Mature Milk Research Articles

Analysis of human milk oligosaccharides from women with gestational diabetes mellitus

Human milk oligosaccharides (HMOs) are bioactive components which play an important role in infant health. HMO composition is vulnerable to changes of maternal conditions including lactation stages and maternal phenotypes. Pregnant diseases such as gestational diabetes mellitus (GDM) are commonly found in women during pragnancy, and may cause disorder in maternal physiological metabolism which is harmful to infants. Unfortunately, anlysis of oligosaccharides from women with GDM is limited. To address this issue, we analyzed HMO compositions and profiles in breast milk from women with GDM using an established 96-well plate permethylation platform and MALDI-TOF-MS. We enrolled 127 women with GDM, and investigated HMO abundances in colostrum, transition milk, and mature milk respectively. We found that GDM affected HMO compositions in breast milk, and the level of fucosylation became higher over the course of lactation for all the women with GDM. Interestingly, the relative abundances of fucosylated HMOs in different lactation stages were affected differentially by GDM, with the most pronounced effect in colostrum. In particular, the relative abundances of H3N1F1 and H3N1F2 sharply decreased over time, showing very low levels in late lactation. These differences in our findings need further investigation to develop optimal feeding for mothers with GDM.

Measurement of New Biomarkers of Immunity and Welfare in Colostrum and Milk of Pigs: Analytical Validation and Changes During Lactation.

Colostrum is a mammary secretion released from the time of farrowing to 36 h post-farrowing. After this time and during all the rest of lactation, the mammary secretion is considered milk. The objectives of this study were: (1) to perform an analytical validation in the colostrum and milk of sows of assays for four analytes related to immunity: total ADA (tADA) and its isoenzymes (ADA1 and ADA2), myeloperoxidase (Mpx), calprotectin, and calgranulin, and two analytes related to welfare: cortisol and alpha-amylase. (2) To evaluate the changes in these analytes during lactation (3) To assess the correlations between these new analytes, as well as with IgG and IgA. In the analytical validation, all the assays were precise and accurate. When changes during lactation were evaluated, the concentration of tADA and ADA2 was found to be higher in colostrum than in milk (p < 0.02), while the activity of Mpx was observed to be higher in mature milk than in colostrum (p < 0.03). Furthermore, cortisol and alpha-amylase activity were found to be higher in colostrum compared to mature milk (p < 0.04 and p < 0.0001, respectively). Regarding the relation between analytes, alpha-amylase showed a significant correlation with both IgG and IgA and calprotectin was correlated with calgranulin and Mpx. Further studies should be performed to elucidate the possible practical application of the analytes evaluated in this study as biomarkers of colostrum and milk in sows.

The skimmed milk proteome of dairy cows is affected by the stage of lactation and by supplementation with polyunsaturated fatty acids

The impact of nutritional modification to increase functional polyunsaturated fatty acids (PUFA), such as n-3 and n-6 fatty acids (FA) or conjugated linoleic acid (CLA), on milk proteome profile during early lactation remains largely unknown. We used an untargeted proteomics approach to investigate the impact of lactation day and PUFA supplementation on the proteome signature in skimmed milk over the course of early lactation. Sixteen Holstein dairy cows received abomasal infusion of saturated FA (CTRL) or a mixture of essential FA and CLA (EFA + CLA group) from − 63 to + 63 days relative to parturition. Using quantitative proteomics, 479 unique proteins were identified in skimmed milk at days 1, 28, and 63 postpartum. The top discriminating proteins between transition milk (day 1) and mature milk (days 28 and 63), including members of complements (i.e. C2 and C5), growth factor (TGFB2), lipoproteins (i.e. APOE and APOD), and chaperones (i.e. ST13 and CLU), are associated with calves’ immune system and gut development. The EFA + CLA supplementation moderately affected a few proteins associated with regulating mammary glands’ lipogenesis through the (re)assembly of lipoprotein particles, possibly under the PPAR signaling pathway. Collectively, skimmed milk proteome is dynamically regulated initially by cow’s metabolic and physiological changes and to a lesser extent by nutritional PUFA modifications.

Comparison of Whey N-Glycoproteins in Yak Colostrum and Mature Milk Based on 4D Label-Free Technology.

