Human milk oligosaccharide mixture

Abstract

This invention relates to a synthetic human lactose oligosaccharide (HMO) mixture comprising 10 wt%–30 wt% acidic HMO and 70 wt%–90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt%–95 wt% fucosylated HMO based on the weight of the neutral HMO, and wherein the mixture comprises at least 19 HMOs.

Description

Technical Field

[0001] This invention relates to synthetic human milk oligosaccharide mixtures and their use in the prevention and treatment of pathogenic infections. Background Technology

[0002] Human milk contains a large amount of non-digestible carbohydrates called human milk oligosaccharides (HMOs). Mature human milk contains approximately 5 to 15 g / L of HMOs. It is estimated that more than 200 structurally different oligosaccharides exist in human milk. The building blocks for the synthesis of human milk oligosaccharides are the monosaccharides D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetylneuraminic acid (Neu5Ac)). Lactose (Galβ1-4Glc) forms the reducing end and can be elongated using N-acetylglucosamine repeating units (Galβ1-3 / 4GlcNAc). The lactose or polylactosamine backbone can be sialylated via α2-3 and / or α2-6 bonds and / or fucosylated via α1-2, α1-3, and / or α1-4 bonds. The structural complexity and abundance of these non-digestible oligosaccharides are unique to human milk, as levels of non-digestible oligosaccharides are much lower in the milk of other mammalian species. HMOs are known to perform a variety of functions, including anti-infection (against bacteria, viruses, fungi, and parasites), signal transduction, anti-inflammatory / immune regulation, and prebiotic effects, with different HMOs having different specific roles.

[0003] Breast milk is recommended for all infants. However, in some cases, breastfeeding is insufficient or less than ideal. In these situations, infant formula is a good alternative. Modern infant formula is formulated to meet the many specific nutritional needs of rapidly growing and developing infants. Previously, infant formula did not contain non-digestible oligosaccharides. Subsequent developments have included prebiotics and non-digestible oligosaccharides to functionally mimic the role of HMOs. One of the most thoroughly studied prebiotic blends is a mixture of galacto-oligosaccharides (GOS) and fructooligosaccharides (lcFOS) in a 9:1 weight ratio. After administering this specific prebiotic mixture to infants, the number of bifidobacteria in the gut microbiota increased and the number of pathogens decreased, making the microbiota more similar to that of breastfed infants (Knol et al., Acta Pædiatrica [Pediatrics], 2005; 94 (Supplement 449); Knol et al., 31-33. Pediatr Gastroenterol Nutr [Pediatric Gastroenterology and Nutrition], Vol. 40, No. 1, January 2005; WO 2005 / 039319).

[0004] Recently, lactooligosaccharides with the same structure as HMOs have been obtained through genetically modified microbial fermentation, and infant formula containing HMOs or mixtures of HMOs has become available. HMOs have been described in the art as reducing the risk of infection by viral and bacterial pathogens. The virulence of enteroviruses and bacteria depends in part on the pathogen's ability to adhere to epithelial surfaces. Two mechanisms by which HMOs modulate the pathogenesis of bacteria and viruses have been proposed. Due to the structural homology between HMOs and epithelial cell surface glycans, epithelial cell surface glycans are used as soluble decoy receptors to prevent early cell attachment. Additionally, HMOs bind to epithelial cell surface receptors to block bacterial or viral adhesion. The prevention of bacterial and viral invasion by mimicking epithelial cell surface glycans with HMOs has been explored.

[0005] In addition, various HMOs have anti-inflammatory effects and act as immunomodulators. Based on this, some have proposed that HMOs may reduce the risk of food allergies. The positive effects of sialylated HMOs on the development of the central nervous system in newborns have also been hotly debated (reviewed in "Prebiotics and Probiotics in Human Milk, Origins and functions of milk-borne oligosaccharides and bacteria", Academic Press (2017) edited by McGuire M., McGuire M. and Bode L).

[0006] Breast milk is recommended for all infants. However, in some cases, due to medical reasons or the mother's choice not to breastfeed, breastfeeding may be insufficient or unsuccessful. Infant formula has been developed for these situations.

[0007] In recent years, because HMOs can be produced through fermentation using genetically modified microorganisms, lactooligosaccharides with the same structure as HMOs, as well as infant formula containing HMOs or mixtures of HMOs, have become available. Efforts are being made to incorporate individual HMOs into nutritional compositions, particularly infant formula, in order to utilize the beneficial effects of HMOs.

[0008] Several compositions for different purposes have been developed using HMO mixtures.

[0009] WO 2004002495 describes oligosaccharide-containing substances or receptors that bind to diarrhea-causing Escherichia coli and / or zoonotic Helicobacter species, and their use in pharmaceutical compositions, nutritional compositions, and other compositions, for example, for the prevention and treatment of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome. WO9956754 relates to compositions containing at least one fucose residue in an α1-2 bond (e.g., 2FL) and their use. In particular, such compositions can be used for the treatment and prevention of gastrointestinal infections (such as diarrhea and enterocolitis). US 2014248415 describes several examples of HMO mixtures (including some of both 2FL and LNnT in various ratios). They can be used for various health benefits, such as immune system maturation, allergies, influenza, and diarrhea.

[0010] WO 9843495 relates to a method for inhibiting infections of Bacteroides, Clostridium, and Escherichia coli in a subject, the method comprising feeding the subject a synthetic nutritional formulation containing an effective antibacterial amount of lactose-N-neotetrasaccharide. By inhibiting the growth of these bacteria, the infant is provided with resistance to gastroenteritis.

[0011] CN 113907144 A describes a combination of five HMOs—53% 2'-FL, 21% 3-FL, 16% LNT, 5% 3'-SL, and 5% 6'-SL—that provide beneficial effects against Staphylococcus aureus. US 2014 / 248415A1 describes a mixture containing four HMOs—10% acidic HMO and 90% neutral HMO—91% of which are fucosylated.

[0012] WO 2009 / 077352 relates to a composition suitable for the prevention of pathogenic infections, the composition comprising a specific synergistic combination of probiotic Bifidobacterium and fucoidylated oligosaccharides.

[0013] WO 2014187464 describes a synthetic HMO mixture for treating the gut microbiota of humans (particularly adults), wherein the mixture contains at least 6 and at most 28 oligosaccharides. Rasmussen et al. in J Nutr Biochem 2017 described the effects of a mixture of 24 HMOs on intestinal function and inflammation in preterm pigs.

[0014] There is a need to develop HMO mixtures that are particularly effective and suitable for the prevention or treatment of a wide range of infections / inflammations.

[0015] There is an ongoing need to develop nutritional solutions that offer combinations of different HMOs that are closer to the natural source of HMOs (i.e., human milk) and help prevent infection in a better way than compositions that contain only a single or limited number of HMO species. Summary of the Invention

[0016] This invention relates to synthetic human lactose oligosaccharide (HMO) mixtures and nutritional compositions comprising said synthetic HMO mixtures. More particularly, this invention relates to novel synthetic HMO mixtures and nutritional compositions comprising said synthetic HMO mixtures, which exert broad preventive and therapeutic effects against infections, preferably pathogenic infections.

[0017] The inventors of this invention have discovered a synthetic HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, which provides beneficial preventive and therapeutic effects against pathogenic infections, preferably against viral, bacterial, fungal, and / or parasitic infections, and more preferably against at least viral and bacterial infections. The inventors have observed that such a synthetic HMO mixture provides improved protection against infection compared to oligosaccharide mixtures containing fewer than six different oligosaccharides, and achieves a full-spectrum effect closer to that of HMOs extracted from breast milk.

[0018] The synthesized HMO mixture according to the present invention comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO. In a preferred aspect, the synthesized HMO mixture comprises 19 HMOs.

[0019] In a preferred aspect, the HMO mixture comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO. In another aspect, the present invention relates to an HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO comprises at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:2.

[0020] In another preferred aspect, the present invention relates to the HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO is selected from 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), sialyllactose-N-tetrasaccharide a (LSTa), sialyllactose-N-tetrasaccharide b (LSTb), sialyllactose-N-tetrasaccharide c (LSTc), and disialyllactose-N-tetrasaccharide (DSLNT), and wherein the neutral HMO is selected from 2'-fucosylated lactose (2'FL), 3'-fucosylated lactose (3'FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and lactose-N-fucosylated pentasaccharide-I (LNFP). Lactose-N-fucosylpentosyl-II (LNFP II), Lactose-N-fucosylpentosyl-III (LNFP III), Lactose-N-fucosylpentosyl-V (LNFP V), Lactose-N-difucosylhexose I (LNDFH I), Lactose-N-neo-difucosylhexose I (LNnDFH I), Lactose-N-difucosylhexose II (LNDFHII), and Lactose-N-neo-difucohexose II (LNnDFH II).

[0021] In a preferred aspect, the HMO mixture comprises, and preferably consists essentially of, 3'SL, 6'SL, LSTA, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFHI, LNnDFH I, and LNDFH II / LNnDFH II. Attached Figure Description

[0022] The invention will now be discussed in more detail with reference to the accompanying drawings.

[0023] Figure 1 The results of RT-qPCR assessment of viral RNA after pre-incubation of SARS-CoV2 WA1 with different concentrations of 2'-FL, GOS-FOS (9:1 ratio), total HMO, a mixture of 19 HMOs, or 6'-SL are shown. Results are shown as a percentage relative to the untreated control and normalized relative to the lowest concentration.

[0024] Figure 2 shows measurements of transepithelial resistance (TEER) and lysine permeability (LY) of Caco-2 cells after exposure to mixtures of 19 HMOs, 5 HMOs, culture medium alone, lactose, cellobiose, or lactoferrin, followed by infection with SA11 rotavirus.

