Bioactive Components In Human Milk Research Articles

Human Milk Oligosaccharides and Their Pivotal Role in Gut-Brain Axis Modulation and Neurologic Development: A Narrative Review to Decipher the Multifaceted Interplay.

Human milk oligosaccharides (HMOs), which are unique bioactive components in human milk, are increasingly recognized for their multifaceted roles in infant health. A deeper understanding of the nexus between HMOs and the gut-brain axis can revolutionize neonatal nutrition and neurodevelopmental strategies. We performed a narrative review using PubMed, Embase, and Google Scholar to source relevant articles. The focus was on studies detailing the influence of HMOs on the gut and brain systems, especially in neonates. Articles were subsequently synthesized based on their exploration into the effects and mechanisms of HMOs on these interconnected systems. HMOs significantly influence the neonatal gut-brain axis. Specific concentrations of HMO, measured 1 and 6 months after birth, would seem to agree with this hypothesis. HMOs are shown to influence gut microbiota composition and enhance neurotransmitter production, which are crucial for brain development. For instance, 2'-fucosyllactose has been demonstrated to support cognitive development by fostering beneficial gut bacteria that produce essential short-chain fatty acids. HMOs serve as crucial modulators of the neonatal gut-brain axis, underscoring their importance in infant nutrition and neurodevelopment. Their dual role in shaping the infant gut while influencing brain function presents them as potential game-changers in neonatal health strategies.

Bioactive Components of Human Milk and Their Impact on Child’s Health and Development, Literature Review

The composition of human breast milk is an ideal combination of substances necessary for the healthy development of an infant’s body while protecting from pathogens and the balanced development of the microbiota. Its composition is dynamic and changes with the age of the child, meeting their current needs. The study provides a thorough overview of human milk components, such as immunological components, growth factors, hormones, carbohydrates, lipids, minerals, and vitamins. Authors focus on capturing the most important aspects of the effects of these substances on a newborn’s body, while also looking for specific connections and describing the effects on given systems. Supplementation and the use of ingredients are also discussed. The purpose of this paper is to present the current state of knowledge about the bioactive components of human milk and their impact on the growth, development, and health of the young child.

Effect of novel human milk oligosaccharides-based human milk fortifier containing lactoferrin, docosahexaenoic acid, and arachidonic acid on the growth of preterm infants with birth weight of 700–1800 g

Background: Premature infants with low birth weight are at risk of growth failure and neurodevelopmental impairment. Human milk oligosaccharides (HMOs) are bioactive components of human milk that may enhance growth and development. The novel HMO-based human milk fortifier (HMoF), which also contains lactoferrin, docosahexaenoic acid (DHA), and arachidonic acid (ARA), can help provide a safer nutritional option for the growth of premature infants. Materials and Methods: We conducted a non-comparative observational study using data from multiple centers that used novel HMO-based HMF (HMoF) in premature infants (n=14) with birth weights of 700–1800 g. The primary outcome of the study was to assess tolerability (in terms of feed tolerance), and secondary outcomes included growth outcomes defined by weight, length, and head circumference during the stay in the NICU and at discharge. Results: A cohort consisting of 14 infants with a mean gestational age of 30.21±2.04 weeks and a birth weight of 1246.36±39.98 g comprised the study. During the study period, incidences of feed interruptions due to feed intolerability were nil, and no infant presented with any signs of adverse effects. The novel HMO-based HMF (HMoF)-fed infant cohort had a mean weight gain of 21.89±5.23 g/day, a mean length gain of 1.01±0.48 cm/week, and mean head circumference gain of 0.89±0.32 cm/week. The mean growth velocity recorded was 16.03±1.73 g/kg/day. Conclusions: Novel HMO-based HMF (HMoF) containing lactoferrin, DHA, and ARA demonstrated acceptable feed tolerance and weight gain without any clinically significant adverse effects. Data indicates that the novel HMoF containing lactoferrin, DHA, and ARA is a safe option for supporting the growth of preterm babies. However, further studies are needed to compare novel HMO-based HMF (HMoF) with standard HMF in a randomized controlled trial.

How Does Human Milk Protect Against Necrotizing Enterocolitis (NEC)? Targeted Validation and Time-Course Analysis of 35 Gene Responses as NEC-Signature in Fetal Intestinal Epithelial Cells.

