Advances in the Biosynthesis and Application of Sialyllactose

Abstract

Human milk oligosaccharides (HMOs), the third most abundant solid component in human milk after lactose and lipids, are recognized as crucial prebiotics that support infant gut health and immune development. Salivary HMOs account for approximately 13% of the total HMOs molar ratio, with 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL) accounting for approximately 2% and 6%, respectively. Sialyllactose (SL) exhibits a range of notable physiological functions, including prebiotic activity, antiviral properties, prevention of necrotizing enterocolitis, immunomodulatory effects, and enhancement of brain development and cognition. Both 3′-SL and 6′-SL have been approved as “Generally Recognized as Safe” (GRAS) by the U.S. FDA and are increasingly incorporated into infant formula. Currently, the biosynthesis of SL is mainly efficiently produced through engineered microorganisms. However, face bottlenecks: low yields, complex downstream processing, and prohibitive costs. Recent advances in synthetic biology and metabolic engineering offer promising avenues to overcome these barriers. This review introduces the synthesis methods, functions, and applications of SL, as well as conducting safety evaluation and regulatory status analysis. We hope this article will enhance understanding of the challenges encountered in the synthetic biology production and application of SL.

Keywords

Sialyllactose / Synthetic biology / 3′-sialyllactose / Human milk oligosaccharides / Biosynthesis / 6′-sialyllactose

Statement of the Use of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this manuscript, the author(s) used ChatGPT in order to refine the English expressions. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.

Acknowledgments

Author Contributions

Conceptualization, H.Z. and H.W.; Methodology, H.Z.; Software, X.C.; Investigation, Z.Z.; Resources, H.Z.; Data Curation, Z.Z. and X.L.; Writing—Original Draft Preparation, H.W.; Writing—Review & Editing, H.Z. and H.W.; Supervision, H.Z.; Project Administration, X.C. and H.W.; Funding Acquisition, H.Z.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated during the current study are available from the corresponding author on reasonable request.

Funding

This research was funded by BYHEALTH Nutrition and Health Research Foundation (NO. TY202101096); China National key Research and development Program from International scientific and technological innovation cooperation between the governments of China and Egypt (grant number 2024YFE0199700).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Sato K, Nakamura Y, Fujiyama K, Ohneda K, Nobukuni T, Ogishima S, et al. Absolute quantification of eight human milk oligosaccharides in breast milk to evaluate their concentration profiles and associations with infants’ neurodevelopmental outcomes. J. Food Sci. 2024, 89, 10152-10170. doi:10.1111/1750-3841.17597.

[2] Smilowitz JT, Lebrilla CB, Mills DA, German JB, Freeman SL. Breast milk oligosaccharides: structure-function relationships in the neonate. Annu. Rev. Nutr. 2014, 34, 143-169. doi:10.1146/annurev-nutr-071813-105721.

[3] Meredith-Dennis L, Xu G, Goonatilleke E, Lebrilla CB, Underwood MA, Smilowitz JT. Composition and Variation of Macronutrients, Immune Proteins, and Human Milk Oligosaccharides in Human Milk from Nonprofit and Commercial Milk Banks. J. Hum. Lact. 2018, 34, 120-129. doi:10.1177/0890334417710635.

[4] Garrido D, Dallas DC, Mills DA. Consumption of human milk glycoconjugates by infant-associated bifidobacteria: mechanisms and implications. Microbiology 2013, 159, 649-664. doi:10.1099/mic.0.064113-0.

[5] Borewicz K, Gu F, Saccenti E, Arts ICW, Penders J, Thijs C, et al. Correlating Infant Fecal Microbiota Composition and Human Milk Oligosaccharide Consumption by Microbiota of 1-Month-Old Breastfed Infants. Mol. Nutr. Food Res. 2019, 63, e1801214. doi:10.1002/mnfr.201801214.

[6] Nguyen TLL, Nguyen DV, Heo KS. Potential biological functions and future perspectives of sialylated milk oligosaccharides. Arch. Pharm. Res. 2024, 47, 325-340. doi:10.1007/s12272-024-01492-3.

