Human milk oligosaccharides (HMOs) have been positively associated with child neurodevelopment in some cohort studies. However, there is a lack of consistency in the association between HMOs and benefits to infants’ brains. Moreover, the quantification methods for HMOs have not yet been standardized. In this study, we developed a quantification method for evaluating eight HMOs (2′‐fucosyllactose [2′‐FL], 3′‐fucosyllactose [3′‐FL], 3′‐sialyllactose [3′‐SL], 6′‐sialyllactose [6′‐SL], lactosialyltetrasaccharide a [LSTa], lactosialyltetrasaccharide b [LSTb], lactosialyltetrasaccharide c [LSTc], and disialyllacto‐N‐tetraose [DSLNT]) in breast milk. After validating the method, we applied it to 1‐month breast milk samples ( n = 150) from the Tohoku Medical Megabank Project Birth and Three‐Generation Cohort Study to assess HMO profiles in breast milk and their possible association with changes in head circumference z‐score (ΔHCZ) and neurodevelopmental scores of children (as measured by the Ages and Stages Questionnaire, Third Edition). The validation demonstrated that the method had relative standard deviation ≤ 12.7% of precision and 79.5–110.9% of accuracy. Using this method, eight HMO levels (2′‐FL, 0–4.74 mg/mL; 3′‐FL, 0.02–1.52 mg/mL; 3′‐SL, 0.07–0.32 mg/mL; 6′‐SL, 0.01–0.70 mg/mL; LSTa, 0.002–0.043 mg/mL; LSTb, 0.02–0.31 mg/mL; LSTc, 0.001–0.47 mg/mL; and DSLNT, 0.09–0.71 mg/mL [min–max, all participants]) and the ratio of low secretors (16.0%) in the Japanese cohort were obtained. The obtained HMO levels in breast milk were subjected to multivariate analysis to screen for HMOs showing a positive association with ΔHCZ and neurodevelopmental scores. The results proposed that ΔHCZ was positively associated with LSTb and 2′‐FL levels, whereas neurodevelopmental scores were positively associated with 2′‐FL levels (among all participants) and 3′‐SL and DSLNT levels (among secretor participants). This study showed that the developed method provides HMO profiles in Japanese breast milk, as well as additional information on the associations between specific HMOs and neurodevelopment, reinforcing the sum of evidence for the role of HMOs in the brain.

The binding ratio of palmitic acid (PA) at the sn-2 position of triacylglycerols in infant formulas is lower than that in breast milk, resulting in higher levels of fecal PA. Even if the ratio is increased to 40–50%, fecal PA levels in formula-fed infants remain higher than those in breast–fed infants. In Japan, infant formulas with 50% or more of PA bound to sn-2 (high sn-2 PA milk) are commercially available; however, their effects on PA excretion have not been investigated. Therefore, this observational study aimed to preliminarily evaluate whether the feeding volume of high sn-2 PA milk is significantly associated with fecal total/soaped PA levels in newborns. Infant formulas were classified as high (≥50% of PA bound to sn-2) or low sn-2 (<50%) PA milk. Associations between feeding volume of high or low sn-2 PA milk and fecal PA levels were evaluated using multiple regression analysis models. The results showed that the feeding volume of low sn-2 PA milk was positively associated with fecal total/soaped PA levels, while there was no significant association between those of high sn-2 PA milk and fecal total/soaped PA levels. Our preliminary study suggests that high sn-2 PA milk may reduce increased fecal PA levels in formula-fed newborns.

(1) Background: Breast milk is the only source of nutrition for breastfed infants, but few studies have examined the relationship between breast milk micronutrients and infant neurodevelopmental outcome in exclusively breastfed infants. The aim of this study was to characterize the association between nicotinamide adenine dinucleotide (NAD)-related compounds in the breast milk of Japanese subjects and infant neurodevelopmental outcome. (2) Methods: A total of 150 mother–child pairs were randomly selected from the three-generation cohort of the Tohoku Medical Megabank in Japan. Infants were exclusively breastfed for up to 6 months. Breast milk was collected at 1 month postpartum, and the quantity of NAD-related substances in the breast milk was quantified. The mothers also completed developmental questionnaires at 6, 12, and 24 months. The relationship between the concentration of NAD-related substances in breast milk and developmental indicators was evaluated via ordinal logistic regression analysis. (3) Results: Nicotinamide mononucleotide (NMN) was quantified as the major NAD precursor in breast milk. The median amount of NMN in the breast milk was 9.2 μM. The NMN concentration in breast milk was the only NAD-related substance in breast milk that showed a significant positive correlation with neurodevelopmental outcome in infants at 24 months. (4) Conclusions: The results suggest that NMN in human milk may be an important nutrient for early childhood development.

The anti-rotavirus components in breast milk and infant formulas play an important role in the prevention of rotavirus infection. The present study examined whether the levels of phospholipids and bovine lactadherin, which are the major components and proteins of the milk fat globule membrane complex, are useful indices of the anti-rotavirus activity of dairy ingredients used in infant formulas. We compared the anti-rotavirus activity of 2 types of dairy ingredients enriched in the milk fat globule membrane complex: high-fat whey protein concentrate (high-fat WPC) and butter milk powder (BMP), using 50% inhibition concentration (IC₅₀) and linear inhibition activity to determine levels of solid contents, total proteins, phospholipids, and bovine lactadherin. Here, we developed a quantification method using full-length isotope-labeled proteins to measure bovine lactadherin levels in these dairy ingredients. The evaluation of anti-rotavirus activity showed that the difference in IC₅₀ was the smallest when the 2 dairy ingredients were compared at the bovine lactadherin level, among other indices in this study. Additionally, no significant difference was observed between the inhibition linearity of 2 dairy ingredients when evaluating only bovine lactadherin levels. These results indicated that the level of bovine lactadherin was more strongly associated with anti-rotavirus activity than the level of phospholipids. Our results suggest that bovine lactadherin levels can be used to estimate the anti-rotavirus activity of dairy ingredients and can be a criterion used in selecting ingredients for infant formulas.

Lactadherin is a major protein in the milk fat globule membrane (MFGM) and is associated with anti-rotaviral effect. Although lactadherin level in breast milk is generally quantified using enzyme-linked immunosorbent assay (ELISA), the many contaminants in the milk samples raise concerns of low accuracy. The aim of this study was to develop a high accuracy method of absolute quantification for breast milk lactadherin using a proteomics-based technique. The developed method uses a full-length expressed stable isotope-labelled protein from the wheat germ cell-free expression system with in-gel digestion and showed much higher accuracy (97.0%–109.9%,) than ELISA (27.2%–31.6%). The developed method revealed lactadherin levels in Japanese breast milk (1 month of postpartum 7.2 [2.2–25.7] mg/100 ml, 6 months of postpartum: 3.8 [1.2–22.8] mg/100 ml). This study has shown that a proteomics approach using a full-length expressed stable isotope-labelled protein with in-gel digestion achieves high accuracy quantification for breast milk lactadherin.