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The effects of acidification on human milk’s cellular and nutritional content



Fortification of human milk for preterm infants is necessary and a common newborn intensive care practice. Currently, acidified human milk as part of a human milk fortifier is being fed to preterm infants. However, there are little data on the acidification effects on mother’s milk. The aim of this study is to determine the effects of acidification on human milk’s cellular and nutritional composition.

Study Design:

One hundred milk samples were collected from eight mothers who had infants in the neonatal intensive care unit. All milk samples were frozen at 4 °C. The frozen samples were thawed and divided into two equal aliquots, control and acidified. The control milk sample had its pH determined while the other sample was acidified to pH 4.5. Each milk sample was examined for pH, white cells, total protein, creamatocrit, lipase activity and free fatty acids.


Mean pH of the human milk control was 6.8±0.1 (M±s.d.) with the acidified milk at 4.5±0.1. Acidification caused a 76% decrease in white cells, a 56% decrease in lipase activity and a 14% decrease in the total protein but a 36% increase in the creamatocrit.


Acidification of human milk causes significant changes of the milk’s cellular and nutritional components that may not be beneficial to preterm infants.


The American Academy of Pediatrics has supported the feeding of human milk for all infants. Its benefits include nutritional, immunological, developmental, psychological, social and economical advantages over formula feedings.1 However, the preterm infant has higher nutritional needs than the term infant. The nutritional content of human milk alone is not sufficient to meet the preterm infant’s needs.2, 3 Nutrient fortification with additional energy, protein, minerals and vitamins of human milk is an acceptable and necessary method for feeding the preterm infant.

Human milk fortifiers have been either liquid or powder that are added to the milk. However, the Centers for Disease Control have recommended that all formulas used with high-risk infants be sterile because of powder formulas being contaminated and causing infections.4 This recommendation has been extended to human milk fortifiers but not to mother’s milk. Liquid formulations are processed to be sterile but powder formulations are not.4 One traditional method to sterilize the liquid formulation was to acidify the product. A commercially available human milk fortifier has been acidified to pH 4.3 but has also caused the milk to be acidified to pH 4.7 ( The effects of acidification on the biological components and nutrients of human milk are unknown. This study aims to evaluate the effects of acidification on the milk’s cellular and nutritional content.


Milk samples

One hundred milk samples were collected from eight mothers in the neonatal intensive care unit and frozen at 4 °C. Milk was collected from day 5 to day 32 of lactation. Almost all the milk was frozen within 24 h after collection. The milk samples were thawed and divided into two aliquots. One aliquot served as the control, and the other was adjusted to pH of 4.5 using <1 ml of citric acid. Each control sample was compared with its acidified counterpart. Our Institutional Review Board reviewed and exempted this study from informed consent.

White cells

A fine smear was prepared using 1 ml of milk by spreading it over a glass slide. The slide was air dried and then dipped in xylene to remove the fat globules. The slide was then stained using Gill Hematoxylin #3 (Richard-Allan Scientific, Model #72604, Kalamazoo, MI, USA). Cells stained with a blue nucleus were counted under a microscope.

Total protein

Total protein concentrations were determined using Thermo Scientific (Rockford, IL, USA) Micro BCA protein assay kit, which uses the Lowry method. This method uses bicinchoninic acid to detect Cu+1 formed from the reduction of Cu 2+ by protein in an alkaline environment.5


Creamatocrit of the human milk samples was determined using the hematocrit centrifuge.6 Each sample was run in triplicates and the results averaged.


Lipase concentrations were measured by using BioAssay QuantiChrom Lipase Assay Kit (Hayward CA, USA) using BALB-DTNB method. This method measured both lipoprotein and bile-salt-stimulated lipase activity. Sulfhydryl groups formed from the lipase cleavage of dimercaptopropanol tributyrate react with 5,5′-dithiobis(2-nitrobenzoic acid) to form a yellow product. The absorbances were measured using a 96-well plate reader and lipase concentrations were calculated.

Free fatty acid

Free fatty acid concentrations were measured by using BioAssay EnzyChrom Free Fatty Acid Assay Kit. Free fatty acids were enzymatically converted to H2O2. The H2O2 reacted with a dye to form a pink product. Colorimetric determination with a 96-well plate allowed reader for calculation of the free fatty acid concentrations.


After acidification, the white cell count decreased a mean of 76%, the total protein decreased 14% and lipase activity decreased 56% but the creamatocrit increased 36% compared with controls. There were no changes in free fatty acids Table 1.

Table 1 The effects of acidification on HM contents (mean±s.d.)