[7] Jantscher-Krenn E, Bode L. Human milk oligosaccharides and their potential benefits for the breast-fed neonate. Minerva Pediatr. 2012, 64, 83-99.

[8] Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez-Munguia B, Newburg DS. Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J. Biol. Chem. 2003, 278, 14112-14120. doi:10.1074/jbc.M207744200.

[9] Nilsen M, Madelen Saunders C, Leena Angell I, Arntzen M, Lødrup Carlsen KC, Carlsen KH, et al. Butyrate Levels in the Transition from an Infant- to an Adult-Like Gut Microbiota Correlate with Bacterial Networks Associated with Eubacterium Rectale and Ruminococcus Gnavus. Genes 2020, 11, 1245. doi:10.3390/genes11111245.

[10] Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 2011, 469, 543-547. doi:10.1038/nature09646.

[11] Albrecht S, Lane JA, Mariño K, Al Busadah KA, Carrington SD, Hickey RM, et al. A comparative study of free oligosaccharides in the milk of domestic animals. Br. J. Nutr. 2014, 111, 1313-1328. doi:10.1017/s0007114513003772.

[12] Craft KM, Townsend SD. The Human Milk Glycome as a Defense Against Infectious Diseases: Rationale, Challenges, and Opportunities. ACS Infect. Dis. 2018, 4, 77-83. doi:10.1021/acsinfecdis.7b00209.

[13] Antunes KH, Fachi JL, de Paula R, da Silva EF, Pral LP, Dos Santos A, et al. Microbiota-derived acetate protects against respiratory syncytial virus infection through a GPR43-type 1 interferon response. Nat. Commun. 2019, 10, 3273. doi:10.1038/s41467-019-11152-6.

[14] Yu ZT, Chen C, Kling DE, Liu B, McCoy JM, Merighi M, et al. The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota. Glycobiology 2013, 23, 169-177. doi:10.1093/glycob/cws138.

[15] Plaza-Díaz J, Fontana L, Gil A. Human Milk Oligosaccharides and Immune System Development. Nutrients 2018, 10, 1038. doi:10.3390/nu10081038.

[16] Cai R, Zhang J, Song Y, Liu X, Xu H. Research Progress on the Degradation of Human Milk Oligosaccharides (HMOs) by Bifidobacteria. Nutrients 2025, 17, 519. doi:10.3390/nu17030519.

[17] Noll AJ, Gourdine JP, Yu Y, Lasanajak Y, Smith DF, Cummings RD. Galectins are human milk glycan receptors. Glycobiology 2016, 26, 655-669. doi:10.1093/glycob/cww002.

[18] Wang B. Sialic acid is an essential nutrient for brain development and cognition. Annu. Rev. Nutr. 2009, 29, 177-222. doi:10.1146/annurev.nutr.28.061807.155515.

[19] Bondue P, Crèvecoeur S, Brose F, Daube G, Seghaye MC, Griffiths MW, et al. Cell-Free Spent Media Obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis Grown in Media Supplemented with 3′-Sialyllactose Modulate Virulence Gene Expression in Escherichia coli O157:H7 and Salmonella Typhimurium. Front. Microbiol. 2016, 7, 1460. doi:10.3389/fmicb.2016.01460.

[20] Craft KM, Townsend SD. Mother Knows Best: Deciphering the Antibacterial Properties of Human Milk Oligosaccharides. Acc. Chem. Res. 2019, 52, 760-768. doi:10.1021/acs.accounts.8b00630.

[21] Lis-Kuberka J, Orczyk-Pawiłowicz M. Development and Well-Being. Sialylated Oligosaccharides and Glycoconjugates of Human Milk. The Impact on Infant and Newborn Protection, Nutrients 2019, 11, 306. doi:10.3390/nu11020306.

[22] Duman H, Bechelany M, Karav S. Human Milk Oligosaccharides: Decoding Their Structural Variability, Health Benefits, and the Evolution of Infant Nutrition. Nutrients 2024, 17, 118. doi:10.3390/nu17010118.

