Publication Vinny Tan

Analysis of the Influence of Processing on Human Milk’s Macronutrient Concentrations Jun Ding, PhD, Megan Yvonne Asula, Vinny Sor Chin Tan Abstract Human breast milk is the optimal source of nutrition for all infants but especially for those born prematurely. As stated by the American Academy of Pediatrics, breast milk, especially in neonatal intensive care units is the optimal food for infants. When a mother is not able to produce enough milk for her infant, the next best source of nutrition is donated human breast milk. However, human breast milk is not a sterile luid and therefore can contain microorganisms that could be transferred to the infants who consume it. Because of this risk donor milk must be processed to ensure its safety, but with processing the milk’s important nutritional components can be altered or removed entirely. The objective of this study was to assess the impact of the processing method on the macronutrients of human milk. During a 1-year period (2013-2014), more than 400 milk samples from individual donors were analyzed for fat, protein, lactose and energy density. Banked donor milk mean values (in weight/ volume) were found to be 1.1% ± 0.04% for protein, 3.3% ± 0.03% for fat, 6.1% ± 0.6% for lactose, and mean total energy was 60 kcal/dL. Amino acids of pooled donor milk were also compared before and after processing. Our data shows that the processing only had a minor effect on the macronutrients of the donor milk. Additionally, the macronutrients were stable and remained constant over time. Introduction It is recommended by the American Academy of Pediatrics, and the World Health Organization (WHO) that mothers exclusively breastfeed from the time an infant is born to the irst 6 months of life for all healthy women and infants, and to continue breastfeeding for up to 2 years and beyond (American Academy of Pediatrics, 2012; WHO United Nations Children’s Fund (UNICEF), 2003). Breastfeeding exclusively for this long is not possible for all women. Only 27% of mothers of premature babies can produce enough milk for their infants (Ewaschuk et al., 2011). This leaves only two options to fulil the required nutritional needs for the infant, one is formula and the other is donated human breast milk. Donor milk is preferable to formula because it contains the same important nutritional components including native human protein needed for the overall growth and development of the infants. Multiple studies have found that preterm infants have a lower risk of developing necrotizing enterocolitis (NEC) when consuming donor breast Jun Ding is a Research Scientist and Lab Manager at Medolac Laboratories. Megan Yvonne Asula and Vinny Sor Chin Tan are both research assistants for Medolac Laboratories. n neonatal INTENSIVE CARE Vol. 28 No. 2 Spring 2015 milk compared to consuming formula (Buescher, 1994; Lucas and Cole, 1990). In addition, Sullivan et al (2010) reported signiicantly lower rates of NEC for premature infants when exclusively fed with human milk that contained human milkbased fortiier compared with a mother’s milk-based diet that includes bovine milk-based products. Human milk banks across the world collect donated human breast milk to feed infants in need. Human breast milk is a highly nutritious liquid that is not only beneicial for infants but also provides an ideal growth medium for many microorganisms. Because of this, breast milk needs to be handled and stored properly otherwise microbial contaminates can be introduced. Microorganisms can be introduced to the donated milk from a variety of sources such as the mothers body, skin, breast-pump or milk containers (Landers and Updegrove, 2010; Lindemann et al., 2004). Therefore, the pasteurization or processing of donor milk is required to minimize the possibility of the transmission of infectious agents from the milk to the infants (Akinbi et al., 2010). Depending on the amount of time and temperature used, exposure to heat may alter or eliminate certain nutritional components which in turn could result in insuficient levels of major and minor nutrients needed to serve the speciic nutritional needs of infants (Modi 2006; Tully et al., 2001). This is especially a concern for premature infants that have a heightened need for the many different important nutritional components found in human breast milk. Given the current trend in increased use of donor breast milk as an alternative nutritional source to mothers own milk and the limited data regarding the effects of processing on the nutrient composition of the milk, we analyzed the nutritional components of pooled donor milk before and after processing. A range of 60 to 180 samples from individual donors were collected, pooled and processed per production lot. Each production lot consisted of 