[0025] List of preferred embodiments 1. A synthetic human lactose oligosaccharide (HMO) mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO, for use in the treatment and / or prevention of infection, preferably pathogenic infection, wherein the mixture preferably comprises 19 HMOs.

[0026] 2. The synthetic HMO mixture for use according to Example 1, wherein these acidic HMOs comprise at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:2.

[0027] 3. The synthetic HMO mixture for use according to Examples 1 and 2, wherein it is used for the treatment and / or prevention of infection, preferably pathogenic infection.

[0028] 4. The synthetic HMO mixture for use according to the foregoing embodiments, wherein the pathogenic infections are selected from viral and / or bacterial infections, preferably pathogenic infections caused by viruses selected from double-stranded RNA viruses, single-stranded positive and antisense RNA viruses, and DNA viruses, and / or bacteria selected from Gram-positive and Gram-negative bacteria.

[0029] 5. The synthetic HMO mixture for use according to any of the foregoing embodiments, wherein it is used for the treatment and / or prevention of infection, preferably pathogenic infection, which is against a virus selected from rotavirus, respiratory syncytial virus (RSV) and SARS-CoV-2 and / or Listeria monocytogenes (RSV). L. monocytogenes ), Escherichia coli, Staphylococcus aureus, Salmonella typhimurium ( S. typhimurium Listeria, Enterococcus faecalis E. faecium ), Enterococcus faecalis ( E. faecalis ), Proteus mirabilis ( P. mirabilis ) and Klebsiella pneumoniae ( K. pneumoniae (i) infection caused by bacteria, preferably Escherichia coli.

[0030] 6. The synthetic HMO mixture for use according to any of the foregoing embodiments, wherein the treatment and / or prevention of infection, preferably pathogenic infection, is more similar to the treatment and / or prevention of infection observed in breastfed infants, and / or is improved compared to the treatment and / or prevention of infection in infants given a composition not containing the HMO mixture.

[0031] 7. A synthetic HMO mixture for use according to any of the foregoing embodiments, wherein the neutral HMOs comprise at least LNT and LNnT, and wherein the weight ratio of LNT to LNnT is between 12:1 and 1:1, preferably between 10:1 and 3:1, and even more preferably between 9:1 and 6:1.

[0032] 8. The synthesized HMO mixture for use according to any of the foregoing embodiments, wherein the HMO is selected from 3'SL, 6'SL, LSTa, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNnDFH I, LNDFH II and LNnDFH II.

[0033] 9. The synthetic HMO mixture for use according to any of the foregoing embodiments, wherein the mixture comprises 2'-FL, 3'FL, 3'-SL, 6'-SL, DFL, LNT, LnNT, LNFP I, LNFP II, LNFP III, LSTc, LNDFH I and DSLNT.

[0034] 10. A synthetic HMO mixture for use according to any of the foregoing embodiments, wherein the HMO mixture is contained in a nutritional composition selected from infant formula, follow-up formula and toddler formula.

[0035] 11. A nutritional composition comprising a synthetic HMO mixture for use according to any one of the foregoing embodiments, wherein the nutritional composition further comprises a Class II non-digestible sugar selected from the group consisting of: fructooligosaccharides, non-digestible dextrins, galacto-oligosaccharides (e.g., β-galacto-oligosaccharides), xylooligosaccharides, arabinose oligosaccharides, arabinose-galacto-oligosaccharides, gluco-oligosaccharides, gentian oligosaccharides, glucomannan oligosaccharides, galacto-manno-oligosaccharides, mannan oligosaccharides, isomaltooligosaccharides, Aspergillus niger oligosaccharides, chitosan oligosaccharides, soybean oligosaccharides, uronic acid oligosaccharides, and mixtures thereof.

[0036] 12. A nutritional composition comprising a synthetic HMO mixture for use according to any one of the foregoing embodiments, wherein the total weight ratio of the synthetic HMO mixture and the short-chain oligosaccharides with a DP between 3 and 6 (DP 3-6) and the long-chain oligosaccharides with a DP of 7 or above in these Class II non-digestible sugars is 5:1 to 12:1, preferably 8:1 to 10:1, and even more preferably about 9:1.

[0037] 13. A synthetic HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO, and wherein the mixture comprises at least 19 HMOs.

[0038] 14. A synthetic HMO mixture comprising or substantially consisting of: 3'SL, 6'SL, LSTTa, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFHI, LNnDFH I, LNDFH II, and LNnDFH II.

[0039] 15. The HMO mixture synthesized according to Example 13, wherein the mixture comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, and wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO.

[0040] 16. The synthesized HMO mixture according to any one of Examples 13 and 15, wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:2.

[0041] 17. The synthesized HMO mixture according to any one of Examples 13 to 16, wherein the synthesized HMO mixture comprises, preferably substantially, the following based on the total dry weight of the synthesized HMO mixture: (i) 1.4 wt% to 2.6 wt%, more preferably 1.6 wt% to 2.4 wt%, even more preferably 1.8 wt% to 2.2 wt% 3'-SL; (ii) 3.5 wt% to 4.7 wt%, more preferably 3.7 wt% to 4.5 wt%, even more preferably 3.9 wt% to 4.3 wt% 6'SL; (iii) 0.8 wt% to 1.6 wt%, more preferably 0.9 wt% to 1.5 wt%, and even more preferably 1.0 wt% to 1.4 wt% LSTa; (iv) 0.7 wt% to 1.4 wt%, more preferably 0.8 wt% to 1.3 wt%, and even more preferably 0.9 wt% to 1.2 wt% LSTb; (v) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, even more preferably 4.3 wt% to 4.6 wt% LSTc; (vi) 7.8 wt% to 11.2 wt%, more preferably 8.0 wt% to 11.0 wt%, and even more preferably 8.5 wt% to 10.5 wt% DSLNT; (vii) 25 wt% to 32 wt%, more preferably 26 wt% to 31 wt%, even more preferably 27 wt% to 30 wt% 2'FL; (viii) 19 wt% to 25 wt%, more preferably 20 wt% to 24 wt%, even more preferably 20.5 wt% to 23 wt% 3'FL; (ix) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, and even more preferably 4.3 wt% to 4.6 wt% DFL; (x) 5 wt% to 10 wt%, more preferably 6 wt% to 9 wt%, even more preferably 7 wt% to 8 wt% LNT; (xi) 0.5 wt% to 1.1 wt%, more preferably 0.6 wt% to 1 wt%, even more preferably 0.7 wt% to 0.9 wt% LNnT; (xii) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP I; (xiii) 3.8 wt% to 4.4 wt%, more preferably 3.9 wt% to 4.3 wt%, and even more preferably 4 wt% to 4.2 wt% LNFP II; (xiv) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP III; (xv) 0.2 wt% to 0.5 wt%, more preferably 0.25 wt% to 0.45 wt%, even more preferably 0.3 wt% to 0.4 wt% LNFP V; (xvi) 4.2 wt% to 5 wt%, more preferably 4.4 wt% to 4.8 wt%, even more preferably 4.5 wt% to 4.75 wt% LNDFH I; (xvii) 0.00001 wt% to 0.02 wt%, more preferably 0.0001 wt% to 0.015 wt%, even more preferably 0.001 wt% to 0.01 wt% LNnDFH I; and (xviii) 0.7 wt% to 1.3 wt%, more preferably 0.8 wt% to 1.2 wt%, and even more preferably 0.9 wt% to 1.15 wt% of a combination of LNDFH II and LNnDFH II.

[0042] 18. A nutritional composition comprising a synthetic HMO mixture according to any one of Examples 13 to 17, wherein the nutritional composition is selected from infant formula, follow-up formula and toddler milk. Detailed Implementation

[0043] definition Throughout this application and as used herein, the following terms have the following meanings.

[0044] An infant is a child under 12 months of age.

[0045] "Infant formula," "follow-up formula," or "toddler formula" means that it involves an artificially made composition, or in other words, it is a synthetic composition (i.e., a synthetic composition that is not breast milk). Therefore, the nutritional composition administered is artificial infant formula, artificial follow-up formula, artificial toddler formula, or synthetic infant formula, synthetic follow-up formula, or synthetic toddler formula. Infant formula refers to an artificially made nutritional composition intended for use in infants aged 0 to 4 to 6 months and intended as a substitute for human milk. Typically, infant formula is suitable as the sole source of nutrition. Such infant formula is also called stage 1 formula.

[0046] Follow-up formula is intended for infants aged 4 to 6 months to 12 months from birth and is designed as a supplemental feed for infants who are weaning and beginning to eat other foods. Infant formula and follow-up formula are subject to strict regulation, such as EU Regulation Nos. 609 / 2013 and 2016 / 127.

[0047] Toddlers are children aged one to three years, also known as toddlers.

[0048] Infant formula refers to an artificially produced nutritional composition designed for use in children aged 12 to 48 months, intended as a supplemental feed for infants.

[0049] "Nutritional composition" means a substance or preparation that meets at least a portion of the nutritional needs of a subject. The nutritional composition according to the invention is preferably selected from infant formula, follow-up formula, and growing milk. This means that the nutritional composition of the invention is not human milk. Alternatively, the term "formula" means that it relates to an artificially made or, in other words, a synthetic composition. Thus, in one embodiment, the nutritional composition is selected from artificial infant formula, artificial follow-up formula, and artificial growing milk, or synthetic infant formula, synthetic follow-up formula, and synthetic growing milk.

[0050] The term "one or more HMOs" refers to one or more human milk oligosaccharides. HMOs are complex carbohydrates found in human breast milk (Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes. Each oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine.

