LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea.

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

The role of Chito-oligosaccharide in regulating ovarian germ stem cells function and restoring ovarian function in chemotherapy mice.

In recent years, the discovery of ovarian germ stem cells (OGSCs) has provided a new research direction for the treatment of female infertility. The ovarian microenvironment affects the proliferation and differentiation of OGSCs, and immune cells and related cytokines are important components of the microenvironment. However, whether improving the ovarian microenvironment can regulate the proliferation of OGSCs and remodel ovarian function has not been reported. In this study, we chelated chito-oligosaccharide (COS) with fluorescein isothiocyanate (FITC) to track the distribution of COS in the body. COS was given to mice through the best route of administration, and the changes in ovarian and immune function were detected using assays of organ index, follicle counting, serum estrogen (E2) and anti-Mullerian hormone (AMH) levels, and the expression of IL-2 and TNF-α in the ovaries. We found that COS significantly increased the organ index of the ovary and immune organs, reduced the rate of follicular atresia, increased the levels of E2 and AMH hormones, and increased the protein expression of IL-2 and TNF-α in the ovary. Then, COS and OGSCs were co-cultured to observe the combination of COS and OGSCs, and measure the survival rate of OGSCs. With increasing time, the fluorescence intensity of cells gradually increased, and the cytokines IL-2 and TNF-α significantly promoted the proliferation of OGSCs. In conclusion, COS could significantly improve the ovarian and immune function of chemotherapy model mice, and improve the survival rate of OGSCs, which provided a preliminary blueprint for further exploring the mechanism of COS in protecting ovarian function.

The Protective and Long-Lasting Effects of Human Milk Oligosaccharides on Cognition in Mammals.

Over the last few years, research indicated that Human Milk Oligosaccharides (HMOs) may serve to enhance cognition during development. HMOs hereby provide an exciting avenue in the understanding of the molecular mechanisms that contribute to cognitive development. Therefore, this review aims to summarize the reported observations regarding the effects of HMOs on memory and cognition in rats, mice and piglets. Our main findings illustrate that the administration of fucosylated (single or combined with Lacto-N-neoTetraose (LNnT) and other oligosaccharides) and sialylated HMOs results in marked improvements in spatial memory and an accelerated learning rate in operant tasks. Such beneficial effects of HMOs on cognition already become apparent during infancy, especially when the behavioural tasks are cognitively more demanding. When animals age, its effects become increasingly more apparent in simpler tasks as well. Furthermore, the combination of HMOs with other oligosaccharides yields different effects on memory performance as opposed to single HMO administration. In addition, an enhanced hippocampal long-term potentiation (LTP) response both at a young and at a mature age are reported as well. These results point towards the possibility that HMOs administered either in singular or combination forms have long-lasting, beneficial effects on memory and cognition in mammals.

Associations between human milk oligosaccharides (HMOs) and eating behaviour in Hispanic infants at 1 and 6 months of age.

BACKGROUND: Human milk oligosaccharides (HMOs) are naturally occurring glycans in human breast milk that act as prebiotics in the infant gut. Prebiotics have been demonstrated to suppress appetite in both adults and children. Therefore, HMOs may affect infant eating behaviour. OBJECTIVE: To determine if HMOs in breast milk are associated with eating behaviour in Hispanic infants. METHODS: Cross-sectional analysis of a prospective cohort of Hispanic mother-infant dyads (1-month, n = 157; 6-months, n = 69). Breast milk samples were screened for 19 HMOs using high pressure liquid chromatography, and eating behaviour was assessed using the Baby Eating Behaviour Questionnaire (BEBQ). We conducted multiple linear regressions to examine associations between HMOs and BEBQ scores, adjusted for maternal pre-pregnancy BMI, infant sex, birthweight, delivery mode and number of breastfeedings per day. We stratified by HMO secretor status-a genetic determinant of the types of HMOs produced. RESULTS: At 1 month, LNnT (lacto-N-neotetraose; P = .04) was negatively associated with food responsiveness in the total sample, while DFLNT (difucosyllacto-N-tetrose; P = .03) and DSLNT (disialyl-LNT; P = .04) were negatively associated with food responsiveness in secretors only. At 6 months, LSTc (sialyllacto-N-tetraose c; P = .01), FLNH (fucosyllacto-N-hexaose; P = .03), LNH (lacto-N-hexaose; P = .006) and DSLNH (disialyllacto-N-hexaose; P = .05) were positively associated with food responsiveness in both the total sample and secretors only. CONCLUSIONS: We found several HMOs that were both positively and negatively associated with infant food responsiveness, which is a measure of drive to eat.