[23] Zhou Q, Lu S, Feng K, Na K, Xiang L, Xiao M, et al. Comparative effect of sialic acid and 3′-sialyllactose on fecal microbiota fermentation and prebiotic activity in ETEC-challenged IPEC-J2 cells. J. Sci. Food Agric. 2025, 105, 6615-6629. doi:10.1002/jsfa.14372.

[24] Zhu Y, Zhang J, Zhang W, Mu W. Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3′-sialyllactose and 6′-sialyllactose. Biotechnol. Adv. 2023, 62, 108058. doi:10.1016/j.biotechadv.2022.108058.

[25] Chen Z, Song Y, Yan Y, Wu Z, Xu J. Simulative Fabrication of Milk Fortified with Sialyloligosaccharides and Its Prospective Applications. J. Agric. Food Chem. 2025, 73, 15835-15846. doi:10.1021/acs.jafc.5c02884.

[26] Zhang Z, Zhang H, Wang Z, You X, Mao Y, Li Z, et al. A Cellular Coupling Strategy Leveraging Pathway Modularization and Cofactor Regeneration for the biosynthesis of 3′-Sialyllactose. J. Agric. Food Chem. 2025, 73, 18380-18389. doi:10.1021/acs.jafc.5c04764.

[27] Chen J, Hansen T, Zhang QJ, Liu DY, Sun Y, Yan H, et al. 1-Picolinyl-5-azido Thiosialosides: Versatile Donors for the Stereoselective Construction of Sialyl Linkages. Angew. Chem. Int. Ed. Engl. 2019, 58, 17000-17008. doi:10.1002/anie.201909177.

[28] Li C, Liu Z, Li M, Miao M, Zhang T. Review on bioproduction of sialylated human milk oligosaccharides: Synthesis methods, physiologic functions, and applications. Carbohydr. Polym. 2025, 352, 123177. doi:10.1016/j.carbpol.2024.123177.

[29] Juge N, Tailford L, Owen CD. Sialidases from gut bacteria: A mini-review. Biochem. Soc. Trans. 2016, 44, 166-175. doi:10.1042/bst20150226.

[30] Abeln M, Albers I, Peters-Bernard U, Flächsig-Schulz K, Kats E, Kispert A, et al. Sialic acid is a critical fetal defense against maternal complement attack. J. Clin. Investig. 2019, 129, 422-436. doi:10.1172/jci99945.

[31] Schmölzer K, Eibinger M, Nidetzky B. Active-Site His 85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac. Chembiochem 2017, 18, 1544-1550. doi:10.1002/cbic.201700113.

[32] Lu N, Ye J, Cheng J, Sasmal A, Liu CC, Yao W, et al. Redox-Controlled Site-Specific α2-6-Sialylation. J. Am. Chem. Soc. 2019, 141, 4547-4552. doi:10.1021/jacs.9b00044.

[33] Perna VN, Dehlholm C, Meyer AS. Enzymatic production of 3′-sialyllactose in milk. Enzyme Microb. Technol. 2021, 148, 109829. doi:10.1016/j.enzmictec.2021.109829.

[34] Guo L, Chen X, Xu L, Xiao M, Lu L. Enzymatic Synthesis of 6′-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo-α-Sialidase from Bacteroides fragilis NCTC9343. Appl. Environ. Microbiol. 2018, 84, e00071-18. doi:10.1128/aem.00071-18.

[35] Guo J, Jia W, Jia S. The multifaceted roles of ST3GAL family in cancer: Mechanistic insights and therapeutic implications. Prog. Biophys. Mol. Biol. 2025, 197, 48-59. doi:10.1016/j.pbiomolbio.2025.06.001.

[36] Agrawal P, Chen S, de Pablos A, Vadlamudi Y, Vand-Rajabpour F, Jame-Chenarboo F, et al. Integrated in vivo functional screens and multiomics analyses identify α2,3-sialylation as essential for melanoma maintenance. Sci. Adv. 2025, 11, eadg3481. doi:10.1126/sciadv.adg3481.