[0051] HMOs can be classified into neutral HMOs or non-acidic HMOs (which can be fucosylated HMOs or non-fucosylated HMOs) and acidic HMOs (which have at least one sialic acid residue in their structure). As used herein, no single HMO in the synthesized HMO mixture belongs to Class II non-digestible oligosaccharides. In the context of this invention, lactose is not considered an HMO.

[0052] Examples of neutral non-fucosylated HMOs include lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), lactose-N-neohexasaccharide (LNnH), p-lactose-N-neohexasaccharide (pLNnH), p-lactose-N-hexasaccharide (pLNH), and lactose-N-hexasaccharide (LNH).

[0053] "Fucosylated oligosaccharides" are oligosaccharides containing fucose residues. Examples of fucosylated HMOs include 2'-fucosyllactose (2'-FL), lactose-N-fucopentose I (LNFP-I), lactose-N-difucohexasose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lactose-N-fucopentose II (LNFP-II), lactose-N-fucopentose III (LNFP-III), and lactose-N-difucohexasose. Lactose-N-difucosylhexose-II (LNDFHII), Lactose-N-fucopentose V (LNFP-V), Lactose-N-difucohexose II (LNDFH-II), Fucosyl-lactose-N-hexose I (FLNH-I), Fucosyl-p-lactose-N-hexose 1 (FpLNH-I), Fucosyl-p-lactose-N-neohexose II (F-pLNnH II), and Fucosyl-lactose-N-neohexose (FLNnH).

[0054] "Sialinated oligosaccharides" are charged oligosaccharides containing sialic acid, that is, oligosaccharides with sialic acid residues. They are acidic. Examples of acidic HMOs include 3'-sialylactose (3'-SL), 6'-sialylactose (6'-SL), 3-fucosyl-3'-sialylactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialic acid-LNH (SLNH), sialic acid-lactose-N-hexasaccharide (SLNH), sialic acid-lactose-N-neohexose I (SLNH-I), sialic acid-lactose-N-neohexose II (SLNH-II), and disialyl-lactose-N-tetrasaccharide (DSLNT).

[0055] As used in this paper, HMO uses the common names corresponding to the molecular names provided in Table 1.

[0056] Table 1. Names of various HMO molecules

[0057] As used in this article, "therapeutic effective dose" is the amount that relieves symptoms, manages, increases or induces detoxification, or provides an individual with the nutritional, physiological or medical benefits associated with it.

[0058] As used herein, the term "synthetic HMO mixture" means an HMO mixture that is chemically distinct from a naturally occurring mixture in mammalian milk in a specific ratio, and that can be obtained by chemical and / or biological methods and / or by blending purified or isolated HMOs derived from mammalian milk. The terms "mixture of HMO" and "HMO mixture" are used interchangeably and both refer to a synthetic HMO mixture.

[0059] HMOs can be isolated from natural sources such as animal milk using chromatographic or filtration techniques, and several HMOs can be produced using biotechnological methods known in the art. Several HMOs (e.g., but not limited to 2'-FL, 3-FL, 3'-Sl, 6'-SL, LNT, LNnT, and DFL) are commercially available and can be obtained from commercial sources such as Chr Hansen, BASF, and DSM-Glycom.

[0060] In this document and its claims, the verb “comprising” and its inflections are used in their non-limiting sense to mean including the portion following the word, but not excluding any portion not specifically mentioned. Unless otherwise stated, all percentages are by weight. Furthermore, unless the context clearly requires the presence of one / a and only one / a type of element, referring to an element by the indefinite article “a / a (a or an)” does not preclude the possibility of more than one / a type of element. Therefore, the indefinite article “a / a (a or an)” generally means “at least one / a type”.

[0061] This invention relates to a synthetic human milk oligosaccharide (HMO) mixture. In a preferred aspect, the synthetic HMO mixture and nutritional compositions comprising the synthetic HMO mixture are used for infants or young children. It has been unexpectedly discovered that the HMO mixture according to the invention can provide an anti-infective composition for the prevention or treatment of pathogenic infections, preferably bacterial and / or viral infections, more preferably at least bacterial or viral infections. It is believed that the HMO mixture can provide broad protection against a wide range of pathogens by specifically modulating the gut microbiota, binding viruses, and improving intestinal barrier function. Additionally, the HMO mixture of the invention acts as a decoy receptor, preventing pathogenic microorganisms from adhering to the cells of a subject. These properties make the HMO mixture according to the invention suitable for the prevention and treatment of pathogenic infections. The HMO mixture has been found to provide improved protection in the prevention and treatment of pathogenic infections compared to oligosaccharide mixtures of fewer than six different oligosaccharides (e.g., 2'-FL, a mixture of five HMOs, or a combination of GOS and FOS), and achieves effects closer to human breast milk.

[0062] In a preferred aspect, the synthesized HMO mixture comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO. In another aspect, the synthesized HMO mixture comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein these neutral HMOs comprise 75 wt% - 95 wt% fucosylated HMO based on the weight of these neutral HMOs. In a preferred aspect, the synthesized HMO mixture according to the invention comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, and preferably comprises 19 HMOs. In a preferred aspect, the synthesized HMO mixture comprises 19 HMOs.

[0063] In another aspect, the present invention relates to a synthetic HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO comprises at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:2. The increase in the content of 6'-SL compared to 3'-SL is considered to beneficially enhance anti-inflammatory properties in the gastrointestinal tract.

[0064] In another preferred aspect, the present invention relates to a synthetic HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO is selected from 3'-sialyl-lactose (3'SL), 6-sialyl-lactose (6'SL), sialyl-lactose-N-tetrasaccharide a (LSTa), sialyl-lactose-N-tetrasaccharide b (LSTb), sialyl-lactose-N-tetrasaccharide c (LSTc), and disialyl-lactose-N-tetrasaccharide (DSLNT), and wherein the neutral HMO is selected from 2'-fucosylated lactose (2'FL), 3'-fucosylated lactose (3'FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and lactose-N-fucosylated pentose-I (LNFP). Lactose-N-fucosylpentaose-II (LNFP II), lactose-N-fucosylpentaose-III (LNFP III), lactose-N-fucosylpentaose-V (LNFP V), lactose-N-difucosylhexose-I (LNDFH I), lactose-N-neo-difucosylhexose-I (LNnDFH I), lactose-N-difucosylhexose-II (LNDFHII), and lactose-N-neo-difucosylhexose-II (LNnDFH II). Therefore, the HMO mixture synthesized according to the present invention comprises 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, and preferably comprises 19 HMOs.

[0065] In a preferred aspect, the synthesized HMO mixture comprises, and preferably substantially consists of, the following: 3'SL, 6'SL, LSTTa, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFPV, LNDFH I, LNnDFH I, and LNDFH II / LNnDFH II.

[0066] Synthetic HMO mixture Therefore, this article provides a synthetic HMO mixture, which preferably comprises 10 wt%-30 wt% acidic HMO and 70 wt%-90 wt% neutral HMO. In a preferred aspect, the synthetic HMO mixture according to the invention comprises 10 wt%-30 wt% acidic HMO and 70 wt%-90 wt% neutral HMO, and preferably comprises 19 types of HMO. In a preferred aspect, the synthetic HMO mixture comprises 19 types of HMO.

[0067] "Synthetic HMO mixture" refers to an HMO mixture that is chemically distinct from a naturally occurring mixture in mammalian milk in a specific ratio, and that can be obtained by chemical and / or biological methods and / or by blending purified or isolated HMOs derived from mammalian milk. Therefore, a synthetic HMO mixture can be a mixture of naturally occurring and purified or isolated HMOs and synthetically prepared HMOs.

[0068] Not wishing to be bound by theory, the inventors believe that the efficacy of synthetic HMO mixtures in the prevention and treatment of infections, preferably pathogenic infections, is likely due to the ratio of individual HMOs and the wide diversity of HMO structures (providing broad-spectrum immune system-related effects and decoy receptors). The inventors have observed that such HMO mixtures provide improved protection against infection compared to oligosaccharide mixtures of fewer than six different oligosaccharides (e.g., 2'-FL, a mixture of five HMOs, or a combination of GOS and FOS), and achieve effects closer to human breast milk. The inventors of the synthetic HMOs have found that the composition of HMO mixtures does not correspond to that of human breast milk, particularly the ratios of some individual HMOs not observed in breast milk during specific lactation periods. Since the underlying causes of pathogenic infections are not always apparent, and identifying the exact pathogen is both expensive and time-consuming, the use of broad-spectrum HMO mixtures beneficially improves infection prevention and treatment.

[0069] HMOs can be obtained by any suitable method. Suitable methods for synthesizing HMOs will be well known to those skilled in the art. For example, methods for producing HMOs have been developed by microbial fermentation, enzymatic methods, chemical synthesis, or combinations of these techniques (Zeuner et al., 2019. Molecules, 24(11), p. 2033).

[0070] In a preferred aspect, the HMO mixture comprises 10 wt% - 30 wt%, more preferably 15 - 25 wt%, even more preferably 20 - 25 wt% acidic HMO based on the total weight of the HMO mixture, and 70 wt% - 90 wt%, more preferably 75 wt% to 85 wt%, even more preferably 75 wt% - 80 wt% neutral HMO.

[0071] Preferably, the neutral HMO in the HMO mixture comprises 75 wt% - 95 wt%, more preferably 80 wt% - 95 wt%, or even more preferably 85 wt% - 90 wt% fucosylated HMO based on the weight of the neutral HMO in the HMO mixture.

[0072] In another embodiment, the HMO mixture preferably contains 60 wt% - 80 wt%, more preferably 65 wt% - 75 wt%, or even more preferably 67 wt% - 72 wt% of fucosylated HMO based on the weight of the HMO mixture.

