ORIGINAL ARTICLE: NUTRITION
Effect of Holder Pasteurisation on Human Milk Glycosaminoglycans Alessandra Coscia, Chiara Peila, Enrico Bertino, yGiovanni V. Coppa, §Guido E. Moro, Orazio Gabrielli, yLucia Zampini, yTiziana Galeazzi, zFrancesca Maccari, and zNicola Volpi
y
ABSTRACT Objectives: The benefits of human milk for preterm infants are mainly the result of its nutritional characteristics and the presence of biologically active compounds. Among these compounds, glycosaminoglycans (GAGs) play an emerging leading role. When mother’s milk is unavailable or in short supply, pasteurised donor milk represents an important nutritional alternative. The aim of this study was to evaluate the effect of Holder pasteurisation on the concentration of different GAGs in preterm human milk. Methods: Milk samples collected from 9 mothers having delivered preterm were divided into 2 parts. One part of each sample was immediately frozen (808C), whereas the other part was pasteurised with the Holder method before being frozen at 808C. Specific analytical procedures were applied to evaluate the amount, composition, and structure of main human milk GAGs. Results: No significative differences were measured between not-treated and pasteurised samples for total GAGs content, relative percentages of chondroitin sulfate and heparan sulfate, and main parameters related to galactosaminoglycans structure, even if a slight decrease of total GAGs content of 18% was observed in treated samples. Conclusions: Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs. Key Words: chondroitin sulfate, glycosaminoglycans, heparan sulfate, Holder pasteurisation, human milk
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F
resh own mother’s milk is the best feeding choice for verylow-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 Received April 1, 2014; accepted September 9, 2014. From the Neonatal Unit, Department of Public Health and Pediatric, University of Turin, Turin, the yDepartment of Clinical Sciences, Polytechnic University of Marche, Ospedali Riuniti, Ancona, the zDepartment of Life Sciences, University of Modena and Reggio Emilia, Modena, and the §Italian Association of Human Milk Banks, Milan, Italy. Address correspondence and reprint requests to Enrico Bertino, MD, Neonatal Intensive Care Unit, Department of Public Health and Pediatric, Turin University, Piazza Polonia 94-10133 Turin, Italy (e-mail: [email protected]). This research has been partly supported by the Iolanda Minoli Foundation, San Giuseppe University Hospital, Milan, Italy. The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000570
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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.
METHODS 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
127
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808C, whereas the other one was pasteurised and then frozen at 808C. The Holder pasteurisation was performed with a Sterifeed Pasteurizer (Medicare Colgate Ltd, Cullompton, UK) heating milk samples at 62.58C for 30 minutes, followed by cooling to 108C 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 608C 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 3.4.21.64], >500 units/ mL; Sigma-Aldrich, St Louis, MO) at 608C for 12 hours. After boiling for 10 minutes, centrifugation, and filtration on 0.45 mm 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 anionexchange 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 48C for 24 hours, the precipitate was recovered by centrifugation at 10,000g for 10 minutes and dried at 608C for 12 hours. The dried precipitate was dissolved in 50 mL 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 4.2.2.4]; Sigma-Aldrich) and separation of generated unsaturated disaccharides by strong-anion-exchange (SAX)-highperformance liquid chromatography with postcolumn derivatisation and fluorimetric detection (18)
Statistical Analyses 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.
RESULTS The donors’ demographic characteristics are reported in Table 1. After human milk GAGs extraction and purification,
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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.
