Cow Milk Oligosaccharides and Their Relevance to Infant Nutrition †

: Cow milk oligosaccharides (CMOs) are complex carbohydrates found in cow milk that resemble the oligosaccharides in human milk and are essential for regulating the immune system and forming the gut flora of infants. As prebiotics, they promote the growth of specific beneficial gut bacteria, such as Lactobacilli and Bifidobacteria , thus promoting the creation of short-chain fatty acids for gut health. Furthermore, CMOs correlate with enhanced infant immune system development, offering safeguards against pathogens and anti-inflammatory benefits. The results of recent CMO research are revealed in this review, together with their biological importance and potential applications. Their relevance to infant nutrition is highlighted, as is their potential to be used as bioactive ingredients in novel functional foods and nutraceuticals. This study also describes upcoming obstacles and opportunities for CMO research, such as understanding their structures and functions, improving extraction methods, and expanding applications to different age groups.


Introduction
Oligosaccharides are crucial biological molecules found in various sources, including glycoproteins, bacteria, fungi, plants, and milk [1][2][3][4][5].This review focuses on cow milk oligosaccharides (CMOs), complex carbohydrates present in cow milk that bear a striking structural resemblance to human milk oligosaccharides (HMOs) [6].One prominent characteristic of cow milk oligosaccharides is their abundant incorporation of Neu5Ac (Nacetylneuraminic acid) [7].These compounds play a pivotal role in shaping the composition of the infant gut microbiota [8] and modulating the immune system.
CMOs function as prebiotics [9][10][11], exhibiting a unique ability to selectively nurture the growth of beneficial gut bacteria, such as Bifidobacteria and Lactobacilli [12].This fosters the production of short-chain fatty acids, which contribute to overall gut health.Furthermore, CMOs have been linked to the enhanced development and function of the infant immune system.They provide defenses against pathogens and exhibit anti-inflammatory properties.Recent CMO structure elucidation also provided deep insights [13].

Cow Milk Oligosaccharides in Ancient Literature and Ayurveda
In ancient literature and Ayurveda, cow's milk was valued for its ability to support the growth of newborns' immune, neurological, and skeletal systems, making it a respected alternative to mother's milk [14].Recent scientific research has revealed that cow milk oligosaccharides play a crucial role in brain development, immunomodulation, human growth stimulation, anti-inflammatory effects, antioxidant properties, and enhancing lactation in women [15,16].Despite historical limitations, cow milk remains potent due to its complex structured oligosaccharides, which are central to numerous vital biological processes for human development.

Classification of Cow Milk Oligosaccharides
Cow milk oligosaccharides (CMOs) exhibit a diverse classification based on their structural characteristics.Notably, the majority of bovine milk oligosaccharides (BMOs) are characterized by their acidic nature, with approximately 70% being sialylated, while a smaller fraction, less than 1%, is fucosylated, as reported by Bruggencate et al. [17].The documentation of neutral oligosaccharides in bovine milk or colostrum was initially published in 1984 by Saito et al. [18].Notably, bovine milk contains fewer types of oligosaccharides compared to human milk, with a higher prevalence of sialylated oligosaccharides and a reduced presence of fucosylated oligosaccharides [19][20][21][22].CMOs can be further categorized into two distinct types: normal and branched.This structural classification system provides a valuable framework for a comprehensive understanding and effective categorization of these significant compounds [16,23,24].

Oligosaccharides Abundance in Cow Milk:
A study conducted by Meng et al. [25] unveiled the presence of 19 different types of oligosaccharides in cow colostrum and 9 in buffalo colostrum.Notably, cow colostrum is rich in neutral disaccharides (m/z 385.15), neutral trisaccharides (m/z 547.21), and acidic oligosaccharides (m/z 635.23).In contrast, buffalo milk contains a higher proportion of neutral oligosaccharides, accounting for 88.88% of the total, compared to 63.16% in cow milk [25].
Figure 1a illustrates that among cow milk samples, the top five milk oligosaccharide components with the highest relative abundances are m/z 547.21, m/z 749.29, m/z 635.23, m/z 385.15, and m/z 426.176.These oligosaccharides constitute 52.22%, 9.96%, 9.85%, 9.11%, and 4.77%, respectively, of the total milk oligosaccharide content.Furthermore, the analysis of IgG oligosaccharides from 13 different animal species, as presented by Raju et al., sheds light on the critical role of cell line selection in producing recombinant IgGs for human therapy.Raju et al. [26], in their research, enhance our understanding of how glycosylation impacts protein therapeutics produced through transgenic technology.As interest grows in utilizing transgenic animals like goats, cows, and sheep for protein therapeutic expression, these data underscore the distinct glycosylation patterns found in IgGs from these species, potentially influencing their biological and pharmacological properties [26].