[0073] In a preferred aspect, the HMO mixture comprises 2'-FL and 3'-FL in a weight ratio between 5:1 and 1:1, preferably between 4:1 and 1:1, and even more preferably between 2:1 and 1:1. The 2'-FL and 3'-FL provided in the claimed weight ratio are considered to provide complementary beneficial effects in interfering with pathogen attachment and to provide complementary immunomodulatory effects.

[0074] In another preferred embodiment, the HMO mixture comprises lactose-N-fucopentose I (LNFP I) and lactose-N-fucopentose III (LNFP III), wherein the HMO mixture comprises LNFP I and LNFP III in a weight ratio between 0.5:3 and 1:1.5, preferably between 1:3 and 1:1, and even more preferably between 1:2 and 1.1. LNFP I and LNFP III provided at the claimed ratio are believed to provide complementary beneficial effects in interfering with pathogen attachment and to provide complementary immunomodulatory effects. While LNFPI has been shown to inhibit pathogens such as enteropathogenic Escherichia coli (EPEC) and protozoan parasite Entamoeba histolytica (… E. histolytica LNFP III adheres to intestinal cells (Bode et al., Advances in Nutrition, Vol. 3, 2012), but LNFP III is associated with certain pathogens (such as Campylobacter jejuni). Campylobacter jejuni ) and enteric Salmonella ( Salmonella enterica It is associated with reduced adhesion of intestinal cells.

[0075] In some embodiments, the neutral HMO in the HMO mixture is further an N-acetylated oligosaccharide, which is at least a mixture of LNT and LNnT. In some specific embodiments, the HMO mixture comprises LNT and LNnT, wherein the HMO mixture comprises LNT:LNnT in a weight ratio between 12:1 and 1:1, preferably between 10:1 and 3:1, and even more preferably between 9:1 and 6:1. LNFPI and LNFP III provided at the claimed ratio are also believed to provide complementary beneficial effects in interfering with pathogen attachment and to provide complementary immunomodulatory effects. LNT reduces the adhesion of protozoan parasites such as Entamoeba histolytica, while LNnT reduces the load of Streptococcus pneumoniae in rabbit lungs.

[0076] In another aspect, the present invention relates to an HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO comprises at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:2.

[0077] In another preferred embodiment, the HMO mixture comprises disialialactolactose-N-tetrasaccharide (DSLNT) and sialylactose-N-tetrasaccharide c (LSTc), wherein the weight ratio of DSLNT to LSTc is between 3:0.5 and 1:1, preferably between 3:1 and 2:1, and even more preferably between 2.5:1 and 2:1. DSLNT and LSTc provided at the claimed ratio are considered to provide complementary beneficial effects in providing effective interference with pathogen attachment. While DSLNT is associated with reduced risk of NEC and is an effective decoy ligand for pathogen binding to host cells (Bode L, Front. Pediatr., Sec. Neonatology, Vol. 6, 2018), LSTc has a protective effect against human JC polyomavirus infection (Neu U et al., Cell Host & Microbe 8(4): 309-319, 2010).

[0078] In another aspect, the present invention relates to a nutritional composition comprising the HMO mixture, wherein, based on the total amount of oligosaccharides present in the nutritional composition, the total ratio of short-chain oligosaccharides (DP 3-6) to long-chain oligosaccharides (DP 7 and above) is 1:99 to 99:1, more preferably 1:19 to 19:1, more preferably 1:1 to 19:1, more preferably 2:1 to 15:1, more preferably 5:1 to 12:1, even more preferably 8:1 to 10:1, and even more preferably about 9:1. In a preferred aspect, the ratio of short-chain oligosaccharides (DP 3-6) to long-chain oligosaccharides is 9:1.

[0079] In a preferred embodiment, the fucosylated HMO of the HMO mixture comprises a combination of 2'-FL, LNFPI, and LNDFH I. In another preferred embodiment, the fucosylated HMO of the HMO mixture comprises a combination of 3'-FL, LNFP III, LNFP V, and LNdFH II. It is believed that HMOs having similar fucosylated bonds and ranging from DP3 to DP6 can provide more effective and sustained protection against pathogens over time.

[0080] In a preferred aspect, the HMO mixture contains at least -1.4 wt% to 2.6 wt%, more preferably 1.6 wt% to 2.4 wt%, even more preferably 1.8 wt% to 2.2 wt% 3'-SL; -3.5 wt% to 4.7 wt%, more preferably 3.7 wt% to 4.5 wt%, and even more preferably 3.9 wt% to 4.3 wt% 6'SL.

[0081] In another preferred aspect, the HMO mixture contains at least -1.4 wt% to 2.6 wt%, more preferably 1.6 wt% to 2.4 wt%, even more preferably 1.8 wt% to 2.2 wt% 3'-SL; -3.5 wt% to 4.7 wt%, more preferably 3.7 wt% to 4.5 wt%, even more preferably 3.9 wt% to 4.3 wt% 6'SL, wherein the weight ratio of 3'SL to 6'SL is between 0.5 : 3 and 1 : 1, preferably between 1 : 3 and 1 : 2, even more preferably about 1 : 2.

[0082] The acidic HMO is preferably selected from 3'-SL, 6'-SL, sialyl-lactose-N-tetrasaccharide a (LSTa), sialyl-lactose-N-tetrasaccharide b (LSTb), sialyl-lactose-N-tetrasaccharide c (LSTc), and disialial-lactose-N-tetrasaccharide (DSLNT). Preferably, the acidic HMO of the HMO mixture is at least 3'-SL and 6'-SL, more preferably at least 3'-SL, 6'-SL, DSLNT, and LSTc.

[0083] Neutral HMOs are preferably selected from 2'-fucosyllactose (2'FL), 3'-fucosyllactose (3'FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), lactose-N-fucosylpentasaccharide-I (LNFPI), lactose-N-fucosylpentasaccharide-II (LNFP II), lactose-N-fucosylpentasaccharide III (LNFP III), lactose-N-fucosylpentasaccharide V (LNFP V), lactose-N-difucosylhexose I (LNDFH I), lactose-N-neodifucosylhexose I (LNnDFH I), lactose-N-difucosylhexose-II (LNDFH II), and lactose-N-neodifucohexose II (LNnDFHII).

[0084] In another preferred aspect, the present invention relates to said HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the acidic HMO is selected from 3'SL, 6'SL, sialyl-lactose-N-tetrasaccharide a (LSTa), sialyl-lactose-N-tetrasaccharide b (LSTb), sialyl-lactose-N-tetrasaccharide c (LSTc), and disialyllactose-N-tetrasaccharide (DSLNT), and wherein the neutral HMO is selected from 2'-fucosylated lactose (2'FL), 3'-fucosylated lactose (3'FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), lactose-N-fucosylated pentose-I (LNFP I), and lactose-N-fucosylated pentose-II (LNFP II). Lactose-N-fucosylpentasylpentasyls ...

[0085] Therefore, the HMO mixture according to the present invention contains 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, and preferably contains 19 types of HMO.

[0086] In a preferred aspect, the HMO mixture comprises, and preferably consists essentially of, 3'SL, 6'SL, LSTA, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFHI, LNnDFH I, and LNDFH II / LNnDFH II.

[0087] In a preferred aspect, the synthesized HMO mixture of the present invention comprises, preferably substantially, the following based on the total dry weight of the HMO mixture: (i) 1.4 wt% to 2.6 wt%, more preferably 1.6 wt% to 2.4 wt%, even more preferably 1.8 wt% to 2.2 wt% 3'-SL; (ii) 3.5 wt% to 4.7 wt%, more preferably 3.7 wt% to 4.5 wt%, even more preferably 3.9 wt% to 4.3 wt% 6'SL; (iii) 0.8 wt% to 1.6 wt%, more preferably 0.9 wt% to 1.5 wt%, and even more preferably 1.0 wt% to 1.4 wt% LSTa; (iv) 0.7 wt% to 1.4 wt%, more preferably 0.8 wt% to 1.3 wt%, and even more preferably 0.9 wt% to 1.2 wt% LSTb; (v) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, even more preferably 4.3 wt% to 4.6 wt% LSTc; (vi) 7.8 wt% to 11.2 wt%, more preferably 8.0 wt% to 11.0 wt%, and even more preferably 8.5 wt% to 10.5 wt% DSLNT; (vii) 25 wt% to 32 wt%, more preferably 26 wt% to 31 wt%, even more preferably 27 wt% to 30 wt% 2'FL; (viii) 19 wt% to 25 wt%, more preferably 20 wt% to 24 wt%, even more preferably 20.5 wt% to 23 wt% 3'FL; (ix) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, and even more preferably 4.3 wt% to 4.6 wt% DFL; (x) 5 wt% to 10 wt%, more preferably 6 wt% to 9 wt%, even more preferably 7 wt% to 8 wt% LNT; (xi) 0.5 wt% to 1.1 wt%, more preferably 0.6 wt% to 1 wt%, even more preferably 0.7 wt% to 0.9 wt% LNnT; (xii) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP I; (xiii) 3.8 wt% to 4.4 wt%, more preferably 3.9 wt% to 4.3 wt%, and even more preferably 4 wt% to 4.2 wt% LNFP II; (xiv) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP III; (xv) 0.2 wt% to 0.5 wt%, more preferably 0.25 wt% to 0.45 wt%, even more preferably 0.3 wt% to 0.4 wt% LNFP V; (xvi) 4.2 wt% to 5 wt%, more preferably 4.4 wt% to 4.8 wt%, even more preferably 4.5 wt% to 4.75 wt% LNDFH I; (xvii) 0.00001 wt% to 0.02 wt%, more preferably 0.0001 wt% to 0.015 wt%, even more preferably 0.001 wt% to 0.01 wt% LNnDFH I; and (xviii) 0.7 wt% to 1.3 wt%, more preferably 0.8 wt% to 1.2 wt%, and even more preferably 0.9 wt% to 1.15 wt% of a combination of LNDFH II and LNnDFH II.

