Background: In neonatal units and human milk banks freezing breastmilk at less than –20°C is the choice for preserving it. Scientific evidence in relation to the loss of nutritional quality during freezing is rare. Our main aim in this study is to determine the effect of freezing time up to 3 months on the content of fat, total nitrogen, lactose, and energy. Our secondary aim is to assess whether ultrasonic homogenization of samples enables a more suitable reading of breastmilk macronutrients with a human milk analyzer (HMA) (MIRIS ^® , Uppsala, Sweden). Methods: Refrigerated breastmilk samples were collected. Each sample was divided into six pairs of aliquots. One pair was analyzed on day 0, and the remaining pairs were frozen and analyzed, one each at 7, 15, 30, 60, and 90 days later. For each pair, one aliquot was homogenized by stirring, and the other by applying ultrasound. Samples were analyzed with the HMA. Results: By 3 months from freezing with the two homogenization methods, we observed a relevant and significant decline in the concentration of fat and energy content. The modification of total nitrogen and lactose was not constant and of lower magnitude. The absolute concentration of all macronutrients and calories was greater with ultrasonic homogenization. Conclusions: After 3 months from freezing at –20°C, an important decrease in fat and caloric content is observed. Correct homogenization is fundamental for correct nutritional analysis.

Women of childbearing age in Western societies are increasingly adopting vegetarian diets. These women are sometimes rejected as milk donors, but little about the composition of their milk is known. The present study aimed to compare the intake, nutritional status, and nutritional composition of human milk from omnivore human milk donors (Donors) and vegetarian/vegan lactating mothers (Veg). Milk, blood, and urine samples from 92 Donors and 20 Veg were used to determine their fatty acid profiles, as well as vitamins and minerals. In a representative sample of both groups, we also determined the lipid class profile as a distribution of neutral and polar lipids, the molecular species of triacylglycerols, and the relative composition of phospholipids in their milk. A dietary assessment was conducted with a five-day dietary record (while considering the intake of supplements). We highlight the following results, expressed as the mean (SE), for the Veg vs. Donors: (1) Their docosahexaenoic acid (DHA) intake was 0.11 (0.03) vs. 0.38 (0.03) g/day; the plasma DHA was 0.37 (0.07) vs. 0.83 (0.06)%; and the milk DHA was 0.15 (0.04) vs. 0.33 (0.02)%. (2) Their milk B12 levels were 545.69 (20.49) vs. 482.89 (4.11) pM; 85% of the Veg reported taking B12 supplements (mean dose: 312.1 mcg/day); and the Veg group showed no differences with Donors in terms of total daily intake or plasma B12. (3) Their milk phosphatidylcholine levels were 26.88 (0.67) vs. 30.55 (1.10)%. (4) Their milk iodine levels were 126.42 (13.37) vs. 159.22 (5.13) mcg/L. In conclusion, the Vegs’ milk was shown to be different from the Donors’ milk, mainly due to its low DHA content, which is concerning. However, raising awareness and ensuring proper supplementation could bridge this gap, as has already been achieved for cobalamin.

The influence of the diet and nutritional status of milk donors on the nutritional composition of donor human milk (DHM) is unknown. The present study aimed to determine the nutritional profile of DHM and the associations between donors’ dietary intake and nutritional status and the micronutrient and lipid composition in DHM. For this purpose, 113 donors completed a food frequency questionnaire, provided a five-day weighed dietary record, and collected milk for five consecutive days. Nutrient determinations in donors’ erythrocytes, plasma, urine, and milk were performed. Multiple linear regressions were conducted for the evaluation of the associations. We highlight the following results: DHM docosahexaenoic acid (DHA) was positively associated with donors’ plasma DHA content and donors’ DHA intake (R² 0.45, p < 0.001). For every 1 g/day DHA intake, an increase of 0.38% in DHA content and 0.78% in total omega-3 content was observed in DHM (R² 0.29, p < 0.001). DHM saturated fatty acids were positively associated with erythrocyte dimethyl acetals, plasma stearic acid, trans fatty acids intake, and breastfeeding duration and negatively associated with erythrocyte margaroleic acid (R² 0.34, p < 0.01). DHM cholecalciferol was associated with plasma cholecalciferol levels and dairy intake (R² 0.57, p < 0.01). Other weaker associations were found for free thiamin, free riboflavin, pyridoxal, dehydroascorbic acid, and the lipid profile in DHM. In conclusion, the diet and nutritional status of donors influence the fatty acid profile and micronutrient content of DHM.

Substantial fat loss may occur during continuous feeding of human milk (HM). A decrease of fat loss has been described following homogenization. Well-established methods of homogenization of HM for routine use in the neonatal intensive care unit (NICU) would be desirable. We compared the loss of fat based on the use of 3 different methods for homogenizing thawed HM during continuous feeding. Sixteen frozen donor HM samples were thawed, homogenized with ultrasound and separated into 3 aliquots (“baseline agitation,” “hourly agitation,” and “ultrasound”), and then frozen for 48 hours. Aliquots were thawed again and a baseline agitation was applied. Subsequently, aliquots baseline agitation and hourly agitation were drawn into a syringe, while ultrasound was applied to aliquot ultrasound before it was drawn into a syringe. The syringes were loaded into a pump (2 mL/h; 4 hours). At hourly intervals the hourly agitation infusion was stopped, the syringe was disconnected and gently shaken. During infusion, samples from the 3 groups were collected hourly for analysis of fat and caloric content. The 3 groups of homogenization showed similar fat content at the beginning of the infusion. For fat, mean (SD) hourly changes of −0.03 (0.01), −0.09 (0.01), and −0.09 (0.01) g/dL were observed for the hourly agitation, baseline agitation, and ultrasound groups, respectively. The decrease was smaller for the hourly agitation group (P < .001). When thawed HM is continuously infused, a smaller fat loss is observed when syringes are agitated hourly versus when ultrasound or a baseline homogenization is used.