Breastfeeding reduces the risk of necrotizing enterocolitis (NEC), one of the most common causes of morbidity and mortality in preterm infants. However, the molecular substrates by which human milk (HM) offers protection against NEC are not well known. Using fetal intestinal epithelial cells treated with known NEC aggravators, namely lipopolysaccharide (LPS) and platelet-activating factor (PAF), we mapped the time-course of changes in targeted expression analysis of 35 NEC-associated genes, so-called the NEC signature. We found, first, that HM treatment fully rescued LPS/PAF-induced fetal intestinal cell death at 12 and 24 h (n = 5). Differential gene expression and bioinformatics revealed that HM did not mitigate inflammatory and cell death signals, but instead promoted cell proliferation and stress response pathways to mitigate LPS/PAF-induced inflammatory cell death. From this, epidermal growth factor (EGF) synthesis emerged as the central player in rescue of the fetal intestinal cell death. Functional validation was supported by reversal of the cellular rescue by HM following EGF knockdown by small interfering RNA. In conclusion, this study suggests that HM might offer protection against NEC through enhancing intestinal EGF production to rescue the inflammatory cell death. Future studies are warranted to verify these HM molecular protective effects in NEC models in vivo. The findings reported herein also support future research avenues to discover new therapeutics to boost intrinsic EGF production in the injured intestinal tissues in neonates with NEC, for example, by bioactive components in human milk, natural compounds, or small molecules.

Breastfeeding: Importance in Early Development of the Immune System and Long-term Health

Breastfeeding supplies the baby and infant with unparalleled natural nutrients. Human breast milk also has several antimicrobial agents and may influence immune system development, as evidenced by prior research on newborn immunisation response and thymus gland development. Human milk is a dynamic supply of nutrients and bioactive ingredients and promotes the healthy growth and development of the human newborn. Infants are more susceptible to infection because their developing immune systems have a number of weaknesses. This review focuses on the direct effect of human milk on innate immunity in infants. Numerous new studies have made the multi-functionality of the bioactive components of human milk very clear. Our knowledge of the potential positive effects of human milk on infants has increased. These effects are not achievable with milk formulae. Human milk contains antimicrobial proteins and peptides that have a broader involvement in innate immune defence than previously thought. A complex combination of the anti-inflammatory and antioxidative substances that human milk supplies to the intestine results in a special environment of improved immune defence with reduced inflammation.

Human Milk Bioactives: Future Perspective.

Human milk is a dynamic, complex fluid that offers much more than nutrition to infants. The macronutrient content of human milk has been well characterized and described. However, human milk is not a simple matrix of protein, carbohydrate, fat, and micronutrients. The National Institutes of Health have defined bioactives in food as elements that "affect biological processes or substrates and hence have an impact on body function or condition and ultimately health." Bioactives are cells, anti-infectious and anti-inflammatory agents, growth factors, and prebiotics that are naturally present in human milk. They may explain the differences in health outcomes observed between breastfed and non-breastfed infants. They influence the development of the immune and gastrointestinal systems, gut microbiota, neurodevelopment, metabolic health, and protection against infection. Human milk oligosaccharides are one bioactive that have been an increasingly popular area of research. This review provides a broad overview of some bioactive components that positively affect the immune system and touches on certain well-known growth factors present in human milk. Future research will look at the interplay of the multitude of bioactive components in human milk as a biological system and beyond singular compounds.

Effect of convection and microwave heating on the retention of bioactive components in human milk

Bioactive substances are very important components of human milk (HM), especially for premature newborns. The effects of convection (CH) and microwave heating (MWH) at 62.5 and 66 °C, on the level of selected bioactive components of HM: lysozyme (LZ), lactoferrin (LF), secretory immunoglobulin A (sIgA), basal lipase (BL), cytokine TGF-2, vitamin C and total antioxidant capacity (TAC) was compared. Regardless of the used heating methods the TAC of HM, determined by TEAC and ORAC-FL assay, proved to be insensitive to temperature pasteurization, in contrary to BL. MWH in the conditions of 62.5 for 5 min and 66 °C for 3 min are ensuring microbiological safety with a higher retention of most of the tested active HM proteins compared to CH. Only in the case of LZ the MWH had a more degradative effect on its concentration. Controlled conditions of MWH preserve the bioactive components of the HM better than CH.