[0088] In other words, when the synthesized HMO mixture is part of a nutrient composition, the composition preferably contains the following amounts of HMO based on the volume of the nutrient composition comprising the HMO mixture according to the invention: (i) 8 to 16 mg / 100 ml, more preferably 9 to 15 mg / 100 ml, and even more preferably 10 to 14 mg / 100 ml 3'-SL; (ii) 20 to 30 mg / 100 ml, more preferably 22 to 26 mg / 100 ml, and even more preferably 23 to 25 mg / 100 ml 6'SL; (iii) 4 to 10 mg / 100 ml, more preferably 5 to 9 mg / 100 ml, and even more preferably 6 to 8 mg / 100 ml LSTa; (iv) 3 to 9 mg / 100 ml, more preferably 4 to 8 mg / 100 ml, and even more preferably 5 to 7 mg / 100 ml LSTb; (v) 20 to 32.5 mg / 100 ml, more preferably 22.5 to 30 mg / 100 ml, and even more preferably 25 to 27.5 mg / 100 ml LSTc; (vi) 50 to 60 mg / 100 ml, more preferably 52 to 56 mg / 100 ml, and even more preferably 53 to 55 mg / 100 ml DSLNT; (vii) 125 to 200 mg / 100 ml, more preferably 140 to 185 mg / 100 ml, and even more preferably 150 to 175 mg / 100 ml 2'FL; (viii) 100 to 150 mg / 100 ml, more preferably 110 to 140 mg / 100 ml, and even more preferably 120 to 130 mg / 100 ml 3'FL; (ix) 38 to 50 mg / 100 ml, more preferably 40 to 48 mg / 100 ml, and even more preferably 42 to 46 mg / 100 ml DFL; (x) 14 to 26 mg / 100 ml, more preferably 16 to 24 mg / 100 ml, and even more preferably 18 to 22 mg / 100 ml LNT; (xi) 3.5 to 5.5 mg / 100 ml, more preferably 4 to 5 mg / 100 ml, and even more preferably 4.5 to 5.5 mg / 100 ml LNnT; (xii) 14 to 24 mg / 100 ml, more preferably 16 to 22 mg / 100 ml, and even more preferably 18 to 20 mg / 100 ml LNFP I; (xiii) 18 to 30 mg / 100 ml, more preferably 20 to 28 mg / 100 ml, and even more preferably 22 to 26 mg / 100 ml LNFP II; (xiv) 14 to 24 mg / 100 ml, more preferably 16 to 22 mg / 100 ml, and even more preferably 18 to 20 mg / 100 ml LNFP III; (xv) 1.7 to 2.3 mg / 100 ml, more preferably 1.8 to 2.2 mg / 100 ml, and even more preferably 1.9 to 2.1 mg / 100 ml LNFP V; (xvi) 20 to 3 mg / 100 ml, more preferably 23 to 31 mg / 100 ml, and even more preferably 25 to 29 mg / 100 ml LNDFH I; (xvii) 0.00001 to 0.02 mg / 100 ml, more preferably 0.0001 to 0.015 mg / 100 ml, and even more preferably 0.001 to 0.01 mg / 100 ml LNnDFH I; and (xviii) A combination of 2 to 10 mg / 100 ml, more preferably 4 to 8 mg / 100 ml, and even more preferably 5 to 7 mg / 100 ml LNDFH II and LNnDFH II.

[0089] Non-digestible carbohydrates In a preferred embodiment, the HMO mixture may be provided together with a type II non-digestible carbohydrate comprising oligosaccharides and / or polysaccharides, preferably comprising at least two types of non-digestible oligosaccharides and / or polysaccharides, particularly two types of non-digestible oligosaccharides and / or polysaccharides from different sources. Type II non-digestible oligosaccharides have been found to provide further beneficial effects in the prevention and / or treatment of infections.

[0090] Preferably, the Class II non-digestible carbohydrates are soluble. As used herein, when referring to polysaccharides, fiber, or oligosaccharides, the term "soluble" means that the substance is at least soluble according to the method described below: L. Prosky et al., J. Assoc. Off. Anal. Chem. [Journal of the Association for Analytical Chemistry] 71, 1017-1023 (1988).

[0091] In one embodiment, the Class II non-digestible sugars are preferably selected from the group consisting of: fructooligosaccharides (e.g., inulin), non-digestible dextrins, galacto-oligosaccharides (e.g., trans-galacto-oligosaccharides), xylooligosaccharides, arabinose oligosaccharides, arabinose galacto-oligosaccharides, gluco-oligosaccharides, gentian oligosaccharides, glucomannan oligosaccharides, galacto-mannino oligosaccharides, mannan oligosaccharides, isomaltooligosaccharides, Aspergillus niger oligosaccharides, chitosan oligosaccharides, soybean oligosaccharides, uronic acid oligosaccharides, and mixtures thereof.

[0092] In one embodiment, the Class II non-digestible sugars preferably comprise a mixture of non-digestible polysaccharides and / or oligosaccharides. The non-digestible oligosaccharides are preferably selected from the group consisting of fructooligosaccharides and galacto-oligosaccharides.

[0093] In one embodiment, the non-digestible sugar is preferably selected from the group consisting of β-galactooligosaccharides, α-galactooligosaccharides, and galactan. According to a more preferred embodiment, the non-digestible oligosaccharide is β-galactooligosaccharide. Preferably, the non-digestible oligosaccharide comprises a galactooligosaccharide having β-(1,4), β-(1,3), and / or β-(1,6) glycosidic bonds and a terminal glucose. Trans-galactooligosaccharides are available, for example, under the trade names Vivinal® GOS (FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar), and Oligomate55 (Yakult). These oligosaccharides are considered to have excellent effects in reducing inflammatory responses in nasal epithelial cells.

[0094] In a preferred embodiment, the average DP of the galacto-oligosaccharide ranges from 2 to 10, preferably 2 to 7, and preferably 3 to 6.

[0095] In one embodiment, the non-digestible sugar preferably comprises fructooligosaccharides. In other contexts, fructooligosaccharides may have names such as fructosaccharides, oligofructoses, polyfructoses, polysaccharides, inulin, and fructans (levan and fructan), and may refer to oligosaccharides containing β-linked fructose units (preferably linked by β-(2,1) and / or β-(2,6) glycosidic bonds) and preferably with a DP between 2 and 200. Preferably, the fructooligosaccharide contains glucose with terminal β-(2,1) glycosidic linkages. Preferably, the fructooligosaccharide contains at least 7 β-linked fructose units. In another preferred embodiment, inulin is used. Inulin is a type of fructooligosaccharide in which at least 75% of the glycosidic bonds are β-(2,1) bonds. Typically, inulin has an average chain length of 8 to 60 monosaccharide units. Suitable fructooligosaccharides for use in compositions according to the method or use of the invention are commercially available under the trade name Raftiline® HP (Orafti). Other suitable sources are Raftilose (Olavti), Fibruloseh and Fibruline (Cosucra), and Frutafit and Frutalose (Sensus).

[0096] In preferred embodiments, the average DP of fructooligosaccharides ranges from 7 to 100, preferably 11 to 60, and more preferably 20 to 50. Such long-chain FOS (lcFOS) are preferred because they are believed to provide beneficial effects on the microbiome and feces, and are considered to replace some of the functions of long-chain HMOs (i.e., those with a DP of 7 and above).

[0097] In one embodiment, the type II sugar is a mixture of galactooligosaccharides and fructooligosaccharides. Preferably, the mixture of galactooligosaccharides and fructooligosaccharides is present in a weight ratio of 1 / 99 to 99 / 1, more preferably 1 / 19 to 19 / 1, more preferably 1 / 1 to 19 / 1, more preferably 2 / 1 to 15 / 1, more preferably 5 / 1 to 12 / 1, even more preferably 8 / 1 to 10 / 1, and even more preferably about 9 / 1. This weight ratio is particularly advantageous when the galactooligosaccharides have a lower average DP and the fructooligosaccharides have a relatively higher DP.

[0098] In a preferred aspect, the total weight ratio of short-chain oligosaccharides with a DP between 3 and 6 (DP 3-6) in the HMO mixture and the Class II non-digestible sugars to long-chain oligosaccharides with a DP of 7 or above is 1 / 99 to 99 / 1, more preferably 1 / 19 to 19 / 1, more preferably 1 / 1 to 19 / 1, more preferably 2 / 1 to 15 / 1, more preferably 5 / 1 to 12 / 1, even more preferably 8 / 1 to 10 / 1, and even more preferably about 9 / 1.

[0099] In a preferred aspect, a nutritional composition is provided comprising a mixture of scGOS, lcFOS and HMO, wherein the ratio of short-chain (DP 3-6) scGOS, lcFOS and HMO to the total long-chain oligosaccharides is about 9:1.

[0100] Unbound by theory, it is believed that providing a balanced mixture of monovalent short-chain oligosaccharides and polyvalent long-chain sugars offers beneficial effects on the immune system and optimal performance in the prevention and treatment of infections.

[0101] The most preferred is a mixture of galacto-oligosaccharides and fructooligosaccharides, wherein the average DP of the galacto-oligosaccharides is less than 10, preferably less than 6, preferably in the range of 2-10, preferably in the range of 2-7, preferably in the range of 3-6, and the average DP of the fructooligosaccharides is greater than 7, preferably greater than 11, even more preferably greater than 20, preferably in the range of 7-100, preferably in the range of 11-60, preferably in the range of 20-50.