Improved Extraction Methods of CMOs and Other Milk Oligosaccharides
Choosing the right extraction method for CMOs depends on various factors.Solidphase extraction (SPE) ensures precision and high purity, ideal for specific oligosaccharides.Graphitized carbon-solid phase extraction enhances BMO extraction without lactose hydrolysis [27].Gel filtering chromatography is suitable for size-based separations.Enzyme digestion isolates lactose-related oligosaccharides effectively.Ultrafiltration is ideal for managing large sample volumes.Hydrophilic interaction-liquid chromatography (HILIC) offers a high resolution and sensitivity for hydrophilic oligosaccharides [28].Bell et al. achieved 95% pure oligosaccharide recovery from fermented whey permeate via lactose hydrolysis and yeast fermentation through nano-filtration [29].The choice depends on research objectives, sample characteristics, and available resources, with various techniques often combined.

Biological Importance of CMOs
CMOs help to protect against infectious agents by promoting beneficial bacteria growth (prebiotic) and by inhibiting pathogen binding to host cell ligands, preventing infections [30].Research by Jakobsen et al. found that BMOs favor the growth of B. longum, ssp.longum and Parabacteroides distasonis while inhibiting Clostridium perfringens and Escherichia coli [31].Milk oligosaccharides also reduce the attachment of enterotoxic Escherichia coli strains in calf intestines [32,33].Perdijk et al. studied sialyllactose from bovine milk and found that it influenced microbiota composition, promoting Bacteroides and Bifidobacteria growth, leading to distinct changes in short-chain fatty acid profiles [34].

Cow and Human Milk Similarities in Supporting Bifidobacteria Growth
Certain cow milk oligosaccharides (CMOs) resemble HMOs, potentially sharing functions [41][42][43].Enriched bovine milk supplements with oligosaccharides enhance gut development and colonization [44].Both cow milk (CM) whey and human milk (HM) contain factors promoting intestinal bifidobacteria growth in infants, with a-LA, LF, and non-protein components playing a role.The specific CM whey factors are still unknown.Different bifidobacteria strains respond differently to CM growth promoters based on NAcGlu or protein reliance.NAcGlu and gastric mucin encourage certain strains' growth, while whey proteins are less effective [45].The study by Paul McJarrow et al. found that sialylated milk oligosaccharides (SMOs) in cow milk, including sialyl lactose and sialyl lactosamine, decrease significantly in concentration from the first to the fifth milking [46].A similar study on the seasonal variation of CMOs was conducted by Zhiqian Liu et al. [47].The variety and abundance of SMOs in cow's milk are notably lower, ranging from 0.035 to 0.042 g per liter (g/L), when contrasted with human milk, where mature milk typically contains 2 to 3 g/L of SMOs.LoCascio et al. found that HMOs mimic complex HMO structures and can serve as selective prebiotics.Bifidobacterium infantis showed a fourfold increase in growth on purified HMOs, outperforming Bifidobacterium breve and Bifidobacterium longum bv.longum.B. infantis utilized 64% of the total HMOs, while B. breve and B. longum bv.longum mainly consumed lacto-N-tetraose, accounting for 35% and 24% of total HMO consumption, respectively [48].

Effects of Sialylated Milk Oligosaccharides
Cowardin et al. introduced gut bacteria from a malnourished infant into germ-free mice and provided them with a diet enriched with cow-derived SMOs.This led to increased cecal succinate levels, elevated tuft cell numbers in the small intestine, and activation of a succinate-induced tuft cell pathway associated with Th2 immune responses [49].Sialic acid, present in breast milk glycoconjugates, is crucial for brain development.Human milk's anti-inflammatory components inhibit certain immune responses, and SMOs may have potential in neoplastic disease treatment.Human milk contains carbohydrate antigens linked to cancers.Modest amounts of deoxyhexonic and arachidonic acids in breast milk aid immunological development.Studies suggest that nursing infants with milk oligosaccharides may offer protection against rheumatoid arthritis, diabetes, and multiple sclerosis [50].

Knowledge Gap
Further research, particularly during the first week of nursing (transition milk phase), is essential to understand CMOs as bioactive components in functional foods and nutraceuticals.This review focuses on early lactation studies related to CMOs, highlighting their bioactive functions and potential in innovative products.Investigating CMOs in cow milk to support infant gut health is a valuable research objective [51,52].

Conclusions
CMOs are emerging as noteworthy players in the realm of nutrition and health.While they may not match the complexity and abundance of their human milk counterparts, they exhibit promising health-promoting properties, particularly in infant nutrition.As research in this area continues to expand, CMOs hold the potential to become valuable components in various applications, benefitting not only infants but also individuals seeking enhanced health and well-being.It is clear that CMOs have a vast array of applications, and further studies are required to unveil their full potential and the extent of their impact on infant health.Overall, this review serves as a valuable resource for researchers, nutritionists, and healthcare professionals interested in CMOs and their implications for human health.

Figure 1 .
Figure 1.(a) Top 5 CMOs' abundance found in cow colostrum by Meng et al. [25]; (b) the quantitative analysis of neutral oligosaccharides was performed using the phenol-sulfuric acid method.The obtained values were determined by assuming an average molecular weight of 150 kDa for IgGs, by Raju et al. [26] (b).