Fresh own mother's milk is the best feeding choice for very-low-birth-weight infants, and several data have confirmed the significant benefits of human milk for preterm infants because of its well-known nutritional characteristics and its biologically active compounds (1,2).
Several human milk glycans (such as glycoproteins, glycolipids, and especially oligosaccharides) have demonstrated specific biological properties (3–5). On the contrary, glycosaminoglycans (GAGs), another family of complex carbohydrates whose presence in human milk was first reported in 1995, have not been extensively studied (6). GAGs are linear heteropolysaccharide compounds with a variable number of repeating disaccharide units that are able to regulate many cellular events and physiological processes (such as cell growth and differentiation, cell–cell and cell–matrix interaction, anti-infective and anti-inflammatory processes) (7–9). GAGs are classified into 4 distinct categories based on their chemical components: hyaluronic acid (HA); galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]); glucosaminoglycans (heparin and heparan sulfate [HS]); and keratan sulfate (10).
Human milk typically contains a 7-fold higher GAG concentration than bovine milk. The main constituent of human milk GAGs is represented by undersulfated CS (∼55% of total GAGs), followed by HS (∼40%), and minor amounts of HA and DS (11). Preterm human milk contains roughly 3 times more GAGs than term milk, but the GAG composition is similar in both types of milk (12). During the first month of lactation, the absolute amount of polysaccharides is constantly and significantly higher in preterm with respect to term milk, with a similar behaviour in the decrease (12).
When mother's milk is unavailable or in short supply, pasteurised donor breast milk offers a safe alternative and is considered the next best choice (13,14). Pasteurisation partially affects the nutritional and immunological properties of breast milk; however, in a clinical perspective, it is well-known that pasteurised human milk maintains a protective effect against necrotising enterocolitis and infections (14). Holder pasteurisation is the most widely used method in human milk banks. This method inactivates some immunological and anti-infectious factors; nevertheless, other key nutritional and biological compounds are not affected by the process (14).
As to our knowledge no data are actually available, and the aim of this study was to evaluate the effects of Holder pasteurisation on the concentration of human milk GAGs.
Preterm milk was chosen because of the higher content of GAGs compared with term samples as reported in literature (12). Nine mothers were included in the study after obtaining an informed consent. The donors’ demographic characteristics were obtained by direct interview. Fresh milk samples were collected in the same morning into sterile, disposable, high-density polyethylene sealed bottles (Flormed, Naples, Italy). Milk expression was obtained by emptying one breast completely by means of an electric breast pump. From the total amount of milk of each mother, a sample of 10 mL of milk was collected and then subdivided into 2 parts. One of the 2 parts of each sample was immediately frozen at −80°C, whereas the other one was pasteurised and then frozen at −80°C.
The Holder pasteurisation was performed with a Sterifeed Pasteurizer (Medicare Colgate Ltd, Cullompton, UK) heating milk samples at 62.5°C for 30 minutes, followed by cooling to 10°C in approximately 20 minutes by immersing into cold water. The study protocol was approved by the ethics committee of the Italian Association of Human Milk Donor Banks.
Sample Preparation and Analysis
Extraction and purification of milk GAGs were performed as previously reported (11,12). Briefly, 2 mL of milk were defatted with acetone. After centrifugation at 10,000g for 10 minutes and drying at 60°C for 24 hours, the pellet was reconstituted with 1 mL of a 20 mmol/L Tris–Cl buffer (pH 7.4) and treated with protease (Proteinase K from Tritirachium album [EC 188.8.131.52], >500 units/mL; Sigma-Aldrich, St Louis, MO) at 60°C for 12 hours. After boiling for 10 minutes, centrifugation, and filtration on 0.45 μm filters, the filtrate was lyophilised. The powder was dissolved in 1 mL of distilled water by prolonged mixing and applied to a column (2 cm × 7 cm) packed with QAE Sephadex A-25 anion-exchange resin (Pharmacia Biotech, Uppsala, Sweden). After washing the resin with 5 volumes of 50 mmol/L NaCl, GAGs were eluted with 2 volumes of 1.2 mol/L NaCl. After adding 3 volumes of ethanol and storing at 4°C for 24 hours, the precipitate was recovered by centrifugation at 10,000g for 10 minutes and dried at 60°C for 12 hours. The dried precipitate was dissolved in 50 μL distilled water and further analysed. The total content of GAGs in milk extracts was determined by carbazole assay for uronic acids performed according to Cesaretti et al (15).