Anti-Pathogenic Functions of Non-Digestible Oligosaccharides In Vitro.

Non-digestible oligosaccharides (NDOs), complex carbohydrates that resist hydrolysis by salivary and intestinal digestive enzymes, fulfill a diversity of important biological roles. A lot of NDOs are known for their prebiotic properties by stimulating beneficial bacteria in the intestinal microbiota. Human milk oligosaccharides (HMOs) represent the first prebiotics that humans encounter in life. Inspired by these HMO structures, chemically-produced NDO structures (e.g., galacto-oligosaccharides and chito-oligosaccharides) have been recognized as valuable food additives and exert promising health effects. Besides their apparent ability to stimulate beneficial microbial species, oligosaccharides have shown to be important inhibitors of the development of pathogenic infections. Depending on the type and structural characteristics, oligosaccharides can exert a number of anti-pathogenic effects. The most described effect is their ability to act as a decoy receptor, thereby inhibiting adhesion of pathogens. Other ways of pathogenic inhibition, such as interference with pathogenic cell membrane and biofilm integrity and DNA transcription, are less investigated, but could be equally impactful. In this review, a comprehensive overview of In vitro anti-pathogenic properties of different NDOs and associated pathways are discussed. A framework is created categorizing all anti-pathogenic effects and providing insight into structural necessities for an oligosaccharide to exert one of these effects.

Therapeutic CMP-Nonulosonates against Multidrug-Resistant Neisseria gonorrhoeae.

Neisseria gonorrhoeae deploys a unique immune evasion strategy wherein the lacto-N-neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac2 and CMP-Neu5Ac9N3, are also substrates for Lst. Incorporation of Leg5,7Ac2 and Neu5Ac9N3 into LOS results in N. gonorrhoeae being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac2 attenuated N. gonorrhoeae colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N3, but not CMP-Neu4,5Ac2, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto-N-neotetraose LOS capped with Kdn and Kdn7N3 bound FH to levels ∼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N3, and CMP-Leg5,7Ac2 administered intravaginally (10 µg/d) to N. gonorrhoeae-colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac2 was the most pH and temperature stable. In addition, Leg5,7Ac2-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac2 was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome (Cmah-/- mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac2 and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant N. gonorrhoeae.

α-L-Fucosidases and their applications for the production of fucosylated human milk oligosaccharides.

α-L-Fucosidases (EC 3.2.1.51), catalyzing the hydrolysis of fucosides and/or the transfer of fucosyl residue, have been characterized and modified into a trans-fucosylation mode or, further, engineered to function as "fucosynthase", which can be employed for the enzymatic synthesis of bioactive glycans, including fucosylated human milk oligosaccharides (HMOs). More than half of HMOs are fucosylated and have attracted ever-increasing interest because of their excellent physiological functions on breast-fed infants. To date, the characterization of novel fucosidases and molecular modification of these enzymes have been extensively studied to efficiently synthesize valuable fucosylated compounds. Herein, we discuss the advantages and challenges of different strategies for the production of HMOs and compare various donor/acceptor substrates used for the synthesis of fucosylated HMOs and their biomimetics. The implementation of trans-fucosylation patterns investigated in this paper via well-designed fucosidase mutants and proper reaction conditions may lead to development of an excellent platform, serving both fundamental studies and industrial-scale processes, for valuable carbohydrates synthesis.Key Points• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.

Effects of human milk oligosaccharides (HMOs) on gastrointestinal health.

Gastrointestinal (GI) health of newborns is maintained by the gastrointestinal microbiome, and the PNEI (psychoneuroendocrinoimmunology) system and can be altered by the delivery mode of the milk to the newborn such as breast versus bottle feeding, exposure to antibiotics in the milk, or exposure to environmental pollutants. The health of GI tract is particularly is maintained by breast feeding since the maternal milk provides micro- and macronutrients essential to growth, as well as multifunctional small oligosaccharides, particularly, 2'-fucosyllactose which maintain the GI health.

Challenges and Pitfalls in Human Milk Oligosaccharide Analysis.