[37] Dimitroff CJ, Pera P, Dall’Olio F, Matta KL, Chandrasekaran EV, Lau JT, et al. Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 1999, 256, 631-636. doi:10.1006/bbrc.1999.0388.

[38] Kim KW, Kim SW, Min KS, Kim CH, Lee YC. Genomic structure of human GM3 synthase gene (hST3Gal V) and identification of mRNA isoforms in the 5′-untranslated region. Gene 2001, 273, 163-171. doi:10.1016/s0378-1119(01)00595-9.

[39] Okajima T, Fukumoto S, Miyazaki H, Ishida H, Kiso M, Furukawa K, et al. Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids. J. Biol. Chem. 1999, 274, 11479-11486. doi:10.1074/jbc.274.17.11479.

[40] Smithson M, Irwin R, Williams G, Alexander KL, Smythies LE, Nearing M, et al. Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer. J. Biol. Chem. 2022, 298, 101594. doi:10.1016/j.jbc.2022.101594.

[41] Britain CM, Bhalerao N, Silva AD, Chakraborty A, Buchsbaum DJ, Crowley MR, et al. Glycosyltransferase ST6Gal-I promotes the epithelial to mesenchymal transition in pancreatic cancer cells. J. Biol. Chem. 2021, 296, 100034. doi:10.1074/jbc.RA120.014126.

[42] Luo Y, Cao H, Lei C, Liu J. ST6GALNAC 1 promotes the invasion and migration of breast cancer cells via the EMT pathway. Genes. Genom. 2023, 45, 1367-1376. doi:10.1007/s13258-023-01445-y.

[43] Dong X, Wang H, Cai J, Wang Y, Chai D, Sun Z, et al. ST6GALNAC1-mediated sialylation in uterine endometrial epithelium facilitates the epithelium-embryo attachment. J. Adv. Res. 2025, 72, 197-212. doi:10.1016/j.jare.2024.07.021.

[44] Murugaesu N, Iravani M, van Weverwijk A, Ivetic A, Johnson DA, Antonopoulos A, et al. An in vivo functional screen identifies ST6GalNAc2 sialyltransferase as a breast cancer metastasis suppressor. Cancer Discov. 2014, 4, 304-317. doi:10.1158/2159-8290.Cd-13-0287.

[45] Jia L, Luo S, Ren X, Li Y, Hu J, Liu B, et al. miR-182 and miR-135b Mediate the Tumorigenesis and Invasiveness of Colorectal Cancer Cells via Targeting ST6GALNAC2 and PI3K/AKT Pathway. Dig. Dis. Sci. 2017, 62, 3447-3459. doi:10.1007/s10620-017-4755-z.

[46] Lu C, Li WL, Ma YR. Study of correlation between polymorphism of ST6GALNAC2 and susceptibility to IgA nephropathy. Exp. Ther. Med. 2015, 9, 2127-2132. doi:10.3892/etm.2015.2404.

[47] Fuseya S, Izumi H, Hamano A, Murakami Y, Suzuki R, Koiwai R, et al. Reduction in disialyl-T antigen levels in mice deficient for both St6galnac3 and St6galnac4 results in blood filling of lymph nodes. Sci. Rep. 2023, 13, 10582. doi:10.1038/s41598-023-37363-y.

[48] Ma R, Ma Q, Zhang L, Ma B, Bao X, Zhang Y, et al. LncRNA ST6GALNAC3 inhibits dermal fibroblast proliferation and migration in cashmere goat hair follicles via the chi-miR-24-3p/ID4 axis. Anim. Biosci. 2025, 38, 1869-1880. doi:10.5713/ab.25.0115.

[49] Dai T, Li J, Liang RB, Yu H, Lu X, Wang G. Identification and Experimental Validation of the Prognostic Significance and Immunological Correlation of Glycosylation-Related Signature and ST6GALNAC4 in Hepatocellular Carcinoma. J. Hepatocell. Carcinoma 2023, 10, 531-551. doi:10.2147/jhc.S400472.