Human milk GAGs were separated and quantified by agarose-gel electrophoresis in barium acetate/1,2-diaminopropane, as reported (16,17). Structural characterisation of galactosaminoglycans, CS/DS, was performed by disaccharide composition after the treatment of GAGs with chondroitinase ABC (from Proteus vulgaris [EC 184.108.40.206]; Sigma-Aldrich) and separation of generated unsaturated disaccharides by strong-anion-exchange (SAX)-high-performance liquid chromatography with postcolumn derivatisation and fluorimetric detection (18)
Data are expressed as mean ± SD. Statistical analysis was performed by analysis of variance and Student–Newman–Keuls test by means of SPSS Statistics software version 19.0 for Windows (IBM SPSS Statistics, Armonk, NY). The level of statistical significance of differences was set at P < 0.05.
The donors’ demographic characteristics are reported in Table 1. After human milk GAGs extraction and purification, uronic acids were determined in a 96-well plate microassay (15). No significant differences were measured between not-treated and pasteurised samples, although a slight decrease of 18% was observed in the total GAG content of the treated milk samples (Table 2).
GAGs were also separated and quantified by agarose-gel electrophoresis able to separate CS, DS, and HS (Fig. 1A and B) (16,17). As previously demonstrated, 2 main species are present in human milk, an undersulfated CS (∼55% of total GAGs) and HS (∼40%) (11). These 2 GAGs were separated and quantified by electrophoresis and the results are illustrated in Table 2. No significant differences were measured between not-treated and pasteurised samples, although a slight decrease of 17% was observed in HS% (and relative increase of CS) in pasteurised milk samples. This is coherent with a ∼18% decrease in total GAGs content, just related to HS, with no modification in the CS structure.
After human milk GAGs extraction, galactosaminoglycans, mainly CS present in human samples, were submitted to enzymatic digestion with a lyase able to degrade these macromolecules, producing variously sulfated unsaturated disaccharides, which is further separated and quantified by high-performance liquid chromatography (18). The results are illustrated in Table 2. In particular, the nonsulfated and monosulfated disaccharides in position C6 and C4 of the N-acetyl-galactosamine unit were detected, according to a previous study (11). The charge density (the sulfate to disaccharide molar concentration ratio), useful to evaluate a possible desulfation process, was calculated and the results are illustrated in Table 2. Because it is evident, no significant differences were observed between not-treated and pasteurised milk samples, confirming that no loss of sulfate groups was produced by pasteurisation treatment.
Our study demonstrates that the concentration and the pattern of human milk GAGs are not affected by Holder pasteurisation. Human milk GAGs, such as oligosaccharides, are indigestible, contain a large number of glycan units, and play a role as inhibitors of viruses and pathogenic bacteria adhesion to the intestinal epithelium. In particular, it has been shown that some cell surface receptors are constituted by GAGs (9), which therefore participate directly in the regulation of the infective process by interacting with pathogens and by competing for their adhesion to the intestinal wall, as already demonstrated for other human milk glycans such as oligosaccharides (19,20). Previous studies have reported that CS isolated from human milk could inhibit the binding of the HIV envelope glycoprotein gp120 to the cellular CD4 receptor (6) and that HA fragments play an important role in promoting an innate antimicrobial effect in intestinal epithelial cells (21). In addition to this, GAGs regulate and activate several growth factors and could help to regulate the gastrointestinal epithelial development (5).
Moreover, CS and other GAGs can stimulate the biochemical pathway that induces the activation of antioxidant enzymes so that they may contribute to the removal of free radicals (22). This is particularly important in preterm infants presenting an immature immune system. Finally, the undigested GAGs, reaching the colon (23), could behave as prebiotics, contributing to the development of bifidogenic flora (24).
Prebiotic, trophic, and antimicrobial effects play a relevant role in feeding vulnerable preterm infants with mother's own milk, and our results support that also pasteurised donor milk has this potential. Three meta-analysis have shown a reduction in necrotising enterocolitis (NEC) incidence in preterm or low-birth-weight babies fed with donor milk compared with those fed with preterm formula (25–27). Although NEC pathophysiology is not completely clear, associated factors are represented by immature gastrointestinal epithelium, impaired immunological defences, enteral feeding, and bacterial colonisation. Human milk GAGs can play a protective role thanks to the activation of antioxidant enzymes, their antimicrobial effect, and their favourable effect on bifidogenic flora selective development (6,21,22,24).
In conclusion, our study confirms that the biological value of human milk associated with the GAGs content is maintained after Holder pasteurisation.
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Keywords:© 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
chondroitin sulfate; glycosaminoglycans; heparan sulfate; Holder pasteurisation; human milk