation increases along the lactation, especially during the first few days, and a similar pattern has been reported for glucose (11,12,31). In contrast, the myoinositol concentration is significantly greater in colostrum than in milk and decreases rapidly during the first days of lactation (12,32). In the present study, the mean concentration of myoinositol in colostrum had a wide range of variation, in agreement with previous results obtained in transitional milk (<15 days of lactation) (15). Further work on this component would be advisable because it seems to play an important role in early neonatal life for the prevention of bronchopulmonary dysplasia and retinopathy in premature infants (33).

Compared with mature milk, colostrum is notably richer in cytokines and other immune-related proteins that provide anti-infectious, anti-inflammatory, and immunomodulatory protection against infection (3–6). Interestingly, several studies have revealed that there is an inverse relation between duration of pregnancy and the concentration of protective factors in colostrum (34–37). Globally, this suggests that the immune components that are unique to preterm colostrum may be especially protective during the first week of life and, particularly, to extremely low birth weight (ELBW) infants, a population at the highest risk for feeding intolerance and nosocomial infection (38,39). Therefore, it would be highly desirable in ELBW infants to start as soon as possible trophic feeds (also known as minimal enteral nutrition) using preterm colostrum if contraindications do not exist. Trophic feeding is defined as the enteral administration of minute volumes of feeds, typically between 1 and 20 mL · kg · day; these amounts of colostrum are nutritionally insignificant and, although there is no definite evidence of their trophic effect on the small intestine, they promote the capacity of full enteral feeding in the preterm infant (40–42). A safe and efficacious alternative method for administering preterm colostrum to ELBW infants, when they cannot be fed enterally during the first days of life, is oropharyngeal administration of their own mother's or donor colostrums, that is well-tolerated by even the smallest and sickest ELBW infants (43).

Although Holder pasteurisation allows a good compromise between microbiological safety and nutritional/biological quality of donor human milk (44–46), it is also well known that this method affects some of the nutritional and biological properties of human milk and eliminates the beneficial microbiota naturally present (47–50). The same seems applicable, and even to a higher extent, to human colostrum. In the present study, most of the cytokines were not affected by the heat processing, but the opposite was observed for the immunoglobulins, probably because of their high molecular weight. Remarkably, the observed increase in the concentration of some cytokines (such as IL-7 or GM-CSF) after pasteurisation could be attributed to their release from cellular and/or fat compartments into the aqueous fraction during the heat treatment. The high nutritional and immunological quality of colostrum and the limited availability of donor colostrum in human milk banks suggest that, when available, it should be dedicated to initiate feeding of the most vulnerable ELBW infants, and that the optimisation of human colostrum and milk pasteurisation should be one of the priorities of human milk banks. In this frame, high-temperature short-time pasteurisation may be an alternative to maintain the nutritional and immunological quality of human colostrum and milk because it retains the protein profile and some of the key active components of donor milk better than the Holder treatment (15).

The number of samples (n = 10) evaluated in the present study was low because of the difficulties in obtaining donor colostrum; however, it represents an initial step that highlights the importance of donation during early breast-feeding to cope with the nutritional and immunological requirements of low-birth-weight preterm infants.