Whey proteins are N-glycosylated proteins that play important roles in a variety of biological processes including immune defense. However, the N-glycosylation of yak colostrum (YC) and mature milk (YM) whey proteins is unknown. Therefore, this study systematically compared and analyzed YC and YM whey N-glycoproteomes using the 4D label-free technique. We identified 162 glycoproteins, 222 glycosylated peptides, and 234 glycosylation sites in YC and YM, of which 59 glycoproteins were differentially expressed in YC and YM. According to gene ontology annotation and KEGG pathway metabolism analysis, the differentially expressed N-glycoproteins were highly enriched in "cell adhesion", "extracellular region", and "calcium binding", and were mainly involved in the extracellular matrix (ECM)-receptor interaction pathway. The immunity-related N-glycoproteins, such as platelet glycoprotein 4 (CD36) and polymeric immunoglobulin receptor (PIGR), were observed to be different between YC and YM. The results revealed the glycosylation sites, composition, and biological functions of YC and YM whey N-glycoprotein, which supplemented our understanding of the N-glycosylation of yak whey proteins.

Goat Milk Exosomes Ameliorate Ulcerative Colitis in Mice through Modulation of the Intestinal Barrier, Gut Microbiota, and Metabolites.

Goat milk is rich in a variety of nutrients that are important for intestinal health and disease prevention. However, the role of exosomes in goat milk remains to be elucidated. This study investigated for the first time the therapeutic efficacy and molecular underlying mechanisms of mature milk exosomes (M-exo) and goat colostrum exosomes (C-exo) on dextran sodium sulfate-induced ulcerative colitis (UC) in mice. The findings demonstrate that M-exo and C-exo significantly improved physiological indices, suppressed the secretion of proinflammatory cytokines, and diminished oxidative stress and apoptosis in UC mice. Moreover, C-exo and M-exo restored the intestinal barrier function, remodeled the gut microbiota, and improved metabolite composition in the feces of colitis mice. In conclusion, goat milk exosomes ameliorate UC in mice, which provides a basis for the development of functional food applications for the prevention and treatment of inflammatory bowel disease.

Pig Milk Exosome Packaging ssc-miR-22-3p Alleviates Pig Intestinal Epithelial Cell Injury and Inflammatory Response by Targeting MAPK14

Inflammatory diseases of the intestinal tract in piglets severely impair the economic performance of pig farms. Pig milk exosomes can encapsulate miRNAs which can then enter the piglet intestine to play an immunomodulatory role. Previously, we comparatively analyzed and identified exosomal miRNAs in the colostrum and mature milk of Bamei and Landrace pigs, and we screened for ssc-miR-22-3p, which is associated with inflammation and immune response; however, the role played by ssc-miR-22-3p in the immune response in IPEC-J2 cells is not yet clear. In this study, we first constructed a pig intestinal inflammatory response model using Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly (I:C)), and we investigated the role of ssc-miR-22-3p targeting MAPK14 in the regulation of LPS and Poly (I:C)-induced inflammatory injury in IPEC-J2 cells by RT-qPCR, cell counting kit-8 (CCK-8), EdU staining, lactate dehydrogenase (LDH) activity assay, and dual luciferase reporter gene assay. We successfully established LPS and Poly (I:C)-induced cell damage models in IPEC-J2 cells. The immune response of IPEC-J2 cells was stimulated by induction of IPEC-J2 cells at 10 μg/mL LPS and 20 μg/mL Poly (I:C) for 24 h. Overexpression of ssc-miR-22-3p decreased cytokine expression and promoted cell viability and proliferation. The functional enrichment analysis revealed that ssc-miR-22-3p targets genes enriched in the pathways of negative regulation of inflammatory response and bacterial invasion of epithelial cells. The validity of the binding site of ssc-miR-22-3p to MAPK14 was tested by a dual luciferase reporter gene. Pig milk exosome ssc-miR-22-3p promotes cell viability and proliferation by targeting MAPK14, and it alleviates LPS and Poly (I:C)-induced inflammatory responses in IPEC-J2 cells.

Proteomic and Transcriptomic Profiling Revealed Vital Molecular Events in the Transition from Goat Colostrum to Mature Milk.