[50] Man D, Jiang Y, Zhang D, Wu J, Ding B, Liu H, et al. ST6GALNAC4 promotes hepatocellular carcinogenesis by inducing abnormal glycosylation. J. Transl. Med. 2023, 21, 420. doi:10.1186/s12967-023-04191-7.

[51] Drolez A, Vandenhaute E, Delannoy CP, Dewald JH, Gosselet F, Cecchelli R, et al. ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier. Int. J. Mol. Sci. 2016, 17, 1309. doi:10.3390/ijms17081309.

[52] InanlooRahatloo K, Parsa AF, Huse K, Rasooli P, Davaran S, Platzer M, et al. Mutation in ST6GALNAC5 identified in family with coronary artery disease. Sci. Rep. 2014, 4, 3595. doi:10.1038/srep03595.

[53] Li M, Ma Z, Zhang Y, Feng H, Li Y, Sang W, et al. Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion. Cancer Cell Int. 2023, 23, 141. doi:10.1186/s12935-023-02983-x.

[54] Huang HC, Chao CC, Wu PH, Chung HY, Lee HY, Suen CS, et al. Epigenetic silencing of the synthesis of immunosuppressive Siglec ligand glycans by NF-κB/EZH2/YY1 axis in early-stage colon cancers. Biochim. Biophys. Acta Gene Regul. Mech. 2019, 1862, 173-183. doi:10.1016/j.bbagrm.2019.01.002.

[55] Wang L, Wu S, He H, Ai K, Xu R, Zhang L, et al. CircRNA-ST6GALNAC6 increases the sensitivity of bladder cancer cells to erastin-induced ferroptosis by regulating the HSPB1/P38 axis. Lab. Investig. 2022, 102, 1323-1334. doi:10.1038/s41374-022-00826-3.

[56] Tan Z, Pan K, Sun M, Pan X, Yang Z, Chang Z, et al. CCKBR+ cancer cells contribute to the intratumor heterogeneity of gastric cancer and confer sensitivity to FOXO inhibition. Cell Death Differ. 2024, 31, 1302-1317. doi:10.1038/s41418-024-01360-z.

[57] Yoshida Y, Kojima N, Kurosawa N, Hamamoto T, Tsuji S. Molecular cloning of Sia alpha 2,3Gal beta 1,4GlcNAc alpha 2,8-sialyltransferase from mouse brain. J. Biol. Chem. 1995, 270, 14628-14633. doi:10.1074/jbc.270.24.14628.

[58] Nara K, Watanabe Y, Maruyama K, Kasahara K, Nagai Y, Sanai Y.Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta1-4Glc beta 1-1 ′Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells. Proc. Natl. Acad. Sci. USA 1994, 91, 7952-7956. doi:10.1073/pnas.91.17.7952.

[59] Zhang X, Dong W, Zhou H, Li H, Wang N, Miao X, et al. α-2,8-Sialyltransferase Is Involved in the Development of Multidrug Resistance via PI3K/Akt Pathway in Human Chronic Myeloid Leukemia. IUBMB Life 2015, 67, 77-87. doi:10.1002/iub.1351.

[60] Yoshida Y, Kojima N, Tsuji S. Molecular cloning and characterization of a third type of N-glycan alpha 2,8-sialyltransferase from mouse lung. J. Biochem. 1995, 118, 658-664. doi:10.1093/oxfordjournals.jbchem.a124960.

[61] Takashima S, Ishida HK, Inazu T, Ando T, Ishida H, Kiso M, et al. Molecular cloning and expression of a sixth type of alpha 2,8-sialyltransferase (ST8Sia VI) that sialylates O-glycans. J. Biol. Chem. 2002, 277, 24030-24038. doi:10.1074/jbc.M112367200.

[62] Eby JM, Barse L, Henning SW, Rabelink MJ, Klarquist J, Gilbert ER, et al. Alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 can support immune responses toward tumors overexpressing ganglioside D3 in mice. Cancer Immunol. Immunother. 2017, 66, 63-75. doi:10.1007/s00262-016-1920-8.