Back to Top | Article Outline


1. Field CJ. The immunological components of human milk and their effect on immune development in infants. J Nutr 2006; 135:1–4.
2. Jiménez E, Fernández L, Delgado S, et al. Assessment of the bacterial diversity of human colostrum by cultural-based techniques. Analysis of the staphylococcal and enterococcal populations. Res Microbiol 2008; 159:595–601.
3. Mathur NB, Dwarkadas AM, Sharma VK, et al. Anti-infective factors in preterm colostrum. Acta Paediatr Scand 1990; 79:1039–1044.
4. Buescher ES, McWilliams-Koeppen P. Soluble tumor necrosis factor-alpha (TNF-alpha) receptors in human colostrum and milk bind to TNF-alpha and neutralize TNF-alpha bioactivity. Pediatr Res 1998; 44:37–42.
5. Buescher ES. Anti-inflammatory characteristics of human milk: how, where and why. Adv Exp Med Biol 2001; 501:207–222.
6. Garofalo R. Cytokines in human milk. J Pediatr 2010; 156:s36–s40.
7. Oddy W. The impact of breastmilk on infant and child health. Breastfeed Rev 2002; 10:5–18.
8. Arnold LDW. Using banked donor milk in clinical settings. In: Caldwell K, ed. Reclaiming Breastfeeding in the United States: Protection, Promotion, Support. Sudbury, MA: Jones & Bartlett; 2002:161–78.
9. Arslanoglu S, Ziegler EE, Moro GE, et al. Donor human milk in preterm infant feeding: evidence and recommendations. J Perinat Med 2010; 38:347–351.
10. Baro C, Giribaldi M, Arslanoglu S, et al. Effect of two pasteurization methods on the protein content of human milk. Front Biosci 2011; E3:818–829.
11. Lonnerdal B, Forsurn E, Hambraeus L. A longitudinal study of the protein, nitrogen, and lactose contents of human milk from Swedish well-nourished mothers. Am J Clin Nutr 1976; 29:1127–1133.
12. van Beusekom CM, Zeegers TA, Martini IA, et al. Milk of patients with tightly controlled insulin-dependent diabetes mellitus has normal macronutrient and fatty-acid composition. Am J Clin Nutr 1993; 57:938–943.
13. Delgado T, Corzo N, Santamaría G, et al. Determination of furosine in milk samples by ion-pair reversed phase liquid-chromatography. Chromatographia 1992; 33:374–376.
14. Cattaneo S, Masotti F, Pellegrino L. Liquid infant formulas: technological tools for limiting heat damage. J Agric Food Chem 2009; 57:10689–10694.
15. Gómez de Segura A, Escuder D, Montilla A, et al. Heating-induced bacteriological and biochemical modifications in human donor milk after holder pasteurisation. J Pediatr Gastroenterol Nutr 2012; 54:197–203.
16. Kullen MJ, Sanozky-Dawes RB, Crowell DC, et al. Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J Appl Microbiol 2005; 89:511–516.
17. Resmini P, Pellegrino L, Batelli G. Accurate quantification of furosine in milk and dairy products by a direct HPLC method. Ital J Food Sci 1990; 2:173–183.
18. Montilla A, Moreno FJ, Olano A. A reliable gas capillary chromatographic determination of lactulose in dairy samples. Chromatographia 2005; 62:311–314.
19. Martín R, Langa S, Reviriego C, et al. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr 2003; 143:754–758.
20. Heikkilä MP, Saris PEJ. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol 2003; 95:471–478.
21. Martín V, Maldonado A, Moles L, et al. Sharing of bacterial strains between breast milk and infant feces. J Human Lact 2012; 28:36–44.
22. Ford JE, Law BA, Marshall VME, et al. Influence of the heat treatment of human milk on some of its protective constituents. J Pediatr 1977; 91:29–35.
23. Landers S, Updegrove K. Bacteriological screening of donor human milk before and after holder pasteurization. Breastfeed Med 2010; 5:117–121.
24. Marín ML, Arroyo R, Jimenez E, et al. Cold storage of human milk: effect on its bacterial composition. J Pediatr Gastroenterol Nutr 2009; 49:343–348.
25. Brown SL, Bright RS, Dwyer DE, et al. Breast pump adverse events: reports to the Food and Drug Administration. J Hum Lact 2005; 21:169–174.
26. Pellegrino L, De Noni I, Resmini P. Coupling of lactulose and furosine indices for quality evaluation of sterilized milk. Int Dairy J 1995; 5:647–659.