Human milk oligosaccharides have been recognized as an important, functional biomolecule in mothers' milk. Moreover, these oligosaccharides have been recognized as the third most abundant component of human milk, ranging from 10-15 g/L in mature milk and up to and over 20 g/L reported in colostrum. Initially, health benefits of human milk oligosaccharides were assigned via observational studies on the differences between breastfed and bottle fed infants. Later, pools of milk oligosaccharides were isolated and used in functional studies and in recent years more specific studies into structure-function relationships have identified some advanced roles for milk oligosaccharides in the healthy development of infants. In other research, the levels, diversity, and complexity of human milk oligosaccharides have been studied, showing a wide variation in results. This review gives a critical overview of challenges in the analysis of human milk oligosaccharides. In view of the myriad functions that can be assigned, often to specific structures or classes of structures, it is very relevant to assess the levels of these structures in the human milk correctly, as well as in other biological sample materials. Ultimately, the review makes a case for a comparative, inter-laboratory study on quantitative human milk oligosaccharide analysis in all relevant biological samples.

Leche materna: características funcionales de los oligosacáridos de la leche materna (Parte 2) / Breast milk: functional characteristics of breast milk oligosaccharides (Part 2)

Durante los primeros meses de vida, los oligosacáridos de la leche materna (HMOs) aportados por la leche materna participan en procesos asociados con la maduración de tejidos y sistemas del tubo digestivo, modulan algunos de sus procesos metabólicos y ejercen efectos prebióticos y antimicrobianos. Otros efectos estudiados son su contribución a la instalación, desarrollo y estimulación de la microbiota residente con predomino de Bifidobacterium y Bacteroides, con efectos protectores frente a posibles colonizaciones o patologías por enteropatógenos (bacterianas, virus o parásitarias) que pueden actuar nivel local en el tubo digestivo, pero también pueden influir a nivel sistémico. Los HMOs modularían el desarrollo de la inmunidad innata y adaptativa, y probablemente previenen el desarrollo de fenómenos de atopia/alergia. Una patología propia de la etapa neonatal de los prematuros es la enterocolitis necrosante y algunos HMOs podrían disminuir el riesgo de su manifestación. Las actividades de los oligosacáridos de la leche materna contribuyen a la adaptación del lactante a los desafíos que plantea su entorno incluyendo la prevención de algunas patologías en edades posteriores, como es el caso de la diabetes tipo 1 y la obesidad.

During the first months of life, breast milk oligosaccharides (HMOs) stimulate development of the gastrointestinal tract in newborns and young infants; they modulate its metabolism and transport capabilities. Additionally, they exert prebiotic and antimicrobial activities and contribute to the development of the resident intestinal microbiota with a predominance of Bifidobacterium and Bacteroides and protect from colonization and infections by enteropathogens (bacteria, virus or parasites). It is highly probable that their activities extend beyond infancy and persist into adult life. HMOs stimulate the development of the innate and adaptive immune systems and decrease the risk of atopy/allergy. Their intake has been associated with a degree of protection against as necrotizing enterocolitis among premature infants. HMOs contribute to the long term adaptation and protection of newborn infants to unfavorable conditions of their environment and in this way may contribute to protect breastfed infants from type 1 diabetes and obesity.

Human Milk Oligosaccharides: Factors Affecting Their Composition and Their Physiological Significance.

Human milk oligosaccharides (HMOs) are elongations of the milk sugar lactose by galactose, N-acetylglucosamine, fucose; and sialic acid. The HMO composition of breast milk is strongly influenced by polymorphisms of the maternal fucosyltransferases, FUT2 and FUT3, and by the stage of lactation. Clinical observational studies with breastfed infant-mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea, and allergies. Observational and basic research data suggest that HMOs influence the establishment of early-life microbiota and mucosal immunity and inhibit pathogens, thereby contributing to protection from infections. Clinical intervention trials with infant formula supplemented with the single HMO, 2'-fucosyllactose (2'FL), or with 2 HMOs, 2'FL and lacto-N-neotetraose (LNnT), demonstrated that they allow for age-appropriate growth and are well tolerated. A priori defined exploratory outcomes related feeding an infant formula with 2 HMOs to fewer reported illnesses of the lower respiratory tract and reduced need for antibiotics during the first year of life compared to feeding a control formula. In parallel, early-life microbiota composition shifted towards that of breastfed infants. Together, HMOs likely contribute to immune protection in part through their effect on early-life gut microbiota, findings that warrant further clinical research to improve our understanding of HMO biology and significance for infant nutrition.

Human Milk Oligosaccharides: Next-Generation Functions and Questions.