As an important nutrient source in large areas of the world, goat milk is favored by more and more consumers; however, the composition, nutritional value, and regulation mechanism of goat milk are not fully characterized. Mammary gland development is as important as detailed annotation of protein composition to address the physiological and nutritional values of goat milk. In the present study, 4353 colostrum and mature goat milk proteins were identified. The abundance of 118 proteins was significantly different between colostrum and mature milk proteins. Our results indicate that the milk protein changes were associated with a network of mammary gene expression changes; importantly, the prime factors include enhanced mammary growth/development, decreased protein translation, attenuated protein folding, and lower lip/carbohydrate metabolism. The present study provides insights into the changes in mammary metabolisms during the transition from colostrum to mature milk, which can help deeply explore the difference and regulation mechanism of active milk protein in colostrum and mature milk and provide references for the identification and functional study of bioactive milk proteins in colostrum.

Study on Vitamin K Levels in Mature Milk of Chinese Lactating Mothers.

This study sought to understand the levels of vitamin K in the mature milk of Chinese lactating mothers, thereby providing a foundation for the development of appropriate intake (AI) of vitamin K for infants aged 0-5 months. Five hundred healthy lactating mothers were selected from the mature milk bank established by the Chinese Maternal and Infant Nutrition and Health Cohort by using a simple random sample procedure. Relevant information about lactating mothers and their infants was obtained by a questionnaire survey. Vitamin K1 and vitamin K2 (MK-4 and MK-7) in mature milk were determined by liquid chromatography-tandem mass spectrometry. The total concentration of vitamin K in mature milk was 4.50 (2.85-6.33) ng/mL. The concentrations of vitamin K1, vitamin K2, MK-4, and MK-7 were 2.81 (1.66-4.39) ng/mL, 1.37 (0.75-2.11) ng/mL, 1.20 (0.58-1.97) ng/mL, and 0.13 (0.08-0.19) ng/mL, respectively. The concentration of vitamin K1 was highest and the concentration of MK-7 was lowest. The concentrations of vitamin K2 and MK-4 in mature milk from the south were significantly higher than those in mature milk from the north. The total vitamin K, vitamin K2, and MK-4 concentrations in mature milk of lactating mothers residing in urban areas were higher than those in rural areas. There was a tendency for the concentration of total vitamin K and vitamin K1 to increase with the mother's age. Moreover, the concentration of MK-4 in mature milk was highest in summer, followed by spring and winter. The levels of vitamin K1 and MK-4 in mature milk were found to be affected by lactation stage; they were highest at 91-120 days and lowest at 31-60 days. Based on the concentration of vitamin K in mature milk found in this study, it is recommended that the appropriate intake of VK for Chinese infants aged 0-5 months is 3.49 μg/d (2.18 μg/d for VK1 and 1.06 μg/d for VK2).

Effects of gestational diabetes mellitus, parity and mode of delivery on polar lipids compositions of mature human milk

Polar lipids in human milk such as glycerophospholipids, sphingomyelin and plasmalogen play important role in growth and development of healthy infants. This work aims to investigate the effects of gestational diabetic mellitus (GDM), parity, delivery mode on the polar lipids’ compositions of human milk. A total of 56 mature human milk samples (1–6 months postpartum) were collected from breastfeeding mothers using electric breast pump from Ningbo, Zhejiang Province of China. Our results showed GDM and parity have limited effect on polar lipid content and composition. Delivery mode was found to significantly affect the polar lipids compositions in human milk with significantly higher total polar lipids in human milk of mothers who had cesarean delivery (938 nmol/mL) as compared to that of mothers who had vaginal delivery (740 nmol/mL). Discriminatory analysis shows human milk of mothers who have cesarean delivery demonstrated up-regulation of glycerophospholipids (GPL) and plasmalogen (PLG) esterified with unsaturated fatty acids (USFAs) [such as plasmenyl-ethanolamine 40:5 and 36:4].