500 to 2,000 gallons of human milk. We compared four different production lots for nutritional components and three production lots for amino acid content before and after processing. The macronutrients were analyzed by 3rd party certiied laboratories as well as our own lab at Medolac Laboratories. In addition, we compared the levels of major nutritional components found in our pooled donor’s breast milk to the national populations pooled milk using previously reported levels in human milk. Methods and Materials Breast milk samples (50mL) were obtained from a partner 19 milk bank, Mothers Milk CO-OP (Lake Oswego, Oregon). Milk was collected from approved donors who answered medical history questions and went through blood testing. The mothers were required to pass blood tests for speciic diseases such as HIV1, 2, HTLV I, II, HBV, HCV, syphilis, Chagas and West Nile virus. The phlebotomy was performed by Labcorp and serology testing was done by the American Red Cross National Blood Testing Laboratories. Overall, donors were healthy before and after delivery and received negative results from the blood tests. Breast milk samples were stored in the donor’s home freezer before being shipped overnight to our lab (Medolac Laboratories) in an insulated cooler. Informed consent was obtained from the participating mothers. For this study samples were collected during a one year period (2013-2014). The milk was collected from all states except for Alaska, Arkansas, California, Hawaii, Maryland and New York. Microbiology Screen All donor milk samples were screened for microorganisms including aerobic bacteria, Staphylococcus aureus and Enterobacteriaceae. Upon receiving, the milk samples were maintained at -20 °C. Donor milk samples were thawed in a water bath and diluted 200-fold with sterile water. A 1-mL aliquot of each diluted sample was plated on the 3M Petriilm for testing (3M, St. Paul, MN). All plates were incubated according to the manufacturer’s instructions. The colonies were counted and the results were expressed in colony-forming units per ml (CFU/ ml). Milk samples within the suggested ranges were pooled and processed. The criteria includes: <105 CFU/ml for aerobic; <104 CFU/ml for Staphylococcus aureus and Enterobacteriaceae (UK National Institute for Health and Clinical Excellence). Once a donation had passed, the milk was pooled in a large tank that contained 60-180 different donations per production lot. A total of four production lots were analyzed for nutritional components as well as three production lots for amino acid content. The analysis was performed on samples before and after processing. Nutrient Analysis Major nutrients analysis Approximately 20 ml of each sample was used to determine the nutrient content. A Calais Mid-range Infrared Milk Analyzer was used to analyze the milk and was previously calibrated for human milk measurements using the Kjeldahl method for protein, Chloramine-T for lactose and Mojonnier for fat. Energies derived from protein and lactose was determined by multiplying the number of grams of each component per 100 ml by a factor of 4 kcal/gram, and energies derived from fat was determined by multiplying the number of grams of fat component per 100 ml by a factor of 9 kcal/gram (Dewey et al., 1984). tested with the AOAC 965.17 method. Cholesterol was tested using the WRE 053 method. Statistical analysis The effects of processing were analyzed through a paired t-test of matched samples collected before and after processing (Microsoft excel, Redmond, WA, USA). Results and discussion Nutrients were consistent among different production lots Nutritional components of human breast milk are derived from three sources: synthesis in the lactocyte, diet, and maternal stores. It has been established that the composition of human milk varies signiicantly among different lactation periods, time of day, length of gestation, and dietary habits (Modi 2006; SalaVila et al., 2005; Yamawaki et al., 2005). To minimize variability, donated human breast milk of different production lots is routinely pooled together (Human Milk Banking Association of North America, 2005). The nutritional components were found to be consistent among the four different production lots tested (Table 1). The mean values for pooled milk samples was 1.1% for protein, 3.1% for fat, and 6.1% for lactose with the exception of production lot A, which had a higher reading for fat (4.2%) and a lower reading for lactose (4.5%). Production lot A also had a higher energy density (71 kcal/dL) which is attributed to the higher fat content. The other production lots had a mean energy density of around 60 kcal/dL (Table 1). The variation observed in production lot A could be due to the different analysis method used by IEH. Smilowitz et al (2014) reported