[63] Zhu M, Xia H, Xu X, Liu Y, Du G, Lv X, et al. A Novel De novo Biosynthetic Pathway for Efficient Synthesis of 6′-Sialyllactose in Escherichia coli. ACS Synth. Biol. 2025, 14, 4016-4024. doi:10.1021/acssynbio.5c00437.

[64] Priem B, Gilbert M, Wakarchuk WW, Heyraud A, Samain E. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology 2002, 12, 235-240. doi:10.1093/glycob/12.4.235.

[65] Lee YC, Chien HC, Hsu WH. Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CH 1 N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase. J. Biotechnol. 2007, 129, 453-460. doi:10.1016/j.jbiotec.2007.01.027.

[66] Lin BX, Zhang ZJ, Liu WF, Dong ZY, Tao Y. Enhanced production of N-acetyl-D-neuraminic acid by multi-approach whole-cell biocatalyst. Appl. Microbiol. Biotechnol. 2013, 97, 4775-4784. doi:10.1007/s00253-013-4754-8.

[67] Endo T, Koizumi S, Tabata K, Ozaki A. Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling. Appl. Microbiol. Biotechnol. 2000, 53, 257-261. doi:10.1007/s002530050017.

[68] Lv X, Chen X, Liu Y, Yuan L, Wu J, Yao J. Efficient Production of 3′-Sialyllactose Using Escherichia coli. J. Agric. Food Chem. 2024, 72, 27314-27325. doi:10.1021/acs.jafc.4c08703.

[69] Fierfort N, Samain E. Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides. J. Biotechnol. 2008, 134, 261-265. doi:10.1016/j.jbiotec.2008.02.010.

[70] Liu Y, Chen X, Lv X, Yuan L, Wu J, Yao J. Engineered Escherichia coli as a Microbial Cell Factory for De Novo 6′-Sialyllactose Biosynthesis. J. Agric. Food Chem. 2025, 73, 15880-15888. doi:10.1021/acs.jafc.5c04082.

[71] Chen Q, Xu X, Sun Z, Wang Y, Liu Y, Li J, et al. Metabolic engineering of Bacillus subtilis for de novo synthesis of 6′-sialyllactose. Syst. Microbiol. Biomanuf. 2025, 5, 223-236. doi:10.1007/s43393-024-00279-3.

[72] Zhu Y, Wu Y, Yu W, Zhang Q, Liu Y, Li J, et al. Engineering Bacillus subtilis for efficient production of 3′-sialyllactose. Syst. Microbiol. Biomanuf. 2024, 4, 294-306. doi:10.1007/s43393-023-00174-3.

[73] Zhu Y, Wan L, Meng J, Luo G, Chen G, Wu H, et al. Metabolic Engineering of Escherichia coli for Lacto-N-triose II Production with High Productivity. J. Agric. Food Chem. 2021, 69, 3702-3711. doi:10.1021/acs.jafc.1c00246.

[74] Schelch S, Eibinger M, Gross Belduma S, Petschacher B, Kuballa J, Nidetzky B. Engineering analysis of multienzyme cascade reactions for 3′-sialyllactose synthesis. Biotechnol. Bioeng. 2021, 118, 4290-4304. doi:10.1002/bit.27898.

[75] Drouillard S, Mine T, Kajiwara H, Yamamoto T, Samain E. Efficient synthesis of 6′-sialyllactose, 6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224. Carbohydr. Res. 2010, 345, 1394-1399. doi:10.1016/j.carres.2010.02.018.

[76] Zhang H, Lv S, Li Z, Xu C, Li H, Liu D. A Method for Low-Cost Synthesis of 3'-Sialyllactose via Three-Strain Coupled Fermentation. CN118127100A, 4 June 2024.

[77] Holck J, Larsen DM, Michalak M, Li H, Kjærulff L, Kirpekar F, et al. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase. N. Biotechnol. 2014, 31, 156-165. doi:10.1016/j.nbt.2013.11.006.