Human milk oligosaccharides (HMOs) are the third most abundant component of human milk. So far, more than 150 different and structurally distinct HMOs have been identified. HMO composition varies substantially between women, but remains fairly constant over the course of lactation in the same woman. Which maternal genetic and environmental factors drive the interindividual variations in HMO composition remains poorly understood, and it is currently unknown whether or not a woman's characteristic HMO composition has evolved to match her own infant's specific needs. A combination of preclinical, cohort, and clinical studies is required to fully assess the many effects, functions, and potential claims associated with HMOs. In some cases, individual HMOs exert a certain effect and, while there might be some redundancy, the effects are often highly structure-specific. In other cases, a combination of different HMOs in specific ratios to each other is required to be effective, and future research needs to assess whether or not the administration of individual HMOs alone may be counterproductive and potentially harmful to the infant's short- and long-term health. Overall, the personalized complexity of HMOs cannot be mimicked in artificial infant formula and provides yet another powerful reason to protect, promote, and support breastfeeding.

Sialylated Oligosaccharides and Glycoconjugates of Human Milk. The Impact on Infant and Newborn Protection, Development and Well-Being.

Human milk not only has nutritional value, but also provides a wide range of biologically active molecules, which are adapted to meet the needs of newborns and infants. Mother's milk is a source of sialylated oligosaccharides and glycans that are attached to proteins and lipids, whose concentrations and composition are unique. Sialylated human milk glycoconjugates and oligosaccharides enrich the newborn immature immune system and are crucial for their proper development and well-being. Some of the milk sialylated oligosaccharide structures can locally exert biologically active effects in the newborn's and infant's gut. Sialylated molecules of human milk can be recognized and bound by sialic acid-dependent pathogens and inhibit their adhesion to the epithelial cells of newborns and infants. A small amount of intact sialylated oligosaccharides can be absorbed from the intestine and remain in the newborn's circulation in concentrations high enough to modulate the immunological system at the cellular level and facilitate proper brain development during infancy. Conclusion: The review summarizes the current state of knowledge on sialylated human milk oligosaccharides and glycoconjugates, discusses the significance of sialylated structures of human milk in newborn protection and development, and presents the advantages of human milk over infant formula.

Non-digestible carbohydrates in infant formula as substitution for human milk oligosaccharide functions: Effects on microbiota and gut maturation.

Human milk (HM) is the golden standard for nutrition of newborn infants. Human milk oligosaccharides (HMOs) are abundantly present in HM and exert multiple beneficial functions, such as support of colonization of the gut microbiota, reduction of pathogenic infections and support of immune development. HMO-composition is during lactation continuously adapted by the mother to accommodate the needs of the neonate. Unfortunately, for many valid reasons not all neonates can be fed with HM and are either totally or partly fed with cow-milk derived infant formulas, which do not contain HMOs. These cow-milk formulas are supplemented with non-digestible carbohydrates (NDCs) that have functional effects similar to that of some HMOs, since production of synthetic HMOs is challenging and still very expensive. However, NDCs cannot substitute all HMO functions. More efficacious NDCs may be developed and customized for specific groups of neonates such as pre-matures and allergy prone infants. Here current knowledge of HMO functions in the neonate in view of possible replacement of HMOs by NDCs in infant formulas is reviewed. Furthermore, methods to expedite identification of suitable NDCs and structure/function relationships are reviewed as in vivo studies in babies are impossible.

Swallow root (Decalepis hamiltonii) pectic oligosaccharide (SRO1) induces cancer cell death via modulation of galectin-3 and survivin.

Swallow root pectic oligosaccharide fraction (SRO1) from swallow root pectic polysaccharide (SRPP) possessed a molecular size of 831 Da. Structural analysis revealed that it is a rhamnogalacturonan I type, bearing arabinogalactan side chain with ß-d-(1→4) galactose along with α-l-Araf (1→5)-α-l-Araf (1→3) structure on α-d-GalA-OAc-(1→2)-α-l-Rha-(1→4)- linear backbone. ß-d (1→4) linked galactose being the specific sugar for galectin-3, SRO1 had potentials in inhibiting galectin-3 mediated cancer progression. SRO1 inhibited galectin-3 mediated agglutination, in vitro, effectively with MIC of 1.08 µg/ mL and down regulated mRNA levels of galectin-3 (∼92%) along with its downstream key protein that inhibits apoptosis - survivin (∼78%) suggesting the capability of SRO1 in inhibiting galectin-3 mediated cancer promoting pathway. This is the first report, which highlights the inhibition of interplay of galectin-3 and survivin by a dietary pectic oligosaccharide.

Digestion of Human Milk Oligosaccharides by Bifidobacterium breve in the Premature Infant.