Characterizing metabolome signature of colostrum, transition and mature milk of indigenous cows (Bos indicus) adapted to high altitude environment of Leh-Ladakh

This study has identified 46 metabolites in colostrum, transition milk and mature milk of unique indigenous high altitude adapted Ladakhi cows using 1D 1H 800 MHz NMR spectroscopy. The multivariate analysis revealed that UDP-galactose, UDP-glucose, citrate, creatine phosphate, myo-inositol, lactose, 2-oxoglutarate, valine, maltose, leucine, dimethylamine, and choline with high VIP scores could differentiate the colostrum, transition and mature milk in separate clusters. . Highly enriched metabolites in colostrum such as UDP-galactose, UDP-glucose play crucial roles in cell growth, differentiation, and defense responses. Similarly, the presence of branched chain amino acids in colostrum could be linked to mammary gland development, N-acetylglucosamine, N-acetyl carnitine, choline etc. in high concentration in colostrum l might be helping in growth and development of neonatal calves of Ladakhi cows under hypoxia environment. Overall, this study has helped to characterize the metabolomic signatures of milk/colostrum of Ladakhi cows adapted to high altitude and cold desert of Leh-Ladakh.

Immune-related miRNAs (181a, 146, 150) Expression in Colostrum and Mature Maternal Milk in normally and Caesarean Delivered Females

Immune-related miRNAs (181a, 146, 150) Expression in Colostrum and Mature Maternal Milk in normally and Caesarean Delivered Females

Comparative analysis of different methods for protein quantification in donated human milk.

Human milk is the best option for feeding newborns, especially premature infants. In the absence of breast milk, milk from a human milk bank can be a suitable alternative. However, the nutritional content of human milk may be insufficient to meet these high requirements and milk fortification is needed. To facilitate the implementation of simpler and faster analyzers in neonatal healthcare facilities, this study focuses on the concordance analysis of two different analyzers, one based on mid-infrared and the other on ultrasound, in comparison to the Bradford method for determining protein concentration in human milk. Mature milk samples from donor mothers were collected and pasteurized at the Human Milk Bank of Barcelona and protein quantification was performed using mid-infrared (MIRIS-HMA), ultrasound (MilkoScope Julie27), and the classical Bradford reference methods. The intraclass correlation coefficient (ICC) with 95% confidence interval and Bland-Altman plots were used to assess the agreement between methods. The mean protein concentration of 142 milk samples calculated using MIRIS-HMA, MilkoScope, and the Bradford assay were 1.38, 1.15, and 1.19 g/100 ml, respectively. The ICC was 0.70 for MIRIS-HMA vs. Bradford and 0.37 for MilkoScope vs. Bradford. MIRIS-HMA obtained a better agreement with the Bradford technique and is a promising method for developing new devices based on MIR transmission spectroscopy principles. This study confirms how MIRIS-HMA can be used to accurately calculate the protein concentration of human milk.

Does Colostrum Bovinum Supplementation Affect Swimming Performance in Endurance-Trained Males? A Randomized Placebo-Controlled Crossover Study.

Background/Objectives: Colostrum Bovinum (COL) is recognized for its unique composition and potential ergogenic and immunological benefits. Unlike mature milk, COL is rich in immunoglobulins, lactoferrin, and various growth factors, making it one of the most potent natural immune stimulants. The purpose of this study was to evaluate the effects of 12-weeks of COL supplementation on swimming-specific performance (SSP) and exercise adaptations in endurance-trained male athletes. Methods: Twenty-eight male triathletes and swimmers (age: 31.1 ± 10.2 years; body mass: 81.9 ± 9.0 kg; height: 1.82 ± 0.06 m) participated in a randomized, double-blind, placebo (PLA)-controlled crossover study and received 25 g∙day-1 of COL or PLA for 12 weeks. The study assessed the effects of COL on SSP (8 × 100 m performed at various intensities) and exercise adaptations [heart rate (HR) and blood lactate concentrations ([La-])]. Four main study visits were conducted-before and after COL (COLPRE and COLPOST) and PLA (PLAPRE and PLAPOST) supplementation. Results: COL had no significant effect on SSP. Still, the total time of the SSP test was about ~3.04 s shorter after COL supplementation, and ~7.13 s longer after PLA supplementation. Neither COL nor PLA supplementation affected HR during the SSP test. Post-exercise blood [La-] was significantly reduced after both COL and PLA supplementation. The analysis of SSP results in the consecutive study visits revealed possible existence of the practice effect. Conclusions: Colostrum Bovinum and high-quality milk protein (PLA) seem to be comparably effective in evoking exercise adaptation in endurance-trained male athletes. Long-term crossover supplementation protocols in athletes must consider the impact of possible practice effect when interpreting the outcomes related to exercise performance, but not biochemical or physiological markers of exercise adaptation.