The protective effects of human milk components, 2′-fucosyllactose and osteopontin, against 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease with high morbidity in infants. Several bioactive components of human milk, such as 2′-fucosyllactose (2′-FL) and osteopontin (OPN), are reported to exhibit anti-inflammatory properties. This study determined that oral administration of 2′-FL and OPN significantly attenuated the 2,4-dinitrochlorobenzene (DNCB)-induced AD symptoms in BALB/c mice. 2′-FL and OPN treatment markedly downregulated the serum IgE level and decreased the numbers of mast cells and eosinophils in the dermal tissues from DNCB-sensitized mice. Interestingly, we found that the combination of OPN with 2′-FL produced a great therapeutic effect on AD in mice. Additionally, the result showed that the components suppressed Th2 response, resulting in an attenuated cutaneous inflammatory response. Furthermore, in vitro study validated that 2′-FL and OPN inhibited Th2 cells differentiation. Together, we conclude that 2′-FL and OPN ameliorates DNCB-induced AD-like skin inflammation, and its supplement might help to prevent AD in infants.

Nutrients and Bioactive Components of Human Milk After One Year of Lactation: Implication for Human Milk Banks.

Specifying the nutrient content and bioactive compounds in milk from long-term lactation for the purpose of finding additional sources of donors' milk for human milk banks. Human milk samples were collected from 43 mothers of term infants (term infant human milk, TIHM) (3-6 weeks of lactation) and 50 mothers who have breastfed for over a year (long-nursing human milk, LNHM). The milk collection time was 24 hours. The analyses of fat, protein, carbohydrate and energy content were performed with human milk analyzer MIRIS (Miris HMA™ ); lactoferrin and vitamin C content using high performance liquid chromatography in reversed phase (RP-HPLC); total antioxidant activity (TAC) and lysozyme by enzyme-linked immunosorbent assay (ELISA); and the activity of glutathione peroxidase (GPx) and catalase (CAT) via spectrophotometric methods. Sociodemographic characteristics of both groups of mothers have been compiled. Higher fat content and energy value was found in LNHM than in TIHM. Protein content in LNHM and TIHM did not differ. Carbohydrate content was lower in LNHM than in TIHM. TAC, CAT and GPx activity were higher in LNHM compared to TIHM. No significant differences in the content of lysozyme, lactoferrin and vitamin C between the studied groups were found. The findings suggest that after 12 months of lactation, human milk still has significant nutritional value, does not lose bioactive components and can be considered for use in human milk banks.

An Expert Panel Statement on the Beneficial Effects of Human Milk Oligosaccharides (HMOs) in Early Life and Potential Utility of HMO-Supplemented Infant Formula in Cow's Milk Protein Allergy.

This review by pediatric gastroenterology and allergy-immunology experts aimed to address the biological roles of human milk oligosaccharides (HMOs) and the potential utility of HMOs in prevention of allergy with particular emphasis on cow’s milk protein allergy (CMPA). The participating experts consider HMOs amongst the most critical bioactive components of human milk, which act as antimicrobials and antivirals by preventing pathogen adhesion to epithelial cells, as intestinal epithelial cell modulators by enhancing maturation of intestinal mucosa and intestinal epithelial barrier function, as prebiotics by promoting healthy microbiota composition and as immunomodulators by modulating immune cells indirectly and directly. Accordingly, the participating experts consider the proposed link between HMOs and prevention of allergy to be primarily based on the impact of HMO on gut microbiota, intestinal mucosal barrier, immunomodulation and immune maturation. Along with the lower risk of respiratory and gastrointestinal infections, HMO-supplemented formulas seem to be promising alternatives in the management of CMPA. Nonetheless, the effects of individual as well as complex mixtures of HMO in terms of clear clinical and immunological effects and tolerance development need to be further explored to fully realize the immunomodulatory mechanisms and the potential for HMOs in prevention of allergic diseases and CMPA.

Structures and Metabolic Properties of Bovine Milk Oligosaccharides and Their Potential in the Development of Novel Therapeutics.

