Impact of Freezing Time on Dornic Acidity in Three Types of Milk: Raw Donor Milk,Mother's Own Milk,and Pasteurized Donor Milk

Introduction

During frozen storage, the physical-chemical properties of breastmilk are modified.^1–3 Dornic acidity is a measure of the acidity of human milk, and it is used as an indirect method for measuring milk quality and bacterial contamination.^4,5 The overall acidity of milk is influenced by its primary acidity, which depends on its casein, organic acid, and phosphate content, and its developed acidity, which is caused by the release of free fatty acids and lactic acid. Fatty acids are cleaved from triglycerides by human milk lipases, and lactic acid is produced by lactose-fermenting bacteria.^4,5

This study aimed to assess changes in Dornic acidity during freezing storage at −20°C in three different types of human milk: raw donor milk, pasteurized donor milk, and mother's own raw milk.

Methods

Three types of human milk were obtained: raw and pasteurized donor milk and mother's own raw milk. The main differences between these types of milk were that only donors were instructed on hygiene measures to be taken before expressing their milk, and how to maintain the cold chain. Raw milk was not frozen or pasteurized. Pasteurized donor milk was pasteurized according to the Holder method.

Each sample was divided into seven aliquots. The first one was analyzed at time 0. The rest were frozen and analyzed at 1, 2, 3, and 4 weeks and at 2 and 3 months.

Dornic acidity was measured following the Brazilian milk bank guidelines. ⁵ A drop of 1% phenolphthalein in ethanol solution was added to 1 mL of milk as an indicator. Then, 0.01 mL of sodium hydroxide ([NaOH] N/9) was titrated until the sample changed from white to light pink, and this change was maintained. Each 0.01 mL needed for the sample to change color accounted for 1° of Dornic acidity. The higher the number of Dornic degrees, the higher the acidity.

The differences at each time point compared with baseline for each type of milk were assessed with Student's t test for paired samples or Wilcoxon's signed-rank test, as appropriate. The normality of the data distribution was tested with the Shapiro–Wilk test. The changes in acidity values during the course of the study were compared using an ordinal model for repeated-measures data.

Results

Forty-three samples of raw donor milk were analyzed. The median Dornic acidity at time 0 was 3°D (interquartile range [IQR] 2–3°D). An increase was found starting in the first week, and was statistically significant (p < 0.05) from the second week until the third month when the median acidity was 5°D (IQR 3–7°D).

In the 16 samples of own mother's raw milk, the median acidity at the start was 3°D (IQR 2–3°D), with an increase that became statistical significant (p < 0.05) after the third week and was maintained throughout the 3 months. The median acidity at the end of the study was 7°D (IQR 4–8°D). Forty samples of pasteurized donor milk were analyzed. The median acidity at time 0 was 3°D (IQR 2–3°D), and at the end of the test period it was 2°D (IQR 2–3°D). This reduction in acidity over the 3 months of storage was statistically significant (p < 0.05) beginning from the first week.

A comparison among the three types of milk revealed that from the first week, there were statistically significant differences between pasteurized donor milk and the raw milk samples (p < 0.01). A comparison between the two types of raw milk yielded no statistically significant differences (p = 0.77; Fig. 1).

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FIG. 1.

Degree of Dornic acidity over freezing time in three types of milk: mother's own raw milk, raw donor milk, and pasteurized donor milk.

Discussion

This study demonstrates that the Dornic acidity of raw milk, whether it is mother's own milk or that of a donor, significantly increases with freezing time. However, the Dornic acidity of pasteurized milk remains stable. Prior studies^2,3 have already described an increase in milk acidity during refrigerated or frozen storage.

Acidic milk is lower in quality for three reasons. First, the bioavailability of calcium and phosphorus is decreased, as these elements bind to casein micelles. ⁶ Milk caseins are proteins that precipitate in an acid medium, destabilizing the micelles and releasing calcium and phosphorus. ⁶ Second, the activity of lipoprotein lipase and gastrin is reduced in acid milk. ⁶ Finally, acidic milk has higher osmotic concentration, which can potentially cause a delay in gastric emptying.

Furthermore, in vitro studies have shown a decrease in the amount of white cells and lymphocyte function and activity impairment in acidic breastmilk. ⁶

There is a positive correlation between Dornic acidity and bacterial content in breastmilk.^4,5 Freshly expressed breastmilk is <4°D. If after expression the cold chain is not maintained, fermenting bacteria can overgrow, and the breastmilk becomes more acidic. During frozen storage, milk bacteria remain latent. ⁷ However, lipoprotein lipase maintains its activity, apparently even at −20°C, and over 3 months, this activity increases. ⁸ Therefore, the rise in Dornic acidity in frozen milk is likely due to an increase in free fatty acids secondary to triglyceride hydrolysis, rather than to the production of lactic acid by fermenting bacteria.^7,8

Until recently, lipids, and specially triglycerides, have been reported to have an antimicrobial function in breastmilk. However, some recent in vitro studies have shown how stored milk can become cytotoxic secondary to free fatty acids damaging the membranes of the intestinal epithelial cells. ⁹

Holder pasteurization destroys human milk lipases, leading to a total loss of enzymatic activity,^8–10 which would explain why acidity did not increase in pasteurized milk. A slight decrease in Dornic acidity with freezing time was found, although this was not clinically significant.

One limitation of this study is that Dornic acidity is only an indirect method of estimating milk quality. However, the results published to date that estimate milk quality using other methods are consistent with those found in this study.^1,2

This study shows how unpasteurized milk deteriorates as freezing time increases. In contrast, pasteurized milk keeps better, as its degree of acidity does not increase over time. The clinical significance of these findings requires further study.