Triacylglycerols in human milk and their association with edible oils in maternal diet: A study of five regions in China

Triacylglycerol (TAG) is the predominant lipid in human milk fat (∼98 %). The association between TAG and maternal diet is interesting but barely understood. In this study, we analyzed the TAG composition in human milk of mothers consuming seven edible oils during three lactation stages in five cities in China. UPLC-Q-TOF-MS was used to identify 147 species of TAGs. TAGs containing palmitic acid (P-TAG) and oleic acid (O-TAG) were significantly higher, whereas medium- and long-chain TAG was significantly lower (P < 0.05) in colostrum compared to transitional and mature milk. PLS-DA plots indicated the total TAG composition varied in different lactation stages and economic regions. Mothers consuming sunflower oil contained significantly lower (P < 0.05) P-TAG and O-TAG in their milk than the peanut oil group. O-P-L was significantly (P < 0.05) lower in rapeseed and olive oil groups. Significantly lower (P < 0.05) contents of TAGs containing linoleic acid (L-TAG) and polyunsaturated fatty acids (PUFA-TAG) were observed in the high oleic acid oil group. All the estimated factors explained 12.20 % – 33.40 % of the variation in different TAGs. These results contribute to a better nutrition strategy of maternal edible oils to promote breastfeeding.

Amino acid requirements of the infant: the amino acid composition of human breast milk.

The recommended amino acid requirements of the infant are based on the amino acid composition of mature human breast milk. The amino acid composition of breast milk is usually determined following either acid or alkaline (for tryptophan) hydrolysis. For accuracy, however, the known effect of hydrolysis time on amino acid composition should be accounted for. Also, ideally the amino acid composition of breast milk should be given in units of digested (assumed to be absorbed) amino acids. A review of the literature is presented which gives mean total amino acid concentrations in mature human milk (n = 26 studies), mean hydrolysis correction factors (n = 3 studies) and mean true ileal amino acid digestibility coefficients (n = 3 studies, suckling piglet). There were differences between the estimates of amino acid concentration corrected for hydrolysis time and digestibility, and current FAO (2013) recommendations that were not corrected for these factors. The values based on the published literature up until 2023 (mg/g true protein) corrected for hydrolysis time and digestibility gave higher values (more than 16% higher) for leucine, lysine and threonine, and considerably higher values (greater than 30%) for histidine and tryptophan. Current recommendations may need revision.

Comprehensive lipidomic profiling of human milk from lactating women across varying lactation stages and gestational ages

An untargeted lipidomic analysis was conducted to investigate the lipid composition of human milk across different lactation stages and gestational ages systematically. A total of 25 lipid subclasses and 934 lipid species as well as 90 free fatty acids were identified. Dynamic changes of the lipids throughout lactation and gestational phases were highlighted. In general, lactation stages introduced more variations in the lipid composition of human milk than gestational ages. Most lipids decreased as the milk progressed from the colostral stage to the mature stage, with some reaching a peak at the transitional stage. Significant variations in lipid composition across gestational ages were predominantly evident during early lactation period. In mature milks, most of the lipids exhibited no discernible statistical differences among gestational ages. This elucidation offers valuable insights and guidance for tailoring precise nutritional strategies for infants with diverse health needs.

Regulatory Effects of Maternal Intake of Microbial-Derived Antioxidants on Colonization of Microbiota in Breastmilk and That of Intestinal Microbiota in Offspring.

In this study, sixteen Sprague Dawley (SD) female rats and eight SD male rats were co-housed to mate. Pregnant SD female rats were fed with a control diet or an MA diet. Breast milk, maternal ileum, and intestinal samples of the offspring were collected at the day of birth and ten days afterwards. The results showed that the impact of MA was more obvious on the microbiota of mature milk (p = 0.066) than on that of colostrum. In addition, MA additive did not significantly affect maternal ileal microbiota, but affected offsprings' colonic microbiota significantly ten days after birth (p = 0.035). From the day of giving birth to ten days afterwards, in addition to the increase in microbial richness and diversity, at genus level, the dominant bacteria of breastmilk changed from Pseudomonas veronii to Bacillus and Lactococcus. Different from breastmilk microbiota, ten days after giving birth, the maternal ileal microbiota and the offsprings' intestinal microbiota were dominated by Lactobacillus. Instead of ileal microbiota, offsprings' colonic microbiota is a key action site of maternal MA additive. Therefore, the current findings have significant implications for the development of maternal feed aimed at modulating the intestinal microbiota of offspring, ultimately leading to improved health outcomes for both mothers and their offspring.

