A method for standardizing the fat content of human milk for use in the neonatal intensive care unit
© Czank et al. 2009
Received: 02 January 2009
Accepted: 16 April 2009
Published: 16 April 2009
Accurately targeting the nutritional needs of the early preterm infant is challenging when human milk is used due to the natural variation in energy composition. The purpose of this study was to develop and evaluate a simple method for reducing the variation in fat and energy content of human milk prior to fortification such that the infant receives a diet of known composition.
Milk was centrifuged at low speed to concentrate the fat into a cream layer and a predetermined volume of skim milk is removed to meet a specific fat concentration. The fat layer is then resuspended to produce reconstituted milk of a specified standard fat content.
Using this method it was possible to reduce the coefficient of variation in fat content of six different samples of donor human milk from 19.3% to 2.6%. As fat globule size may be associated with fat absorption, the effect that centrifugation and resuspension had on human milk fat globule distribution was assessed by laser diffraction particle sizing. No difference in the particle distribution of the treated and untreated human milk was observed.
This method is accurate and simple, allowing for integration alongside current milk bank and NICU practices for use with both donor human milk and mother's own milk.
The benefits of using mother's own milk and donor human milk for premature and sick infants in the neonatal intensive care unit (NICU) are well known [1, 2]. In particular, the use of human milk in the NICU is associated with decreasing the likelihood of infection and in turn reducing the length of stay in hospital and associated costs [3, 4]. Unfortunately, human milk is not adequate to meet the nutritional needs of the early premature infant  and it is common practice to fortify the human milk prior to enteral feeding . Fortification provides essential vitamins and minerals at necessary levels not ordinarily found in human milk, and is especially required to meet the protein and energy needs vital for adequate growth. Current recommendations of reasonable nutrient intakes state that protein:energy ratios of between 2.5–3.4 g protein/100 kcal of energy are required for extremely low birth weight (ELBW) infants and 2.6–3.8 g protein/100 kcal for very low birth weight (VLBW) infants . However, given that the energy content of human milk varies widely , the desired protein:energy ratio may not be met, because the pre-fortification energy level of the human milk has not been standardized.
The total energy content of breast milk can be considered as the sum of the individual energy contributing components. Nutritionally, fat, lactose and protein are the most abundant energy sources contributing 9, 4 and 4 kcal/g (37.7, 16.7, 16,7 kJ/g) respectively . Fat is the most variable component of human milk (40 ± 16 g/l , Coefficient of Variation (CV):40%) compared to lactose (63 ± 2 g/l, (CV):3.1%) and protein (9.2 ± 1.8 g/l , CV:19%) and varies between mothers, throughout the day and during a breast expression . Often assumed human milk energy and protein content are used, which in turn may result in either a nutritional deficit once fortified or, conversely, a nutritional excess. The consequences of either under- or over- nutrition during this critical period of developmental programming may predispose the infant to a range of chronic disease states later in life [13–19].
As fat is the most variable nutritional component and contributes over half of the energy to breast milk , adjustment of the fat content to a specified level is a prerequisite to providing fortified human milk of a known energy content to meet the protein:energy needs of the preterm infant. The method described here allows for standardizing the energy content of human milk prior to fortification, such that all infants will receive a standard level of energy from breast milk.
Samples were obtained from a store of breast milk donated to the Perron Rotary Expressed Milk Bank (PREM Bank), Subiaco, Western Australia. Mothers had given prior consent for their milk to be used in research. All samples were collected by the mother and immediately frozen prior to transportation to the milk bank and research laboratory.
Quantitation of fat content
Samples of breast milk were thawed and 30 to 50 ml portions aliquoted into vessels. Skim milk and cream were separated by centrifugation at either 4°C or 10°C at a relative centrifugal force (RCF) and time needed to give a range between 125 to 12500 g.min (eg: 125 × g for 1 minute to 2500 × g for 5 minutes, respectively).
Skim milk volume adjustment and resuspension of breast milk
Milk fat globule size distribution
Freshly expressed breast milk from a term mother was aliquoted into sterile 5 ml containers and either frozen at -20°C or stored at 4°C overnight. Frozen milk was then thawed and both the thawed and milk stored at 4°C mixed and 1.0 ml samples of mixed milk taken prior to centrifugation. Milk was then centrifuged at the 3750 g.min at 4°C and the cream layer resuspended and another 1.0 ml sample taken from the reconstituted milk. Particle size was determined using a Mastersizer 2000 fitted with Hydro SM sample dispersion system (Malvern Instruments). Absorbance was adjusted to meet a target weighted residual of 1%, a dispersant refractive index of 1.33 was used and sample added to the dispersion unit until an obscuration target of 10–15% was achieved. Averaged data from ten repeated scans was analyzed with Dispersion Technology Software V4.02 (Malvern Instruments).
All values were calculated in Microsoft Excel 2003 and expressed as mean ± standard deviation unless otherwise stated.