27. Marconi E, Messia MC, Amine A, et al. Heat-treated milk differentiation by a sensitive lactulose assay. Food Chem 2004; 84:447–450.
28. Lawrence RA, Lawrence RM. Lawrence RA, Lawrence RM. Biochemistry of human milk. Breastfeeding: A Guide to the Medical Profession 6th ed.St Louis:Mosby; 2005. 98–152.
29. Montilla A, Calvo MM, Santa-María G, et al. Correlation between lactulose and furosine in UHT heated milk. J Food Prot 1996; 59:1061–1064.
30. Villamiel M, Arias M, Corzo N, et al. Use of different thermal indices to assess the quality of pasteurized milks. Z Lebensm Unters Forsch A 1999; 208:169–171.
31. Coppa GV, Gabrielli O, Pierani P, et al. Changes in carbohydrate-composition in human-milk over 4 months of lactation. Pediatrics 1993; 3:637–641.
32. Pereira GR, Baker L, Egler J, et al. Serum myoinositol concentrations in premature infants fed human milk, formula for infants, and parenteral nutrition. Am J Clin Nutr 1990; 51:589–593.
33. Hallman M, Saugstad OD, Porreco RP, et al. Role of myoinositol in regulation of surfactant phospholipids in the newborn. Early Hum Dev 1985; 10:245–254.
34. Castellote C, Casillas R, Ramírez-Santana C, et al. Premature delivery influences the immunological composition of colostrum and transitional and mature human milk. J Nutr 2011; 141:1181–1187.
35. Montagne P, Cuilliere ML, Mole C, et al. Immunological and nutritional composition of human milk in relation to prematurity and mother's parity during the first 2 weeks of lactation. J Pediatr Gastroenterol Nutr 1999; 29:75–80.
36. Araujo ED, Goncalves AK, Cornetta M, et al. Evaluation of the secretory immunoglobulin A levels in the colostrum and milk of mothers of term and preterm infants. Braz J Infect Dis 2005; 9:357–362.
37. Koenig A, Diniz EMD, Barbosa SFC, et al. Immunologic factors in human milk: the effects of gestational age and pasteurization. J Hum Lact 2005; 21:439–443.
38. LaGamma E, Brown L. Feeding practices for infants weighing less than 1500 g at birth and the pathogenesis of necrotizing enterocolitis. Clin Perinatol 1994; 21:271–306.
39. Westerbeek E, van den Berg A, Lafeber HN, et al. The intestinal bacterial colonization in preterm infants: a review of the literature. Clin Nutr 2006; 25:361–368.
40. Hay WW. Strategies for feeding the preterm infant. Neonatology 2008; 94:245–254.
41. Thompson FM, Catto-Smith AG, Moore D, et al. Epithelial growth of the small intestine in human infants. J Pediatr Gastroenterol Nutr 1998; 26:506–512.
42. Cummins AG, Thompson FM. Effect of breast milk and weaning on epithelial growth of the small intestine in humans. Gut 2002; 51:748–754.
43. Rodriguez NA, Meier PP, Groer MW, et al. A pilot study to determine the safety and feasibility of oropharyngeal administration of own mother's colostrum to extremely low-birth-weight infants. Adv Neonatal Care 2010; 10:206–212.
44. Human Milk Banking Association of North America (HMBANA). Guidelines for the Establishment and Operation of a Donated Human Milk Bank. Ft Worth, TX: HMBANA; 2009.
45. UK Association for Milk Banking. Guidelines for the Establishment and Operation of Human Milk Banks in the UK. 3rd ed. London: Royal College of Paediatrics and Child Health; 2003.
46. Hartmann BT, Pang WW, Keil AD, et al. Australian Neonatal Clinical Care Unit. Best practice guidelines for the operation of a donated human milk bank in an Australian NICU. Early Hum Dev 2007; 83:667–673.
47. Arslanoglu S, Moro GE, Ziegler EE. Donor human milk in preterm infant feeding: evidence and recommendations. World Association of Perinatal Medicine (WAPM) Working Group on Neonatal Nutrition Recommendations. J Perinat Med 2010; 38:347–351.
48. Moro G, Arslanoglu S. Heat treatment of human milk. J Pediatr Gastroenterol Nutr 2012; 54:165–166.
49. Wight NE. Donated human milk for preterm infants. J Perinatol 2001; 21:249–254.
50. Goelz R, Hihn E, Hamprecht K, et al. Effects of different CMV-heat-inactivation-methods on growth factors in human breast milk. Pediatr Res 2009; 65:458–461.

colostrum; immune compounds; milk; pasteurisation; thermal indices

© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,