Among the many bioactive components in human milk, the free oligosaccharides (OS) have been intensely studied in recent decades due to their unique ability to selectively modulate the infant gut microbiota, in addition to providing numerous other health benefits. In light of the demonstrated value of these compounds, recent studies have set out to characterize the structures and properties of the similar and more widely-available OS in the dairy industry. This mini review gives a brief overview of the common analytical techniques used to characterize bovine milk OS and highlights several recent, key studies that have identified valuable physiological and metabolic effects of these molecules in vivo. Although traditionally considered indigestible by human enzymes, evidence now suggests that milk OS are partially absorbed in the intestines and likely contribute to the development of molecular structures in the brain. Furthermore, aside from their prebiotic effects, these compounds show promise as therapeutics that could alleviate numerous metabolic abnormalities, including undernutrition, obesity, and excessive intestinal permeability. The need for novel treatments to address these and related health issues is motivating the development of scalable techniques to produce large quantities of milk OS for use as food ingredients. The safety and tolerability of high dosages of bovine milk OS have been demonstrated in two independent human studies, which potentially opens the door for further research aiming to utilize these molecules to alleviate common metabolic health issues.

Bioactive components in human milk are differentially associated with rates of lean and fat mass deposition in infants of mothers with normal vs. elevated BMI.

To model breastfed infant growth and body composition patterns over the first 4months with multiple bioactive components of human milk (HM) and clinical factors (including maternal BMI status), which are related to growth. Longitudinal observation of infant growth and body composition from 0 to 4months among 41 predominantly breastfed infants (25 mothers of Normal-weight and 16 mothers with overweight/obesity). Fasted morning HM samples were collected at 5 time-points. Macronutrients, leptin, adiponectin, ghrelin, insulin, cytokines and n-6:n-3 esterified fatty acid ratio were measured. Infant weight-for-length Z-score (WLZ) trajectory, fat-free mass (FFM) gain, fat mass gain and %fat gain were modelled controlling for clinical covariates. HM insulin negatively associated with WLZ trajectory among infants of NW mothers (P=0.028), but not associated with WLZ trajectory among infants of OW/Ob mothers. HM glucose (P<0.001) was associated with slower rates of infant FFM gain. Infants of mothers with OW/Ob exhibited slower rates of FFM gain. HM protein, adiponectin and insulin concentrations, and n-6:n-3 ratio were all significant predictors in the model of infant fat mass gain (P<0.03). Any amount of formula supplementation was associated with faster fat gain (P=0.002). The model of %fat gain was similar to that of fat mass gain, excepting HM adiponectin was not a significant covariate, and a trend for maternal OW/Ob to correlate with faster %fat gain (P=0.056). Bioactive components in HM may contribute to regulation of partitioning of body composition, and these contributions may differ between mothers of normal-weight vs. with OW/Ob.

Human milk composition and infant growth.

This review highlights relevant studies published between 2015 and 2017 on human milk composition and the association with infant growth. High-quality studies investigating how human milk composition is related to infant growth are sparse. Recent observational studies show that human milk concentrations of protein, fat, and carbohydrate likely have important influence on infant growth and body composition. Furthermore, some observational studies examining human milk oligosaccharides and hormone concentrations suggest functional relevance to infant growth. For human milk micronutrient concentrations and microbiota content, and other bioactive components in human milk, the association with infant growth is still speculative and needs further investigation. The included studies in this review are all limited in their methodological design and methods but have interesting potential in understanding infant growth. Available evidence on human milk composition in relation to infant growth is sparse. This review summarizes recent publications investigating human milk composition; including micro- and macronutrients, human milk oligosaccharides, hormones and other bioactive components, and the association with infant weight, length, body mass index, and body composition.