Is it possible to obtain substitutes for human milk oligosaccharides from bovine milk, goat milk, or other mammal milks?

Considering the current level of chemical and biological synthesis technology, it was a sensible selection to obtain milk oligosaccharides (MOs) from other mammals as the potential substitute for human MOs (HMOs) that possessed various structural features in the infant formula. Through a comprehensive analysis of the content, structure, and function of MOs in six distinct varieties of mammal milk, it has been shown that goat milk was the most suitable material for the preparation as a human milk substitute. Goat MOs (GMOs) had a relatively high content and diverse structural features compared to those found in other mammalian milks. The concentration of GMOs in colostrum ranged from 60 to 350mg/L, whereas in mature milk, it ranged from 200 to 24,00mg/L. The acidic oligosaccharides in goat milk have attracted considerable attention due to their closeness in acidic content and structural diversity with HMOs. Simultaneously, it was discovered that some structures, like N-glycolylneuraminic acid, were found to have a certain content in GMOs and served essential functional properties. Moreover, studies focused on the extraction of MOs from goat milk indicated that the production of GMOs on an industrial scale was viable. Furthermore, it is imperative to do further study on GMOs to enhance the preparation process, discover of new MOs structures and bioactivity evaluation, which will contribute to the development of both the commercial production of MOs and the goat milk industry.

The changes in HMOs of GDM mothers over lactation: Regulation on the gut microbiota development in offspring

This study systematically described the differences in both human milk oligosaccharides (HMOs) and infant gut microbiota between mothers with gestional mellitus (GDM) and healthy mothers (H) over lactation. A total of 56 HMOs were identified, of which 49 found in H and 39 in GDM. Not only the number of both fucosylated and sialylated oligosaccharides but also the level of LNT and 3′-GL were higher in H than in GDM. Infants of GDM had higher abundance of pro-inflammatory microbiota such as Escherichia-Shigella and Parabacteroides and lower abundance of Bifidobacterium breve than infants of H. LNT, LNnT were found to be positively related with both Bifidobacterium breve in colostrum and Bifidobacterium longum in the mature milk of all mothers. In sum, the HMOs profile and gut microbiota differed between GDM and H; while the correlations between HMOs and gut microbiota of infants were the same in all mothers. These findings underscore the potential role of HMOs in shaping infant gut microbiota and emphasize the importance of targeted nutritional interventions.

Comparative Analysis of Angora Rabbit Colostrum and Mature Milk Using Quantitative Proteomics.

Colostrum intake is a crucial determinant of survival in newborn rabbits. Neonates rely entirely on passive immunity transfer from their mothers while suckling colostrum. The goal of this study was to explore the protein differences of rabbit milk during different lactation periods. Our findings showed that the daily milk yield exhibited an increasing trend from the 2nd to the 21st day of lactation. A data-independent acquisition proteomics approach identified a total of 2011 proteins. Significantly, different abundances were found for 525 proteins in the colostrum and the mature milk samples. Eleven differentially abundant proteins (DAPs) were examined using parallel reaction monitoring, which verified the reliability of the proteomic data. Gene Ontology analysis revealed that these DAPs were primarily associated with glycosyltransferase activity, macromolecule transmembrane transporter activity, and regulation of acute inflammatory response. The dominant metabolic pathways of the DAPs involve the complement and coagulation cascades. A protein-protein interaction analysis identified apolipoprotein B, apolipoprotein A1, triose phosphate isomerase 1, and albumin as the hub proteins responsible for distinguishing differences between biological properties in rabbit colostrum and mature milk. These findings enhance our comprehension of the rabbit milk proteome, particularly in expanding our knowledge regarding the requirements of neonatal rabbits.