OBJECTIVE: The aim of this study was to measure consumption and absorption of human milk oligosaccharides (HMOs) in a cohort of premature infants treated with probiotic Bifidobacterium breve. METHODS: Twenty-nine premature infants (median gestational age 28 weeks, range 23-32 weeks) cared for in the neonatal intensive care unit of the King Edward and Princess Margaret Hospital in Perth, Australia, were treated with B breve at a dose of 1.66 billion organisms per day. Samples of feces, urine, and milk were obtained at initiation of the probiotic and again 3 weeks later. 16S ribosomal RNA from the feces was analyzed by next-generation sequencing. Quantitation of HMO content of the milk, urine, and feces was performed using nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry. RESULTS: There was heterogeneity in colonization with bifidobacteria. "Responders" received milk with higher percentages of fucosylated HMOs and had higher percentages of bifidobacteria and lower percentages of Enterobacteriaceae in their feces than "nonresponders." Several individual HMOs in the milk were associated with changes in fecal bifidobacteria over time. Changes over time in milk, fecal, and urine HMOs suggested heterogeneity among HMO structures in consumption by microbes in the gut lumen and absorption from the intestine. CONCLUSIONS: Colonization of the premature infant intestinal tract with probiotic B breve is influenced by prebiotic HMOs. B breve is a selective consumer of HMOs in the premature infant.

Late seed maturation: drying without dying.

Besides the deposition of storage reserves, seed maturation is characterized by the acquisition of functional traits including germination, desiccation tolerance, dormancy, and longevity. After seed filling, seed longevity increases up to 30-fold, concomitant with desiccation that brings the embryo to a quiescent state. The period that we define as late maturation phase can represent 10-78% of total seed development time, yet it remains overlooked. Its importance is underscored by the fact that in the seed production chain, the stage of maturity at harvest is the primary factor that influences seed longevity and seedling establishment. This review describes the major events and regulatory pathways underlying the acquisition of seed longevity, focusing on key indicators of maturity such as chlorophyll degradation, accumulation of raffinose family oligosaccharides, late embryogenesis abundant proteins, and heat shock proteins. We discuss how these markers are correlated with or contribute to seed longevity, and highlight questions that merit further attention. We present evidence suggesting that molecular players involved in biotic defence also have a regulatory role in seed longevity. We also explore how the concept of plasticity can help understand the acquisition of longevity.

Role of sialyl 6-sulfo Lewis X in antitumor immunity against oral squamous cell carcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) reportedly play a pivotal role in antitumor immunity against oral squamous cell carcinoma (OSCC); however, mechanisms governing TIL recruitment to OSCC tissues remain to be clarified. This study was undertaken to assess a potential association between TILs and high endothelial venule (HEV)-like vessels that express sialyl 6-sulfo Lewis X (LeX). METHODS: OSCC tissue sections (n=41) were subjected to immunohistochemistry for sialyl 6-sulfo LeX and CD34 to allow quantitation of HEV-like vessels. Triple immunohistochemistry for sialyl 6-sulfo LeX and either CD3 and CD20 or CD4 and CD8 was conducted to determine which lymphocyte subset is more closely associated with HEV-like vessels. RESULTS: HEV-like vessels expressing sialyl 6-sulfo LeX were detected in 27 of 41 (65.9%) OSCC cases, and these vessels were more frequently found in early disease (T1/T2 stages) compared with advanced (T3/T4) stages. The number of T cells attached to the inner surface of these HEV-like vessels was significantly greater than that of B cells, while the number of CD4+ helper T cells and CD8+ cytotoxic T cells did not differ significantly. Interestingly, sialyl 6-sulfo LeX was also expressed on the membrane of a fraction of OSCC cells, and CD8+ cytotoxic T cells were almost exclusively found attached to these carcinoma cells. CONCLUSIONS: Sialyl 6-sulfo LeX is displayed not only on HEV-like vessels but also on OSCC cells and may potentially function in antitumor immunity against OSCC.

Regulatory Aspects of Human Milk Oligosaccharides.

Human milk oligosaccharides are key components of human milk and appear in various compositions and concentrations in all human milks. In regulatory sense human milk oligosaccharides are classified as novel foods or novel food ingredients requiring safety assessment. In addition, if any health messages are intended to be used also health claim regulations apply. This chapter reviews the regulatory settings and studies human milk oligosaccharides are required to fulfill to be able to enter markets in European Union or United States or elsewhere. Examples include Lacto-N-neotetraose and 2-fucosyllactose with safety assessment in European Union and United States.