DISCUSSION 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
TABLE 1. Demographic characteristics of milk donors Mother no. Age, y Delivery type (vaginal/caesarean) Duration of gestation, wk Duration of lactation at time of milk collection, days Birth weight of the infant, g
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1 31 Caesarian 31 68
2 40 Vaginal 26 40
3 38 Caesarian 36 31
4 37 Vaginal 30 38
5 32 Cesarean 33 25
6 38 Caesarian 30 74
7 33 Cesarean 31 32
8 37 Cesarean 33 39
9 32 Vaginal 34 38
1100
850
1760
1320
1650
1210
1000
2100
1500
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Effect of Holder Pasteurisation on Human Milk Glycosaminoglycans
TABLE 2. Total glycosaminoglycans content of milk samples measured by uronic acid assay, relative percentages of CS and HS determined by agarose-gel elctrophoresis and charge density related to galactosaminoglycans (mainly CS) structures are illustrated for not-treated compared with pasteurised human milk samples Uronic acids, mg/mL
Electrophoresis (CS% and HS%) Not-treated
Samples 1 2 3 4 5 6 7 8 9 Mean SD SE
Pasteurised
CS charge density
Difference
Not-treated
Pasteurised
Difference
CS%
HS%
CS%
HS%
CS%
HS%
Not-treated
Pasteurised
Difference
1.22 0.88 1.14 1.28 0.82 3.82 6.72 1.16 0.52 1.95 2.03 0.68
1.05 0.80 0.72 0.66 0.63 3.23 4.36 1.39 1.52 1.60 1.32 0.44
0.17 0.08 0.42 0.62 0.19 0.59 2.36 0.23 1.00 0.36 0.90 0.30
36.6 49.6 59.7 41.9 48.1 46.9 53.4 43.9 57.0 48.6 7.3 2.4
63.4 50.4 40.3 58.1 51.9 53.1 46.6 56.1 43.0 51.4 7.3 2.4
65.4 64.5 65.0 59.8 69.2 76.5 67.6 52.9 70.0 65.7 6.6 2.2
34.6 35.5 35.0 40.2 30.8 23.5 32.4 47.1 30.0 34.3 6.6 2.2
28.8 14.9 5.3 17.9 21.1 29.6 14.2 9.0 13.0 17.1 8.3 2.8
28.8 14.9 5.3 17.9 21.1 29.6 14.2 9.0 13.0 17.1 8.3 2.8
0.41 0.39 0.16 0.17 0.31 0.72 0.24 0.47 0.54 0.38 0.18 0.06
0.50 0.33 0.32 0.35 0.47 0.38 0.19 0.36 0.27 0.35 0.09 0.03
0.10 0.07 0.16 0.18 0.16 0.34 0.05 0.12 0.27 0.03 0.19 0.06
Single values, mean, SD, and SE are reported for data along with their differences. No significant differences were observed between the 2 groups of samples. CS ¼ chondroitin sulfate; HS ¼ heparan sulfate; SD ¼ standard deviation; SE ¼ standard errors.
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.
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 lowbirth-weight babies fed with donor milk compared with those fed
CS DS HS
Mix
CS
CS
1
2
3
4
5
6
7
8
9
CS
CS
Mix
CS DS HS
O Mix Mix
CS
CS
1
2
3
4
5
6
7
8
9
CS
CS
Mix
FIGURE 1. Agarose-gel electrophoresis run of not-treated samples (A) and pasteurised samples (B).
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REFERENCES 1. American Academy of Paediatrics. Breastfeeding and use of human milk. Pediatrics 2012;129:e827–41. 2. Schanler RJ, Lau C, Hurst NM, et al. Randomized trial of donor human milk versus preterm formula as substitutes for mothers’ own milk in the feeding of extremely premature infants. Pediatrics 2005;116:400–6. 3. Coppa GV, Gabrielli O. Human milk oligosaccharides as prebiotics. In: Versalovic J, Wilson M, eds. Therapeutic Microbiology. Washington, DC: ASM Press; 2008:131–46. 4. Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 2012;22:1147–62. 5. Newburg DS. Glycobiology of human milk. Biochemistry 2013;78: 771–85. 6. Newburg DS, Linhardt RJ, Ampofo SA. Human milk glycosaminoglycans inhibit HIV glycoprotein gp120 binding to its host cell CD4 receptor. J Nutr 1995;125:419–24. 7. Jackson RJ, Bush SJ, Cardin AD. Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes. Physiol Rev 1991;71:481–539. 8. Raman R, Sasiseskharan V, Sasiseskharan R. Structural insights into biological roles of protein-glycacosaminoglycan interactions. Chem Biol 2005;12:267–77. 9. Sasiseskharan R, Raman R, Prabhakar V. Glycomics approach to structure function relationships of glycosaminoglycans. Annu Rev Biomed Eng 2006;8:181–231. 10. Gandhi NS, Mancera RL. The structure of glycosaminoglycans and their interactions with proteins. Chem Biol Drug Des 2008;72:455–82. 11. Coppa GV, Gabrielli O, Buzzega D, et al. Composition and structure elucidation of human milk glycosaminoglycans. Glycobiology 2011; 21:295–303. 12. Coppa GV, Gabrielli O, Zampini L, et al. Glycosaminoglycan content in term and preterm milk during the first month of lactation. Neonatology 2012;101:74–6. 13. Italian Association of Human Milk BanksArslanoglu S, Bertino E, Tonetto P, et al. . Guidelines for the establishment and operation of a donor human milk bank. J Matern Fetal Neonatal Med 2010;23 (suppl 2):1–20. 14. Arslanoglu S, Corpeleijn W, Moro G, et al. Donor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr 2013;57:535–42.