that fat, protein and lactose in human milk was 3.2%, 1.0%, 6.2%, which is comparable to our observation. The current nutrient recommendation for preterm infants is 120 kcal with 3.5 to 4 g protein/day (American Academy of Pediatrics, 2009). Preterm infants are fed 150 ml/ kg of breast milk, therefore the maximum protein intake would be 3.3 g/kg making fortiier a required supplement for preterm infants. Vitamin C and other macronutrients in pooled donor milk samples were also analyzed. The macronutrients were consistent among samples except for potassium, which was about 10-fold higher in production lot B than what was observed in production lots C and D (Table 2). Though nutritional components in breast milk vary among mothers and among pumping sessions, there is consistency in nutritional concentrations when breast milk is combined into large pools. Table 1: Major nutrient analysis of production lots before processing. Data is mean ± SD. Amino acid analysis Samples Aa Bb Cb Db Amino acids were analyzed by AAA Service Laboratory (Damascus, Oregon) using a Hitachi ion-exchange high-pressure liquid chromatography amino acid analyzer with a post-column, ninhydrin derivatization instrument (Hitachi L8900, Hitachi HighTech Trading Corp, Minato-ku, Japan). Protein % 1.2 ± 0.05 1.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.04 Fat % 4.2 ± 0.04 3.3 ± 0.03 3.1 ± 0.03 2.7 ± 0.03 Lactose % 4.5 ± 0.5 6.4 ± 0.6 6.4 ± 0.6 5.9 ± 0.6 71 64 64 58 Others Samples were sent to IEH and Euroins to be analyzed. Vitamin C was tested by AOAC 984.26 (IEH) or AOAC 967.22 method (Euroins). Calcium, copper, iron, magnesium, manganese, potassium, sodium, and zinc were tested with modiied EPA 6020 (IEH) or AOAC 965.17/AOAC 985.01 method (Euroins). Iron and sodium were tested with the WRE 063 method. Phosphorus was 20 Energy kcal/dL a Analyses were performed by IEH. b Analyses were performed by Euroins. Nutrients were retained after processing The medical community is interested in understanding the effect of processing on the nutritional composition of donated human breast milk, especially the possibility that nutrients could be altered or eliminated completely. However the major nutrients of pooled breast milk before and after processing were not n neonatal INTENSIVE CARE Vol. 28 No. 2 Spring 2015 signiicantly different. The mean content for post-processing samples was 1.03% ± 0.03% for protein, 3.01% ± 0.26% for fat, and 6.20% ± 0.26% for lactose, compared to pre-processing samples of 1.06% ± 0.03% for protein, 3.07% ± 0.13% for fat, and 6.54% ± 0.15% for lactose (Table 3). Little to no difference was observed therefore our processing does not affect the major nutrients of human breast milk. On the contrary, a signiicant reduction in fat, energy content (Garcia-Lara et al., 2013), and protein (Akinbi et al., 2010; Koenig et al., 2005) of donor human breast milk was observed after Holder pasteurization. Table 2: Macronutrient analysis of production lots before processing. Samples B C D Vitamin C our processing enables infants who have severe gastrointestinal disease or who are recovering from major gastrointestinal surgery, which is commonly seen among preterm babies, to receive suficient glutamine to meet demands. A previous study has evaluated the effect of Holder pasteurization (62.5°C for 30 minutes) on free amino acid content in pooled donor breast milk (Valentine et al., 2010). There were signiicant increases in arginine and leucine, signiicant decreases in aspartate and no signiicant difference in lysine after pasteurization. To our knowledge, no data has been published on the effect of Holder pasteurization or the processing of human milk on total amino acid proile. <4.4 ppm <4.4 ppm <4.4 ppm Calcium 0.03% 0.02% 0.02% Copper <1 ppm <1 ppm <1 ppm Amino acid (mg/ml) PRE POST Iron <2 ppm <2 ppm <2 ppm ALA (A) 0.24 ± 0.00 0.27 ± 0.00 0.21 ± 0.00 0.20 ± 0.00 Table 4: Amino acid concentration of three production lots before and after processing. Data is mean ± SD (n = 3). Magnesium 0.003% 0.003% 0.003% ARG (R) Manganese <0.5 ppm <0.5 ppm <0.5 ppm ASP (D) 0.68 ± 0.01 0.68 ± 0.02 Phosphorus 0.01% 0.01% 0.01% GLU (E) 1.09 ± 0.03 1.32 ± 0.09 0.16 ± 0.00 0.17 ± 0.01 Potassium 0.33% 0.04% 0.03% GLY (G) Sodium 0.01% 0.01% 0.01% HIS (H) 0.16 ± 0.00 0.15 ± 0.00 Zinc 1 ppm 1 ppm 1 ppm ILE (I) 0.38 ± 0.01 0.39 ± 0.01 Cholesterol 0.01% NA 0.01% LEU (L) 0.92 ± 0.03 0.99 ± 0.04 Saturated Fat 1.30% NA 1.16% LYS (K) 0.45 ± 0.01 0.36 ± 0.01 MET (M) 0.10 ± 0.00 0.11 ± 0.00 PHE (F) 0.31 ± 0.01 0.29 ± 0.02 PRO (P) 0.60 ± 0.01 0.66 ± 0.01 SER (S) 0.32 ± 0.00 0.34 ± 0.00 THR (T) 0.32 ± 0.01 0.34 ± 0.00 TYR (Y) 0.25 ± 0.01 0.25 ± 0.01 VAL (V) 0.39 ± 0.01 0.41 ± 0.01 Table 3: Nutritional comparison of three production lots between pre- and post-processing. Pre-processing samples