[0102] Preferably, the composition according to the invention comprises 2.5 wt% to 20 wt%, more preferably 2.5 wt% to 15 wt%, even more preferably 3.0 wt% to 10 wt%, and most preferably 5.0 wt% to 7.5 wt% of total non-digestible oligosaccharides based on the total dry weight of the composition (i.e., the total wt% of both the HMO mixture and the Class II non-digestible oligosaccharides). When in liquid form, the composition for the method or use of the invention preferably comprises 0.35 wt% to 2.5 wt%, more preferably 0.35 wt% to 2.0 wt%, and even more preferably 0.4 wt% to 1.5 wt% of total non-digestible oligosaccharides based on 100 ml of the composition.

[0103] Nutritional composition The HMO mixture according to the invention is preferably provided as a nutritional composition, preferably in the form of infant formula, follow-up formula, or toddler formula. Similarly, the HMO mixture and the type II non-digestible oligosaccharide according to the invention are preferably provided as a nutritional composition, preferably in the form of infant formula, follow-up formula, or toddler formula. The nutritional composition according to the invention is not natural cow's milk or natural milk from another mammal. The nutritional composition of the invention preferably comprises digestible carbohydrates, proteins, and lipids, wherein the lipids preferably provide 30% to 60% of the total calories, preferably 35% to 55%, the proteins provide 5% to 15% of the total calories, more preferably 6% to 12%, even more preferably 7% to 9%, and the digestible carbohydrates provide 25% to 75% of the total calories, more preferably 40% to 60%. The non-digestible oligosaccharide has a caloric density of 2 kcal / g and preferably accounts for 0.4% to 7% of the total calories. The nutritional composition preferably contains 3 g to 7 g lipids / 100 kcal, more preferably 4 g to 6 g lipids / 100 kcal, more preferably 4.5 g to 5.5 g lipids / 100 kcal; it preferably contains 1.25 g to 4 g protein / 100 kcal, more preferably 1.5 g to 3.0 g protein / 100 kcal, more preferably 1.8 g to 2.2 g protein / 100 kcal, and it preferably contains 6 g to 20 g digestible carbohydrates / 100 kcal, more preferably 10 g to 15 g digestible carbohydrates / 100 kcal.

[0104] Preferably, when the nutritional composition is in ready-to-use form, its energy density is 45 to 75 kcal / 100 ml, more preferably 60 to 70 kcal / 100 ml, and even more preferably 65 to 70 kcal / 100 ml. This density ensures an optimal balance between hydration and calorie intake. The osmotic pressure of the compositions of the present invention is preferably between 150 and 420 mOsmol / L, more preferably between 260 and 320 mOsmol / L.

[0105] The nutritional composition is preferably a solid product, more preferably a powder. Suitably, the nutritional composition is in powder form and can be reconstituted with water to form a ready-to-use liquid. Alternatively, the nutritional composition may be a ready-to-use liquid or a liquid concentrate that should be diluted with water to become a ready-to-use liquid.

[0106] The nutritional composition preferably contains digestible carbohydrates. Based on calories, the nutritional composition preferably contains 6 g to 20 g of digestible carbohydrates per 100 kcal, more preferably 10 g to 15 g per 100 kcal. When in liquid form (e.g., as a ready-to-use liquid), the nutritional composition preferably contains 4 g to 15 g of digestible carbohydrates per 100 ml, more preferably 7 g to 10 g per 100 ml. Based on dry weight, the nutritional composition preferably contains 30 wt% to 85 wt%, more preferably 40 wt% to 65 wt% of digestible carbohydrates. In other words, when the nutritional composition is in powder form, the digestible carbohydrates are preferably present in an amount of 40 g to 85 g per 100 g dry weight, more preferably 40 g to 65 g per 100 g dry weight. Preferred sources of digestible carbohydrates are one or more of lactose, glucose, sucrose, fructose, galactose, maltose, starch, and maltodextrin. Lactose is the main digestible carbohydrate found in human milk. Lactose advantageously has a low glycemic index. The nutritional composition preferably contains lactose. The nutritional composition preferably contains digestible carbohydrates, wherein at least 35 wt%, more preferably at least 50 wt%, more preferably at least 75 wt%, even more preferably at least 90 wt%, and most preferably at least 95 wt% of the digestible carbohydrates are lactose.

[0107] The nutritional composition preferably contains protein. The protein concentration in the nutritional composition is determined by the sum of protein, peptides, and free amino acids. Preferably, the nutritional composition contains 1.25 g to 4 g protein / 100 kcal, even more preferably 1.5 g to 3.0 g protein / 100 kcal, even more preferably 1.8 g to 2.2 g / 100 kcal. A lower protein concentration is advantageously closer to human milk because human milk contains a lower amount of protein based on total calories compared to cow's milk. Based on ready-to-use liquid products, the nutritional composition preferably contains 0.8 to 2.5 g / 100 ml, more preferably 1.0 g to 2.0 g / 100 ml, even more preferably 1.2 to 1.5 g / 100 ml. Based on dry weight, the nutritional composition preferably contains 6 to 18 wt%, more preferably 7 to 15 wt%, even more preferably 8 to 11 wt% protein. In other words, when the nutritional composition is in powder form, the protein is preferably present in an amount of 6 to 18 g / 100 g dry weight, more preferably 7 to 15 g, and even more preferably 8 to 11 g / 100 g dry weight. The protein source is preferably selected in such a way that it meets the minimum requirements for essential amino acid content and ensures satisfactory growth. Therefore, protein sources based on bovine milk proteins (such as whey protein, casein, and mixtures thereof) and proteins based on soybean, rice, or peas are preferred. When whey protein is used, the protein source is preferably based on acidified or sweet whey, modified sweet whey, whey protein isolates, or mixtures thereof.

[0108] The nutritional composition of the present invention preferably comprises lipids. The lipids are preferably present in an amount of 3 g to 7 g / 100 kcal, more preferably 4 g to 6 g lipid / 100 kcal, and most preferably 4.5 g to 5.5 g lipid / 100 kcal. When in liquid form (e.g., as a ready-to-use liquid), the nutritional composition preferably contains 2.2 g to 4.5 g lipid / 100 ml, more preferably 2.5 g to 4.0 g, and even more preferably 3.0 to 3.75 g / 100 ml. Based on dry weight, the nutritional composition preferably contains 16 wt% to 32 wt%, more preferably 18 wt% to 30 wt%, and even more preferably 20 wt% to 28 wt% lipids. In other words, when the nutritional composition is in powder form, the lipids are preferably present in an amount of 16 g to 32 g / 100 g dry weight, more preferably 18 to 30 g, and even more preferably 20 to 28 g / 100 g dry weight of the composition.

[0109] The lipids preferably comprise plant lipids. The presence of plant lipids advantageously achieves an optimal fatty acid profile high in polyunsaturated fatty acids, such as essential linolenic acid and α-linolenic acid, and is more similar to human milk fat. Lipids derived solely from non-human mammalian milk (e.g., cow's milk) do not provide an optimal fatty acid profile. In non-human mammalian milk, the amount of essential fatty acids is too low. Preferably, the nutritional composition comprises at least one, preferably at least two, plant lipid sources selected from the group consisting of: linseed oil (or flaxseed oil), rapeseed oil (such as colzaoil, low-erucic acid rapeseed oil, and canola oil), sunflower oil, high-oleic sunflower oil, safflower oil, high-oleic safflower oil, olive oil, coconut oil, soybean oil, palm oil, and palm kernel oil.

[0110] In addition, animal fats (such as milk fat) are preferably present in the nutritional composition. Such lipid sources can provide additional desirable components, such as butyric acid (BA) and caproic acid (CA), as well as β-palmitic acid (sn2-PA). Components such as butyric acid are known to have synergistic effects on the intestinal barrier, immune system, and antipathogenic effects when combined with human milk oligosaccharides. Preferably, the nutritional composition contains at least 0.5 wt% butyric acid based on total fatty acids, more preferably at least 0.7 wt% to 2 wt%.

[0111] Additionally, egg oil and / or fish oil and / or microbial oil (such as oils from fungi and algae) may be present. Such oils are suitable sources of long-chain polyunsaturated fatty acids (such as docosahexaenoic acid (DHA), arachidonic acid (ARA), and / or eicosapentaenoic acid (EPA)). Preferably, the nutritional composition comprises n3 LC-PUFAs, such as EPA and / or DHA, more preferably DHA. Preferably, the nutritional composition comprises at least 0.05 wt%, preferably at least 0.1 wt%, more preferably at least 0.2 wt% of DHA based on total fatty acids. Preferably, the nutritional composition comprises no more than 2.0 wt%, preferably no more than 1.0 wt% of DHA based on total fatty acids. The nutritional composition preferably comprises ARA. Preferably, the nutritional composition comprises at least 0.05 wt%, preferably at least 0.1 wt%, more preferably at least 0.2 wt% of ARA based on total fatty acids.

[0112] Preferably, the nutritional composition contains additional ingredients such as vitamins, minerals, trace elements, nucleotides, and other micronutrients known in the art.

[0113] In some respects, the nutritional composition may contain selected Bifidobacteria (Bifidobacterium spp.) Bifidobacterium ) and / or Lactobacillus spp. LactobacillusThe nutritional composition comprises a group of lactic acid-producing bacteria, particularly those belonging to the genus *Bifidobacterium*. Breastfed infants have a high concentration of *Bifidobacterium* in their gut microbiota. Adding one or more strains of *Bifidobacterium* to the nutritional composition further improves the gut microbiota and its activity, thus providing additional beneficial effects against pathogenic infections. More preferably, the nutritional composition contains *Bifidobacterium breve* (…). Bifidobacteriumbreve Bifidobacterium longum subsp. ( Bifidobacteriumlongum spp. longum Bifidobacterium longum infantis subspecies ( Bifidibacteriumlongum spp infantis ) and / or Bifidobacterium bifidum ( Bifidobacterium bifidum Such strains of Bifidobacterium species are commercially available or can be isolated from the infant's gut microbiota. The amount of Bifidobacterium and / or Lactobacillus is preferably 10. 4 Up to 10 11 CFU / gram of nutrient composition dry weight.