[78] Li C, Li M, Hu M, Miao M, Zhang T. Metabolic Engineering of Escherichia coli for High-Titer Biosynthesis of 3′-Sialyllactose. J. Agric. Food Chem. 2024, 72, 5379-5390. doi:10.1021/acs.jafc.3c09335.

[79] Coppa GV, Gabrielli O, Pierani P, Catassi C, Carlucci A, Giorgi PL. Changes in carbohydrate composition in human milk over 4 months of lactation. Pediatrics 1993, 91, 637-641. doi:10.1542/peds.91.3.637.

[80] Burzyńska P, Sobala ŁF, Mikołajczyk K, Jodłowska M, Jaśkiewicz E. Sialic Acids as Receptors for Pathogens. Biomolecules 2021, 11, 831. doi:10.3390/biom11060831.

[81] Beerens H, Romond C, Neut C. Influence of breast-feeding on the bifid flora of the newborn intestine. Am. J. Clin. Nutr. 1980, 33, 2434-2439. doi:10.1093/ajcn/33.11.2434.

[82] Yang M, Jiang Z, Zhou L, Chen N, He H, Li W, et al. 3′-Sialyllactose and B. infantis synergistically alleviate gut inflammation and barrier dysfunction by enriching cross-feeding bacteria for short-chain fatty acid biosynthesis. Gut Microbes 2025, 17, 2486512. doi:10.1080/19490976.2025.2486512.

[83] Perdijk O, van Baarlen P, Fernandez-Gutierrez MM, van den Brink E, Schuren FHJ, Brugman S, et al. Corrigendum: Sialyllactose and Galactooligosaccharides Promote Epithelial Barrier Functioning and Distinctly Modulate Microbiota Composition and Short Chain Fatty Acid Production In Vitro. Front. Immunol. 2019, 10, 762. doi:10.3389/fimmu.2019.00762.

[84] Cheng L, Kiewiet MBG, Logtenberg MJ, Groeneveld A, Nauta A, Schols HA, et al. Effects of Different Human Milk Oligosaccharides on Growth of Bifidobacteria in Monoculture and Co-culture with Faecalibacterium prausnitzii. Front. Microbiol. 2020, 11, 569700. doi:10.3389/fmicb.2020.569700.

[85] Ding J, Ouyang R, Zheng S, Wang Y, Huang Y, Ma X, et al. Effect of Breastmilk Microbiota and Sialylated Oligosaccharides on the Colonization of Infant Gut Microbial Community and Fecal Metabolome. Metabolites 2022, 12, 1136. doi:10.3390/metabo12111136.

[86] Bajic D, Wiens F, Wintergerst E, Deyaert S, Baudot A, Van den Abbeele P. HMOs Exert Marked Bifidogenic Effects on Children’s Gut Microbiota Ex Vivo, Due to Age-Related Bifidobacterium Species Composition. Nutrients 2023, 15, 1701. doi:10.3390/nu15071701.

[87] Sun XL. The role of cell surface sialic acids for SARS-CoV-2 infection. Glycobiology 2021, 31, 1245-1253. doi:10.1093/glycob/cwab032.

[88] Duan Q, Yu B, Huang Z, Luo Y, Zheng P, Mao X, et al. Sialyllactose Attenuates Inflammation and Injury of Intestinal Epithelial Cells upon Enterotoxigenic Escherichia coli Infection. Int. J. Mol. Sci. 2025, 26, 3860. doi:10.3390/ijms26083860.

[89] Sodhi CP, Wipf P, Yamaguchi Y, Fulton WB, Kovler M, Niño DF, et al. The human milk oligosaccharides 2′-fucosyllactose and 6′-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling. Pediatr. Res. 2021, 89, 91-101. doi:10.1038/s41390-020-0852-3.

[90] Yu ZT, Chen C, Newburg DS. Utilization of major fucosylated and sialylated human milk oligosaccharides by isolated human gut microbes. Glycobiology 2013, 23, 1281-1292. doi:10.1093/glycob/cwt065.