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15. Cesaretti M, Luppi E, Maccari F, et al. A 96-well assay for uronic acid carbazole reaction. Carbohydr Polymers 2003;54:59–61. 16. Volpi N, Maccari F. Electrophoretic approaches to the analysis of complex polysaccharides. J Chromatogr B Analyt Technol Biomed Life Sci 2006;834:1–13. 17. Volpi N, Maccari F. Detection of submicrogram quantities of glycosaminoglycans on agarose-gels by sequential staining with toluidine blue and Stains-All. Electrophoresis 2002;23:4060–6. 18. Volpi N. Advances in chondroitin sulfate analysis: application in physiological and pathological States of connective tissue and during pharmacological treatment of osteoarthritis. Curr Pharm Des 2006; 12:639–58. 19. Newburg DS. Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans. J Anim Sci 2009;87 (suppl 13):26–34. 20. Coppa GV, Zampini L, Galeazzi T, et al. Human milk oligosaccharides inhibit the adhesion to Caco-2 cells of diarrheal pathogens: Escherichia coli, Vibrio cholerae and Salmonella fyris. Pediatr Res 2006;59:377– 82. 21. Hill DR, Kessler SP, Rho HK, et al. Specific-sized hyaluronan fragments promote expression of human b-defensin 2 in intestinal epithelium. J Biol Chem 2012;287:30610–24. 22. Egea J, Garcia AG, Verges J, et al. Antioxidant antiinflammatory and neuroprotective actions of chondroitin sulphate and proteoglycans. Osteoarthritis Cartilage 2010;18:S24–7. 23. Volpi N, Gabriello O, Carlucci A, et al. Human milk glycosaminoglycans in feces of breastfed newborns: preliminary structural elucidation and possible biological role. Breastfed Med 2014;9:105–6. 24. Ventura M, Canchaya C, Fitzgerald GF, et al. Genomics as a means to understand bacterial phylogeny and ecological adaptation: the case of bifidobacteria. Antonie Leeuwenhoek 2007;91:351–71. 25. Mc Guire W, Anthony MY. Donor human milk versus formula for preventing necrotising enterocolitis in preterm infants: systematic review. Arch Dis Child Fetal and Neonatal Ed 2003;88:F11. 26. Boyd CA, Quigley MA, Brocklehurst P. Donor breast milk versus infant formula for preterm infants: systematic review and meta-analysis. Arch Dis Child Fetal Neonatal Ed 2007;92:F169. 27. Quigley M, McGuire W. Formula versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database Syst Rev 2014;22:4.
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Effect of Holder Pasteurisation on Human Milk Glycosaminoglycans Alessandra Coscia, Chiara Peila, Enrico Bertino, yGiovanni V. Coppa, §Guido E. Moro, Orazio Gabrielli, yLucia Zampini, yTiziana Galeazzi, zFrancesca Maccari, and zNicola Volpi
y
ABSTRACT Objectives: The benefits of human milk for preterm infants are mainly the result of its nutritional characteristics and the presence of biologically active compounds. Among these compounds, glycosaminoglycans (GAGs) play an emerging leading role. When mother’s milk is unavailable or in short supply, pasteurised donor milk represents an important nutritional alternative. The aim of this study was to evaluate the effect of Holder pasteurisation on the concentration of different GAGs in preterm human milk. Methods: Milk samples collected from 9 mothers having delivered preterm were divided into 2 parts. One part of each sample was immediately frozen (808C), whereas the other part was pasteurised with the Holder method before being frozen at 808C. Specific analytical procedures were applied to evaluate the amount, composition, and structure of main human milk GAGs. Results: No significative differences were measured between not-treated and pasteurised samples for total GAGs content, relative percentages of chondroitin sulfate and heparan sulfate, and main parameters related to galactosaminoglycans structure, even if a slight decrease of total GAGs content of 18% was observed in treated samples. Conclusions: Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs. Key Words: chondroitin sulfate, glycosaminoglycans, heparan sulfate, Holder pasteurisation, human milk
(JPGN 2015;60: 127–130)
F
resh own mother’s milk is the best feeding choice for verylow-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 Received April 1, 2014; accepted September 9, 2014. From the Neonatal Unit, Department of Public Health and Pediatric, University of Turin, Turin, the yDepartment of Clinical Sciences, Polytechnic University of Marche, Ospedali Riuniti, Ancona, the zDepartment of Life Sciences, University of Modena and Reggio Emilia, Modena, and the §Italian Association of Human Milk Banks, Milan, Italy. Address correspondence and reprint requests to Enrico Bertino, MD, Neonatal Intensive Care Unit, Department of Public Health and Pediatric, Turin University, Piazza Polonia 94-10133 Turin, Italy (e-mail: [email protected]). This research has been partly supported by the Iolanda Minoli Foundation, San Giuseppe University Hospital, Milan, Italy. The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000570
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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.