Bacteria and Bioactivity in Holder Pasteurized and Shelf-Stable Human Milk Products

BackgroundHistorically, Holder pasteurization has been used to pasteurize donor human milk available in a hospital setting. There is extensive research that provides an overview of the impact of Holder pasteurization on bioactive components of human milk. A shelf-stable (SS) human milk product, created using retort processing, recently became available; however, to our knowledge, little has been published about the effect of retort processing on human milk. ObjectiveWe aimed to assess the ability of retort processing to eliminate bacteria and to quantify the difference in lysozyme and secretory immunoglobulin A (sIgA) activity between Holder pasteurized (HP) and SS human milk. MethodsMilk samples from 60 mothers were pooled. From this pool, 36 samples were taken: 12 samples were kept raw, 12 samples were HP, and 12 samples were retort processed to create an SS product. All samples were analyzed for total aerobic bacteria, coliform bacteria, Bacillus cereus, sIgA activity, and lysozyme activity. Raw samples served as the control. ResultsOne raw sample and 3 HP samples contained B. cereus at the time of culture. There were no detectable bacteria in SS samples at the time of culture. Raw samples had significantly greater lysozyme and sIgA activity than HP and SS samples (P < 0.0001). HP samples retained significantly more lysozyme and sIgA activity (54% and 87%, respectively) than SS samples (0% and 11%, respectively). ConclusionsHuman milk processed using Holder pasteurization should continue to be screened for the presence of B. cereus. Clinicians should be aware of the differences in the retention of lysozyme and sIgA activity in HP and SS products when making feeding decisions for medically fragile or immunocompromised infants to ensure that patients are receiving the maximum immune protection.

Human milk: composition and health benefits.

Breastfeeding is widely acknowledged as the normal and unequalled method for feeding infants due to its associated health benefits, both for the infant and the mother. The World Health Organization recommends that infants are exclusively breastfed up to the completion of six months of age, with breastfeeding continuing to be an important part of the diet until the infant is at least two years old. The several health benefits associated with breastfeeding are driven by the combined action of the nutritional and bioactive components in human milk and the magnitude of the majority of the ascertained biological effects is directly dependent on breastfeeding duration.This review briefly summarizes the current knowledge on the composition of human milk and provides an overview on its functional effects on health outcomes, focusing on the latest researchresults.

Human milk insulin is related to maternal plasma insulin and BMI: but other components of human milk do not differ by BMI.

BackgroundThe impact of maternal BMI and insulin sensitivity on bioactive components of human milk (HM) is not well understood. As the prevalence of obesity and diabetes rises, it is increasingly critical that we understand how maternal BMI and hormones associated with metabolic disease relate to concentrations of bioactive components in HM.MethodsThis longitudinal cohort design followed 48 breastfeeding mothers through the first four months of lactation, collecting fasting morning HM samples at 2-weeks and 1, 2, 3, and 4-months, and fasting maternal blood at 2-weeks and 4-months. Insulin, glucose, adipokines leptin and adiponectin, appetite regulating hormone ghrelin, marker of oxidative stress 8OHdG, and inflammatory cytokines (IL-6, IL-8, and TNF-a) were measured in HM and maternal plasma.Results26 normal weight (NW) (BMI=21.4±2.0 kg/m2), and 22 overweight/obese (OW/Ob) (BMI=30.4±4.2 kg/m2) were followed. Of all HM analytes measured, only insulin and leptin were different between groups - consistently higher in the OW/Ob group (leptin: p<0.001; insulin: p<0.03). HM insulin was 98% higher than maternal plasma insulin at 2-weeks and 32% higher at 4-months (p<0.001). Maternal fasting plasma insulin and HOMA-IR were positively related to HM insulin at 2-weeks (p<0.001, R2≥0.38, n=31), and 4-months (p≤0.005, R2≥0.20, n=38).ConclusionsThe concentrations of insulin in HM are higher than in maternal plasma and are related to maternal BMI and insulin sensitivity. With the exception of leptin, there were minimal other differences observed in HM composition across a wide range in maternal BMI.