[0114] The synthetic HMO mixtures according to the invention are preferably provided as nutritional compositions in the form of infant formula, follow-up formula, or toddler formula. The nutritional compositions can be advantageously used as complete nutrition for infants. This means that the composition administered is not human milk. Infant formula, follow-up formula, or toddler formula means that it involves an artificially manufactured composition, or in other words, it is a synthetic composition. In the context of the invention, toddler formula may also be referred to as growing milk. The nutritional compositions of the invention are preferably intended or used to provide nutrition to infants or toddlers.

[0115] Infant formula is designed for use in infants from birth to approximately 4 to 6 months of age and is intended as a substitute for human milk. Typically, infant formula is suitable as the sole source of nutrition. Such infant formula is also known as stage 1 formula. In the context of this invention, this is referred to as a nutritional composition or infant formula for the first 6 months of life.

[0116] Follow-up formula is intended for infants aged 4 to 6 months to 12 months and is intended as a supplemental feed for infants who are weaning and beginning to eat other foods. In the context of this invention, this is referred to as a nutritional composition or follow-up formula for infants aged 6 to 12 months.

[0117] Infant formula refers to an artificially manufactured nutritional composition intended for use in children aged 12 to 47 months (inclusive), or in other words, for children aged 1 to 3 years, intended as a supplemental feed. In the context of this invention, this is referred to as a nutritional composition or infant formula for the age of 12 to 47 months (for the age of 1 to 3 years).

[0118] Infant formula and follow-up formula are subject to strict regulation, such as EU Regulation Nos. 609 / 2013 and 2016 / 127 and Codex Alimentarius for Infant Formula CODEXSTAN 72-1981. Toddler formula preferably follows the guidelines for follow-up formula.

[0119] The nutritional composition is preferably an infant formula or a follow-up formula.

[0120] Observations of human milk show that the amount of HMOs decreases with increasing infant age during lactation. In preferred embodiments, infant formula contains more HMOs than the same amount or volume of older infant formula, and older infant formula contains more HMOs than the same amount or volume of toddler formula. Since toddlers consume less formula in their total diet than infants (who may be the sole source of nutrition), in some embodiments, it is preferable to provide an increased amount of HMOs in toddler formula to provide the desired daily HMO intake.

[0121] application The HMO mixtures and nutritional compositions containing the HMO mixtures of the present invention are preferably used to provide nutrition to infants or young children, and more preferably to infants.

[0122] Preferably, the nutritional composition of the invention is provided to human subjects during the first three years of life. Preferably, the nutritional composition is used in methods for providing nutrition to human subjects during the first 12 months of life, optionally during the first three years of life, or for providing nutrition to human subjects during the first 12 months of life, optionally during the first three years of life. In one aspect, the nutritional composition comprising an HMO mixture is a first infant formula for the first six months of life, wherein the formula comprises an HMO mixture according to the invention.

[0123] This invention particularly relates to the use of HMO mixtures for the prevention and / or treatment of infections, preferably pathogenic infections. The invention further relates to reducing the risk of infections, preferably pathogenic infections. The invention further relates to the use of HMO mixtures for the prevention and / or treatment of pathogenic infections, preferably viral and / or bacterial infections, more preferably at least viral and bacterial infections in human subjects. The HMO mixtures contain a variety of different HMOs, providing a variety of properties and biological activities, including effects on pathogen binding, regulation of pathogen cell binding, and regulation of immune responses. Advantageously, the HMO mixtures prevent and / or treat more than one pathogenic infection; that is, the mixtures advantageously provide prevention and / or treatment against a wide range of pathogenic infections. Synthetic HMO mixtures further advantageously prevent the development of resistance to the prevention and / or treatment of pathogenic infections. By providing a broad range of HMO structures, escape of pathogens from specific HMOs is prevented. Therefore, the HMO mixtures advantageously provide long-term prevention and / or treatment against pathogenic infections.

[0124] Without being bound by theory, it is believed that the synthesized HMO mixtures according to the present invention exhibit greater resistance to the evolution of microbial pathogenicity. In this way, the HMO mixtures will possess future applicability and resistance to pathogen mutations, thereby providing long-term beneficial effects. Furthermore, it is believed that providing a broad spectrum of HMOs also allows for the provision of a mixture to meet the needs of subjects with varying susceptibility and genetic diversity responding to HMO structures.

[0125] In one aspect, the present invention relates to synthetic HMO mixtures for the prevention and / or treatment of infections, preferably pathogenic infections, and nutritional compositions comprising the HMO mixtures, wherein the prevention and / or treatment of infection is improved compared to the prevention and / or treatment of infection observed with oligosaccharide mixtures of fewer than six different oligosaccharides. In a preferred embodiment, the prevention and / or treatment of infection is improved compared to the prevention and / or treatment of infection observed with oligosaccharide mixtures of fewer than six different oligosaccharides (including 2'-FL, GOS, and FOS) and / or with five HMO mixtures.

[0126] In another aspect, the present invention relates to HMO mixtures, preferably pathogenic infections, for the prevention and / or treatment of infections, wherein the prevention and / or treatment of infection is comparable to and / or improved compared to the prevention and / or treatment of infection by HMOs in human breast milk. Therefore, the effects of the HMO mixtures and the nutritional compositions containing the HMO mixtures are at least closer to the effects of human breast milk.

[0127] In one aspect, the present invention relates to non-therapeutic uses and methods comprising administering an HMO mixture and a nutritional composition comprising the HMO mixture for improving the resistance of human subjects (preferably healthy infants and / or full-term infants) to infection, preferably pathogenic infection.

[0128] In another preferred aspect, the present invention relates to HMO mixtures for use in therapy and nutritional compositions comprising the HMO mixtures. The present invention relates to therapeutic use, wherein the human subject is preferably selected from premature infants, (very) low birth weight infants, infants born by cesarean section, and infants receiving antibiotic treatment. The latter group of infants has a dysbiosis and is particularly susceptible to pathogenic infections.

[0129] In a preferred aspect, the HMO mixture and the nutritional composition comprising the HMO mixture are used for the prevention and / or treatment of infections. More preferably, the use for the prevention and / or treatment of pathogenic infections includes the prevention and / or treatment of at least viral and / or bacterial pathogenic infections.

[0130] In certain respects, HMO mixtures are used for the prevention and / or treatment of infections caused by viruses selected from double-stranded RNA viruses, single-stranded positive and antisense RNA viruses, and DNA viruses, and / or bacteria selected from Gram-positive and Gram-negative bacteria. In one respect, the pathogenic bacteria are selected from Listeriaceae, Enterobacteriaceae, and Staphylococciceae families.

[0131] In another specific aspect, the HMO mixture is used for the prevention and / or treatment of infections, preferably pathogenic infections, caused by viruses selected from rotavirus, respiratory syncytial virus (RSV) and SARS-CoV-2 and / or bacteria selected from Listeria, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis and Klebsiella pneumoniae, preferably Escherichia coli.

[0132] In other words, the present invention relates to the use of HMO mixtures in the manufacture of nutritional compositions for the prevention and / or treatment of infections, preferably pathogenic infections, in human subjects.

[0133] In some jurisdictions, this can also be expressed as the use of HMO mixtures to improve the resistance of human subjects (preferably healthy infants, full-term infants or young children) to infection, preferably pathogenic infection, particularly for non-therapeutic improvement of the resistance of human subjects (preferably healthy infants, full-term infants or young children) to infection, preferably pathogenic infection.

[0134] Example Example 1: HMO mixture An example HMO mixture is provided.

[0135]

[0136] Synonyms for: LNDFH III Test compounds The test compounds used in Examples 2 through 5 meet the following specifications: - Total HMO The fraction represents all HMOs that can be found in mature human milk, at the corresponding weight ratio observed in mature human breast milk, and with a DP between 3 and 28 or higher. This HMO fraction is obtained by removing lactose and mineral content from the total carbohydrate-mineral fraction extracted from mature, merged human milk (see Chia LW, Mank M, et al., Cross-feeding between). Bifidobacterium infantis and Anaerostipes caccae Onlactose and human milk oligosaccharides [Bifidobacterium infantis and Corynebacterium fecalis crossfeeding between lactose and human milk oligosaccharides]. Benef Microbes. 2021; 12(1):69-83.

[0137] 19 types of HMOs The synthetic mixture was produced by recombination of defined single neutral and acidic HMO fractions with a degree of polymerization (DP) between 3 and 6. These acidic and neutral HMO fractions were also obtained from total carbohydrate and mineral fractions of human milk by preparative SEC, as described by Chia et al. The 19 HMO mixtures used were prepared according to the amounts of the mixture in Example 1 above.

[0138] For a ratio of 9:1 GOS: A mixture of FOS, scGOS is derived from Vivinal GOS, and lcFOS is derived from RaftilineHP. 5 types of HMO The mixture of 48.5% 2'-fucosyllactose, 11.6% 3-fucosyllactose, 26.0% lactose-N-tetrasaccharide, 4.5% 3'-sialyllactose and 5.2% 6'-sialyllactose was obtained from Rheinbritbach-Köhhansen GmbH, Germany.