METHODS 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
127
Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Bertino et al
JPGN
808C, whereas the other one was pasteurised and then frozen at 808C. The Holder pasteurisation was performed with a Sterifeed Pasteurizer (Medicare Colgate Ltd, Cullompton, UK) heating milk samples at 62.58C for 30 minutes, followed by cooling to 108C 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 608C 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 3.4.21.64], >500 units/ mL; Sigma-Aldrich, St Louis, MO) at 608C for 12 hours. After boiling for 10 minutes, centrifugation, and filtration on 0.45 mm 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 anionexchange 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 48C for 24 hours, the precipitate was recovered by centrifugation at 10,000g for 10 minutes and dried at 608C for 12 hours. The dried precipitate was dissolved in 50 mL 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 4.2.2.4]; Sigma-Aldrich) and separation of generated unsaturated disaccharides by strong-anion-exchange (SAX)-highperformance liquid chromatography with postcolumn derivatisation and fluorimetric detection (18)
Statistical Analyses 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.
RESULTS The donors’ demographic characteristics are reported in Table 1. After human milk GAGs extraction and purification,
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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.
DISCUSSION 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
TABLE 1. Demographic characteristics of milk donors Mother no. Age, y Delivery type (vaginal/caesarean) Duration of gestation, wk Duration of lactation at time of milk collection, days Birth weight of the infant, g
128
1 31 Caesarian 31 68
2 40 Vaginal 26 40
3 38 Caesarian 36 31
4 37 Vaginal 30 38
5 32 Cesarean 33 25
6 38 Caesarian 30 74
7 33 Cesarean 31 32
8 37 Cesarean 33 39
9 32 Vaginal 34 38
1100
850
1760
1320
1650
1210
1000
2100
1500
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Volume 60, Number 1, January 2015
Effect of Holder Pasteurisation on Human Milk Glycosaminoglycans
TABLE 2. Total glycosaminoglycans content of milk samples measured by uronic acid assay, relative percentages of CS and HS determined by agarose-gel elctrophoresis and charge density related to galactosaminoglycans (mainly CS) structures are illustrated for not-treated compared with pasteurised human milk samples Uronic acids, mg/mL
Electrophoresis (CS% and HS%) Not-treated
Samples 1 2 3 4 5 6 7 8 9 Mean SD SE
Pasteurised
CS charge density
Difference
Not-treated
Pasteurised
Difference
CS%
HS%
CS%
HS%
CS%
HS%
Not-treated
Pasteurised
Difference
1.22 0.88 1.14 1.28 0.82 3.82 6.72 1.16 0.52 1.95 2.03 0.68
1.05 0.80 0.72 0.66 0.63 3.23 4.36 1.39 1.52 1.60 1.32 0.44
0.17 0.08 0.42 0.62 0.19 0.59 2.36 0.23 1.00 0.36 0.90 0.30
36.6 49.6 59.7 41.9 48.1 46.9 53.4 43.9 57.0 48.6 7.3 2.4
63.4 50.4 40.3 58.1 51.9 53.1 46.6 56.1 43.0 51.4 7.3 2.4
65.4 64.5 65.0 59.8 69.2 76.5 67.6 52.9 70.0 65.7 6.6 2.2
34.6 35.5 35.0 40.2 30.8 23.5 32.4 47.1 30.0 34.3 6.6 2.2
28.8 14.9 5.3 17.9 21.1 29.6 14.2 9.0 13.0 17.1 8.3 2.8
28.8 14.9 5.3 17.9 21.1 29.6 14.2 9.0 13.0 17.1 8.3 2.8
0.41 0.39 0.16 0.17 0.31 0.72 0.24 0.47 0.54 0.38 0.18 0.06
0.50 0.33 0.32 0.35 0.47 0.38 0.19 0.36 0.27 0.35 0.09 0.03
0.10 0.07 0.16 0.18 0.16 0.34 0.05 0.12 0.27 0.03 0.19 0.06
Single values, mean, SD, and SE are reported for data along with their differences. No significant differences were observed between the 2 groups of samples. CS ¼ chondroitin sulfate; HS ¼ heparan sulfate; SD ¼ standard deviation; SE ¼ standard errors.
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.
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 lowbirth-weight babies fed with donor milk compared with those fed
CS DS HS
Mix
CS
CS
1
2
3
4
5
6
7
8
9
CS
CS
Mix
CS DS HS
O Mix Mix
CS
CS
1
2
3
4
5
6
7
8
9
CS
CS
Mix
FIGURE 1. Agarose-gel electrophoresis run of not-treated samples (A) and pasteurised samples (B).
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