[91] Flesner M, Lueckenotte A, Vogelsmeier A, Popejoy L, Canada K, Minner D, et al. Advanced Practice Registered Nurses’ Quality Improvement Efforts to Reduce Antipsychotic Use in Nursing Homes. J. Nurs. Care Qual. 2019, 34, 4-8. doi:10.1097/ncq.0000000000000366.

[92] Yamabe M, Kaihatsu K, Ebara Y. Binding inhibition of various influenza viruses by sialyllactose-modified trimer DNAs. Bioorg Med. Chem. Lett. 2019, 29, 744-748. doi:10.1016/j.bmcl.2018.12.064.

[93] Zhu L, Wang M, Li H, Luo T, Deng Z, Li J, et al. Supplementation of 3′-Sialyllactose During the Growth Period Improves Learning and Memory Development in Mice. J. Agric. Food Chem. 2024, 72, 24518-24529. doi:10.1021/acs.jafc.4c06106.

[94] Wang HX, Chen Y, Haque Z, de Veer M, Egan G, Wang B. Sialylated milk oligosaccharides alter neurotransmitters and brain metabolites in piglets: An in vivo magnetic resonance spectroscopic (MRS) study. Nutr. Neurosci. 2021, 24, 885-895. doi:10.1080/1028415x.2019.1691856.

[95] Wang B, Brand-Miller J. The role and potential of sialic acid in human nutrition. Eur. J. Clin. Nutr. 2003, 57, 1351-1369. doi:10.1038/sj.ejcn.1601704.

[96] Asakuma S, Akahori M, Kimura K, Watanabe Y, Nakamura T, Tsunemi M, et al. Sialyl oligosaccharides of human colostrum: Changes in concentration during the first three days of lactation. Biosci. Biotechnol. Biochem. 2007, 71, 1447-1451. doi:10.1271/bbb.60529.

[97] Zhao C, Pu J. Influence of Host Sialic Acid Receptors Structure on the Host Specificity of Influenza Viruses. Viruses 2022, 14, 2141. doi:10.3390/v14102141.

[98] ten Bruggencate SJ, Bovee-Oudenhoven IM, Feitsma AL, van Hoffen E, Schoterman MH. Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides. Nutr. Rev. 2014, 72, 377-389. doi:10.1111/nure.12106.

[99] Boll EJ, Lopez DV, Terne M, Hessing S, Parschat K, Jensen SR. Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro. Front. Immunol. 2024, 15, 1359499. doi:10.3389/fimmu.2024.1359499.

[100] Castillo-Courtade L, Han S, Lee S, Mian FM, Buck R, Forsythe P. Attenuation of food allergy symptoms following treatment with human milk oligosaccharides in a mouse model. Allergy 2015, 70, 1091-1102. doi:10.1111/all.12650.

[101] Zhang P, Li C, Ma X, Ye J, Wang D, Cao H, et al. Glycopolymer with Sulfated Fucose and 6′-Sialyllactose as a Dual-Targeted Inhibitor on Resistant Influenza A Virus Strains. ACS Macro Lett. 2024, 13, 874-881. doi:10.1021/acsmacrolett.4c00221.

[102] Turck D, Bohn T, Castenmiller J, De Henauw S, Hirsch-Ernst KI, Maciuk A, et al. Safety of 6′-sialyllactose (6′-SL) sodium salt produced by a derivative strain (Escherichia coli NEO6) of E. coli W (ATCC 9637) as a Novel Food pursuant to Regulation (EU) 2015/2283. EFSA J. 2023, 21, e08224. doi:10.2903/j.efsa.2023.8025.

[103] Turck D, Bohn T, Castenmiller J, De Henauw S, Hirsch-Ernst KI, Maciuk A, et al. Safety of the extension of use of 2′-fucosyllactose/difucosyllactose (2′-FL/DFL) mixture and lacto-N-tetraose (LNT) as novel foods in food supplements for infants pursuant to Regulation (EU) 2015/2283. EFSA J. 2022, 20, e07140. doi:10.2903/j.efsa.2022.7140.