Impact of Maternal Diet and Exercise on Human Milk Insulin

The impact of maternal phenotype, diet and exercise on bioactive components in human milk (HM) remains an area of active research. We aimed to determine if maternal diet, specifically the nature of her carbohydrate intake, correlated with the fasting concentrations of insulin in her breast milk. Maternal 3‐day diet records were collected at 2‐weeks and 4‐months postpartum from 48 exclusively breastfeeding women. Diets were analyzed by a registered dietitian at the Colorado Clinical and Translational Sciences Institute using the Nutrient Data System for Research (NDSR) software (2012). Fasting maternal blood and a mid‐feed HM sample were collected in the morning hours at 2‐weeks and 4‐months. Skim milk was generated via centrifugation and HM and maternal plasma insulin concentrations were measured via chemiluminescent ELISA. At 4‐months, mothers completed a Godin Leisure‐Time Exercise Questionnaire to characterize degree of physical activity. The cohort exhibited a wide range of caloric and fiber intakes. Maternal circulating insulin concentrations were highly correlated with HM insulin at both time points (p&lt;0.0001). Increased consumption of insoluble fiber at 2‐weeks was associated with decreased concentrations of HM insulin (p=0.049, n=48, R2=0.08). While this association did not remain significant at 4‐months, increased consumption of added sugars and %kcal from carbohydrates at 4‐months were both positively associated with HM insulin concentrations (n=37; p=0.075, R2=0.07 and p=0.044, R2=0.08, respectively). These relationships between dietary carbohydrate quality and fasting HM insulin remained significant after controlling for maternal pre‐pregnancy BMI, age, total calories and maternal insulin concentrations. Maternal exercise score was not related to HM insulin.The quality of carbohydrate consumed at 2‐weeks and 4‐months postpartum was associated with HM insulin concentrations, suggesting that modifiable factors, such as diet, may impact bioactive components in HM. Recommendations to encourage fiber intake and limit added sugars may impact HM components; potential benefits to maternal health and infant outcomes warrant further investigation.Support or Funding InformationUniversity of Colorado Center for Women's Health Research; The Thrasher Research Fund; NIH/NIDDK: T32‐DK007658–21; NIH/NICHD F32‐HD0978068; The Colorado Clinical and Translational Sciences Institute NIH/NCATS UL1‐TR001082.

Bioactive Components in Human Milk Along the First Month of Life: Effects of Iodine Supplementation during Pregnancy

Background/Aims: Human milk is considered the most suitable food for infants. The potential benefits of breastfeeding can be explained by the presence of different growth and neurotrophic factors in human milk. This study was designed to detect some biomarkers in human milk, which could be involved in the infant neurodevelopment and in the regulation of the maturation of neonatal intestine (brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP), fibroblast growth factor 21 (FGF21), lysophosphatidic acid (LPA) and autotaxin (ATX)), and compare them on the basis of the consumption of iodine supplements or multivitamins. Methods: A prospective study included 37 healthy breastfeeding mothers, divided into 3 different groups: (1) 10 mothers who did not take supplements, (2) 17 mothers who took potassium iodine (KI) 200 µg/day and (3) 10 mothers who took a multivitamin supplement. Results: The concentrations of BDNF, GDNF, GFAP, FGF21, LPA and ATX in human milk were not significantly different in women who took a multivitamin or KI supplement compared with those who did not take any supplement. Conclusions: The presence of neurotrophic factors in human milk is neither modified by the consumption of supplements nor by their type.

Human Milk Components Modulate Toll-Like Receptor-Mediated Inflammation.

Toll-like receptor (TLR) signaling is central to innate immunity. Aberrant expression of TLRs is found in neonatal inflammatory diseases. Several bioactive components of human milk modulate TLR expression and signaling pathways, including soluble toll-like receptors (sTLRs), soluble cluster of differentiation (sCD) 14, glycoproteins, small peptides, and oligosaccharides. Some milk components, such as sialyl (α2,3) lactose and lacto-N-fucopentaose III, are reported to increase TLR signaling; under some circumstances this might contribute toward immunologic balance. Human milk on the whole is strongly anti-inflammatory, and contains abundant components that depress TLR signaling pathways: sTLR2 and sCD14 inhibit TLR2 signaling; sCD14, lactadherin, lactoferrin, and 2'-fucosyllactose attenuate TLR4 signaling; 3'-galactosyllactose inhibits TLR3 signaling, and β-defensin 2 inhibits TLR7 signaling. Feeding human milk to neonates decreases their risk of sepsis and necrotizing enterocolitis. Thus, the TLR regulatory components found in human milk hold promise as benign oral prophylactic and therapeutic treatments for the many gastrointestinal inflammatory disorders mediated by abnormal TLR signaling.

MALDI Q-TOF CID MS for diagnostic ion screening of human milk oligosaccharide samples.

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.