Optimal relative centrifugal force for readily resuspending the cream layer
Fat content of skim milk after centrifugation
Development of an equation for skim milk volume adjustment to standardize breast milk fat content
V 1 = Initial volume of milk (ml)
V 2 = Volume of skim milk to be adjusted (ml)
V 3 = Final volume of milk after adjustment (ml)
C 1 = Initial fat content (g/l)
C 2 = Content of fat in skim milk to be added or removed (g/l)
C 3 = Desired fat content (g/l)
Total grams of fat (F T ) is defined as a function of volume and content
Similarly, the total grams of fat in the skim milk (F S ) is defined as:
The total grams of fat in the final adjusted volume (F F ) is defined as:
Proof of concept
Initial fat content, volume of skim milk adjusted and final fat content of milk from six mothers to which the fat standardization applied
Human milk sample
Initial fat content (g/l)
Volume of skim milk adjusted (ml)a
Final fat content of reconstituted milk (g/l)
Percentage difference from target fat content
Breast milk fat globule distribution in reconstituted breast milk after centrifugation and fat resuspension
Discussion and conclusion
The basis of this method was to use low speed centrifugation to concentrate the fat globules into a cream layer, followed by the adjustment of the underlying skim milk and then resuspension of the cream layer. In order to accurately adjust the fat content of whole milk to a specified fat amount, an equation was developed for calculating the amount of skim milk to be either removed or added. Given the relatively low RCF used for this procedure it is likely that some fat would remain in the skim and it was therefore necessary to account for the amount of fat remaining in the skim when performing this calculation.
The samples chosen for the proof of concept studies had relatively low variation of 19.3% between samples, which was reduced to 2.6% by employing the method described. For these studies, an assumed value skim milk fat content of 17 g/l was used, which was derived from the average of 72 skim milk samples centrifuged at optimal RCF. Measurement of the fat content of the skim milk would decrease the variation between samples, but was not considered to be clinically important for either the milk bank or NICU. Nonetheless, assuming a skim milk fat content did result in a large decrease in the variability of fat between samples and contributed to less than 2.2% error in the final fat content of the reconstituted milk.
The effect that centrifugation and resuspension of the milk had on the fat globule size distribution was also investigated. Low temperatures are recommended for preventing microbial growth in human milk , however it is not known how low temperatures affect the solidity of the cream layer and the ease of which the fat globules can be resuspended. Centrifuging at temperatures between 4°C and 10°C did not appear to affect the resuspension process. Subsequently, later centrifugation procedures were performed at 4°C to minimize any microbial growth. Fat globule size also may be important to infant gastric emptying and ability to absorb fat from the gut .
Centrifugation results in concentration of the fat globules into a dense cream layer at the top of the vessel, leading to the possibility of coalescence occurring and in turn altering the milk fat globule distribution. To test this hypothesis, samples of fresh and frozen milk, before and after manipulation were analyzed using laser diffraction particle sizing, a technology that has been succesfully used for studying the particle distribution of bovine milk . Results were similar to that of previous findings for human milk [26, 27] which employed more classical techniques such as coulter counters. Centrifugation and resuspension of milk did not alter the fat globule distribution, suggesting that coalescence does not occur under these conditions. The effect of centrifugation on milk proteins would be insignificant because much higher centrifugal forces are required for casein sedimentation  and the separation of small molecules (eg. lactose, oligosaccharides, peptides, hormones) from complex biological solutions cannot be achieved using centrifugation alone.
The method presented here has the potential for incorporation with current human milk banking protocols. While this study used a spectrophotometric assay for quantifying fat content of human milk, it was also demonstrated that the results from the simpler and quicker creamotocrit method correlated well with those derived from the more advanced spectrophotometric method. It is unlikely that most milk banks or NICUs would have access to a spectrophotometer, and the creamotocrit is an accurate and cost-efficient alternative for determining fat content of human milk. Using a creamotocrit it would be possible to determine milk fat content in the NICU or milk bank, standardize the fat content of the milk prior to pasteurization, followed by appropriate fortification.
The validation of this method involved using assumed values of protein and lactose. Human milk composition is challenging to quantify outside the laboratory environment. Consequently, the concentrations of nutritional components in human milk are often assumed, contributing to inaccurate nutrition of the preterm infant. In recent years, human milk analysis equipment such as the MilkoScan (FOSS International) have become available that simultaneously determines protein, lactose and fat content in human milk. The equation presented here can be expanded to include these components in relation to total energy of the milk. Ideally all the variables including fat, protein, lactose and specified energy content can be inputted into the expanded equation and in combination with current fortification regimes, a standardized fortified human milk of known energy and protein content can be prepared that precisely meets the infant's nutritional recommendations. The method is also versatile, allowing for batch processing by employing a large capacity centrifuge or alternatively, for prescriptive use for standardizing the fat content of donor milk or mother's own milk to meet the needs of a particular infant. Finally, the simplicity of this method ensures that with minimal training, non-laboratory trained staff can utilize it to standardize the energy content of breast milk for use in the NICU.
The authors would like to acknowledge the technical assistance of Mr. Jorge Martinez. This study was funded by the Rotary Clubs of Thornlie and Belmont (Western Australia), The Womens and Infant Research Foundation (Western Australia), The Perron Charitable Trust and Medela AG (Switzerland).
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