are randomly picked from frozen samples, post-processing data are from Eurofins reports. Data is mean ± SD (n ≥ 3). Samples Fat % Protein % Lactose % Energy kcal/dL PRE 3.07 ± 0.13 1.06 ± 0.03 6.54 ± 0.15 57.67 ± 1.53 POST 3.01 ± 0.26 1.03 ± 0.03 6.20 ± 0.26 58.00 ± 0.00 The protein quality and quantity of human milk are an important factor for infant growth and development (Zhang et al., 2013) and the amino acid proile is recognized as an indicator of the overall protein quality (Raiten et al., 1998). Amino acids are required for protein synthesis facilitate the uptake of other nutrients and also enhance the infants’ immune system against potential pathogenic bacteria, viruses and yeasts (Carratu, 2004; Ogechi and Irene, 2013). The complete characterization and quantiication of proteins especially amino acids in human milk serves as an appropriate nutritional guide for understanding and deining an infant’s protein and amino acid requirements. Therefore, amino acid levels in pooled samples of three production lots were measured before and after processing (Table 4). Minor increases was found in Alanine (0.24 ± 0.00 vs 0.27 ± 0.00; p < 0.001) and a decrease of 20% was found in Lysine, which is an important biological indicator of the nutritional value of milk (0.45 ± 0.01 vs 0.36 ± 0.01; p < 0.001). In a previous evaluation of amino acid stability during manipulation of human breast milk, Silverstre et al. (2006) observed a signiicant 30% decrease in lysine content after Holder Pasteurization. Overall, our processing only had a minor effect on the total amino acid levels in pooled donor breast milk (Table 4). Plasma glutamine levels fall during critical illness or following major surgery and glutamine deiciency may limit tissue recovery in these situations (Newsholme, 2001). The retention of glutamate from n neonatal INTENSIVE CARE Vol. 28 No. 2 Spring 2015 Signiicant diferences between pre- and post-processing samples were labeled in bold. A p value ≤ 0.001 was considered signiicant. Table 5: Nutritional comparisons between original processed samples and samples been stored at room temperature for 5 to 7 months. Data is mean ± SD (n = 4). Samples Protein % Fat % Lactose % Energy kcal/dL Processed samples 1.04 ± 0.00 3.08 ± 0.00 6.19 ± 0.00 58.00 ± 0.82 5-7 months later 1.06 ± 0.00 3.03 ± 0.00 6.51 ± 0.00 57.75 ± 2.87 Nutrients were stable over time There is a concern about the milks stability over time. Nutritional composition of four different production lots was analyzed after production and after being stored at room temperature for 5 to 7 months. Protein and fat were both stable over time and a slight increase in lactose was observed (Table 5). Nutritional composition of the processed pooled donor milk is not affected by storage at room temperature and is ready for use when needed. To our knowledge, our data demonstrate that our product is the only room temperature stable donated human breast milk in the world. Traditionally, donor breast milk is stored frozen and must be defrosted to use, which allows more time for microorganisms to grow and results in a delay before feeding. Room temperature stable milk is more convenient for the NICU since no defrosting is needed. 21 Conclusion Human breast milk has the optimal levels of many different nutrients for neonates although addition of protein fortiication is needed for preterm babies weighing less than 1,500 g, as recommended by the American Academy of Pediatrics. Mother’s own milk remains the optimum feeding choice, followed by donor breast milk and as a last resort, infant formula (Akinbi et al., 2010). Breast milk from individual mothers differs in composition with each pumping and among different mothers, but the nutritional components of multiple lots of pooled breast milk showed consistency over time. Although concerns have been expressed that processing human breast milk could potentially affect or alter nutritional components of the breast milk, our results show that little to no change occurs after processing. In addition, the concentrations of amino acids in breast milk is consistent before and after processing, with one exception, lysine, which was decreased by 20%. Though mother’s own milk is the best nutritional source for nutrition for infants, there is consensus in the medical community that donor human breast milk is a better alternative than formula. Our pasteurization and processing method does not affect the nutritional composition of the milk. Donor breast milk provides the necessary nutritional food that is needed for infants, although protein and manual fortiication is required for very low birth weight preterm infants. References • Akinbi H, Meinzen-Derr J, Auer C, Ma Y, Pullum D, Kusano R, Reszka KJ, Zimmerly K. 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