Early in the 1900, acidification of infant formula feedings was promoted to improve the digestion of cows’ milk. The process was to reduce bacterial infection. With the improvement of infant formula processing and sterilization, acidification became unnecessary.7 However, the use of acidification of a human milk fortifier has been developed to assist with sterilization of the product. But this fortifier also acidifies the milk itself.

From our study, we found that acidification of human milk caused cellular changes. The white cells in the acidified human milk decreased. We speculate that the white cells were lysed during the acidification process. Also, it has been reported that an acid pH environment can impair lymphocyte function and activity.8 Whether cellular in vitro changes in the milk may affect the infant’s host defense system and increase the incidence of infection is unknown.

Changing the human milk pH to 4.5 renders a non-physiological feeding to a preterm infant. Mother’s milk has been reported to range from pH 7 to 7.4 but never to pH 4.5. The colostrum or the first milk produced during the early days of lactation is alkalotic at pH 7.45. Then the pH of the milk remains between 7.0 and 7.1 until 3 months postpartum. Later, the pH increases to 7.4 by 10 months.9 There are no published studies in peer-reviewed journals on the effect of acidified human milk on preterm infant’s growth and acid–base status. A preliminary report in abstract form of the liquid acidified fortifier causing growth issues and acidosis compared with a powder control fortifier in preterm infants has been reported.10 Though the liquid acidified fortifier had more protein than the control fortifier, there were essentially no differences in growth. We speculate that the growth issue with the liquid acidified fortifier was due to the reported acidosis. The acidosis may have caused a negative nitrogen balance from the increased urinary ammonia and nitrogen excretion.11 Similarly, the use of acidified infant formulas with high protein on preterm infants has resulted in loss of weight, dehydration and metabolic acidosis.12, 13 In term infants, the effect of an acidic feeding has been reported to produce metabolic acidosis and was associated with the pH of the feeding.7

Acidification causes the milk’s lipase activity to be reduced. Human milk lipase helps convert the triglyceride into free fatty acids, which are important for fat digestion. Human milk contains two lipases, a lipoprotein lipase and a bile salt-activated lipase. The lipoprotein lipase is not stable at pH below 5.14 In our study, the pH of the milk was at 4.5 and we speculate that this lipase would not be functional at this lower pH. The preservation of the milk lipases is important for fat digestion and absorption in preterm infants.15

In our study, we found that the creamatocrit of human milk increased in the acidified milk and that there was a trend toward lowered free fatty acids. The creamatocrit measured mainly the triglycerides in human milk.16 Thus, in acidified human milk there were more triglycerides and less free fatty acids compared with controls. These findings of the creamatocrit and free fatty acids support the effect of decreased lipase activity in the acidified milk.

The total protein content of the acidified milk was decreased in our study. We speculate that the acid environment caused the casein proportion of the milk to denature and precipitate.17, 18 Whether acidification will affect other proteins and enzymes in human milk has yet to be determined.

Acidifying feedings to pH 4.5 for infants may not be beneficial for several reasons. First, the pH of the gastric contents in preterm infants is greater than 4 over 90% of the time.19 The ingested milk is able to buffer the gastric pH.20 The higher pH may be beneficial both in terms of preserving the bioactive components of the breast milk and reducing the acid-induced esophageal inflammation during the frequent reflux experienced by preterm infants. However, acidifying milk removes its buffering capacity. A non-buffered feeding may result in a lower than normal gastric pH, a decrease in the immunological components in breast milk and an increase for the risk of acid gastroesophageal reflux. Second, acidified feedings may cause less gastrin production as its secretion is related to the feeding’s pH; the more acidic the feeding will cause less gastrin production.21 Less gastrin production from acidified feedings may cause partial digestion of protein especially if the feedings are high in protein. Furthermore, we have shown that the acidifying mother’s milk caused a decrease in its lipase activity with probably less hydrolysis of the milk’s triglycerides to free fatty acids. Again, we speculate that this will cause partial fat malabsorption with less caloric intake for the infant and more acidic fecal excretion. More research is required to evaluate the clinical and physiological effects of acidic feedings on preterm infants.

In summary, acidification of mother’s milk caused decreases in white cells, lipase activity and the total protein but an increase in the creamatocrit. There were no changes in the free fatty acids level.


Acidification of human milk will decrease the milk’s cellular and nutritional content and may not be beneficial for the preterm infant.


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Correspondence to G M Chan.

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Erickson, T., Gill, G. & Chan, G. The effects of acidification on human milk’s cellular and nutritional content. J Perinatol 33, 371–373 (2013).

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  • acidification
  • human milk
  • total protein
  • lipase
  • creamatocrit
  • free fatty acids

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