[0139] Example 2: The effect of HMO mixtures on SARS-CoV-2 infection To evaluate the potential of the 19 HMO mixtures from Example 1 to neutralize or provide anti-infective effects against positive single-stranded RNA viruses (e.g., SARS-CoV2 WA1 (USA-WA1 / 2020 isolate; BEI Resources, catalog number NR-52281)), human lung epithelial cancer cells (A549-hACE2 / TMPRSS2; Invivogen, a549-hace2tpsa) overexpressing the major entry receptor for SARS-CoV-2 angiotensin-converting enzyme 2 (ACE2) and its entry cofactor (membrane-bound protease TMPRSS2) were cultured according to the following protocol: Virus pre-incubation protocol: Pre-incubation of SARS-CoV2 WA1 with HMO prior to infection of A549-hACE2 / TMPRSS2 cells.

[0140] Cell inoculation A549-hACE2 / TMPRSS2 cells were seeded in 96-well plates (Costar, 3610) in medium (DMEM Glutamax, 10% FBS, 100 U / mL penicillin-streptomycin, 0.5 µg / mL puromycin, 300 µg / mL hygromycin B, 100 mM sodium pyruvate) and incubated overnight at 37°C and 5% CO2.

[0141] Test compounds The HMO mixture was prepared into a 16 mg / ml stock solution in sterile water and then serially diluted 2-fold in infection medium (DMEM Glutamax, 2% FBS, 100 U / mL penicillin-streptomycin) to obtain working dilutions of 16, 8, 4, 2, 1 and 0.5 mg / ml.

[0142] The ability of the following compounds to inhibit SARS-CoV2 infection was evaluated at the above 6 concentrations and in triplicate: -2'-FL - A mixture of GOS:FOS at a weight ratio of 9:1 -Total HMO -19 HMO mixtures -6'-SL - Untreated control Virus incubation Transfer the test compound to a plate containing the virus and add additional infection medium to ensure the test concentration range is 0.5–16 mg / mL. Incubate the plate at 37°C and 5% CO2 for 1 hour.

[0143] Infection with A549-hACE2 / TMPRSS2 cells The culture medium was removed from the A549-hACE2 / TMPRSS2 cell plates and replaced with HMO-treated SARS-CoV-2 to achieve a multiplicity of infection (MOI) of 0.1. The plates were incubated at 37°C and 5% CO2 for 1 h. Then, the culture medium was removed from the plates; all wells were washed once with 1X PBS and replenished with 100 µl of fresh culture medium. The plates were incubated at 37°C and 5% CO2 for 48 h.

[0144] Assay for viral inhibition To evaluate the efficacy of different test compounds in inhibiting viral infection, viral RNA (vRNA) was quantified by RT-qPCR using oligonucleotides and probes from the N gene of SARS-CoV-2 (IDT; 10006830, 10006831, and 10006832) with a Taqman Fast Virus 1-step kit (Thermo Fisher Scientific, 4444434). Data analysis was performed using GraphPad Prism v9.1, where data were calculated as a percentage of the untreated control and then normalized relative to the effect of the lowest compound concentration.

[0145] result The reduction in viral RNA was associated with a decrease in the presence of virus-infected cells. Within the assessed concentration range, 2'-FL and 6'-SL showed no virus-neutralizing efficacy after re-incubation of SARS-CoV2 WA1 with these HMOs. GOS:FOS and a mixture of 19 HMOs showed virus-neutralizing efficacy after pre-incubation at higher concentrations, while the total HMO mixture showed strong antiviral activity at lower concentrations. Figure 1 ).

[0146] Overall, a mixture of 19 HMOs was found to be effective in neutralizing the virus, with effects comparable to a mixture of total HMOs and improvements compared to single fucoidylated oligosaccharides or sialylated oligosaccharides.

[0147] Example 3: The effect of HMO mixtures on rotavirus infection To assess the effect of HMO mixtures on double-stranded RNA virus infection, the effect of HMOs on rotavirus infection was evaluated in vitro using the Caco-2 epithelial cell barrier model.

[0148] Caco-2 cells were used according to established methods. In short: cells were cultured in MEM (minimum essential medium) supplemented with 10% FCS heat-inactivated, 100 units / ml penicillin, 0.1 mg / ml streptomycin, 2 mM sodium pyruvate, and non-essential amino acids at a density of 0.3 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1.13 cm⁻¹. 2 ThinCert inserts (polyethylene terephthalate membrane (Greiner, Monroe, NAT, USA), 0.4 μm pore density) were placed in 12-well plates. Caco-2 cells were maintained in a humidified atmosphere of 37°C, 95% air, and 5% CO2. Transepithelial resistance (TEER) was measured as a quantitative marker of barrier integrity. After 3 weeks of culture, confluent monolayers were obtained, and the mean TEER, measured using a Millicell resistance system voltammometer (Millipore, Temecula, CA, USA), exceeded 400 W / cm². 2 .

[0149] After 3 weeks of culture, the cells differentiated well and formed a barrier layer on ThinCert insert plates. Subsequently, the cells were incubated with different test compounds (i.e., a mixture of 19 HMOs, a mixture of 5 HMOs, culture medium only, lactose, cellobiose, or lactoferrin) in serum-free medium at a concentration of 50 mg / ml for 4 h, followed by the addition of rotavirus (RV) strain SA11 with an MOI of 0.1.

[0150] On the second day, 20 hours post-infection (pi), transepithelial electrical resistance (TEER) and firefly yellow (LY) permeability were measured to investigate barrier integrity. For TEER measurements, TEER values ​​were measured using a Millicel-ERS voltammometer connected to a pair of chopstick electrodes. For cell-side tracer flux assays, membrane-impermeable firefly yellow (LY) (Sigma, St. Louis, Missouri, USA) was added to assess barrier leakage. Firefly yellow at a concentration of 32 μg / ml was added to the top compartment of a ThinCert plate and incubated for 48 h. Cell-side flux was determined by measuring fluorescence intensity in the basal outer compartment using a fluorescence spectrophotometer (FlexStation 3, Molecular Devices, San Jose, CA, USA) (set at excitation and emission wavelengths of 410 and 520 nm, respectively). LY diffusion was assessed at 48 h post-infection (pi, post-infection; RV, rotavirus; LY, firefly yellow).

[0151] Results and conclusions: Exposure to a 5% mixture of five HMOs almost completely prevented the reduction of rotavirus-induced TEER at 20 h p, indicating a protective effect against infection. Figure 2A Exposure to a 5% mixture of 19 HMOs showed similar preventative effects, with the lower amplitude reflecting the slightly lower concentrations of the five HMO structures present in the 19-HMO mixture. Using different control sugars (e.g., 5% lactose or cellobiose (no effect)) indicated that the observed effects were specific to oligosaccharides in both the five HMOs and the 19-HMO mixture. Barrier leakage results ( Figure 2B The results showed that pre-incubating Caco-2 cells with 5% of 5 types of HMOs and 5% of 19 types of HMOs prevented barrier disruption and LY diffusion to the basal lateral compartment.

[0152] Overall, the data show that the 19 HMO mixtures according to the present invention are effective against rotavirus infection.

[0153] Example 4: The impact of HMOs on RSV infection To assess the effect of HMO mixtures on antisense single-stranded RNA viruses, in vitro infection assays were performed using the RSV-A2 strain. Pre-incubation of HEp-2 cells or RSV-A2 with HMOs before infection was compared to evaluate antiviral activity. Hep2 cells were exposed to the following conditions, as discussed separately in more detail: I. Pre-incubation of HEp-2 cells with HMO before viral infection.

[0154] II. Pre-incubation of RSV-A2 with HMO before infecting HEp-2 cells.

[0155] I. Hep-2 cells were pre-incubated with HMOs, followed by viral infection. Cell inoculation HEp-2 cells were seeded in white [Glenore 655098] 96-well plates in assay medium (EMEM [Sigma M2279]) supplemented with 2% heat-inactivated fetal bovine serum (HI-FBS) [Gibco 10500064], 1% penicillin / streptomycin [Gibco 15140122] and 1% L-glutamine [Gibco 25030024] and incubated at 37°C and 5% CO2 for 5 hours.

[0156] Treatment of HEp-2 cells The HMO mixture was prepared in sterile water, and ribavirin (a compound that blocks viral RNA synthesis and caps viral mRNA) was prepared in 1% DMSO at a 10-fold test concentration in a round-bottom 96-well plate [Corning 3788], followed by serial 2-fold dilutions.

[0157] The ability of the following compounds to inhibit RSV-A2 virus infection was evaluated: -2'-FL - A mixture of GOS:FOS at a weight ratio of 9:1 -Total HMO -5 HMO mixtures -19 HMO mixtures -6'-SL The test compound was then transferred to HEp-2 cell plates. Additional assay medium was added to ensure the final HMO concentration ranged from 0.13 to 16 mg / mL. The cell plates were incubated at 37°C and 5% CO2 for 24 hours.

[0158] Viral infection After incubation for 24 hours under the test conditions, RSV-A2 was added to HEp-2 cell plates containing HMOs to achieve a final multiplicity of infection (MOI) of 0.5. The cell plates were then incubated at 37°C and 5% CO2 for another 72 hours.

[0159] Assay for viral inhibition Viral ToxGlo™ [Promega G8943] was added to the plates to evaluate cell metabolism following viral infection and HMO treatment. After 20 minutes of incubation, luminescence was measured on each plate using a spectrophotometer. Data were analyzed using GraphPad Prism software to determine the concentration that effectively produced 50% viral inhibition, expressed as EC50 of ribavirin and HMO. 50 Value (in relevant cases).

[0160] result The experiment was conducted in duplicate. The percentage of viral inhibition is shown in Table 1. For some test compounds, the assessed concentration range was too low to accurately determine the EC50. 50 The values ​​show the EC values ​​of a mixture of 2'-FL and 19 HMOs. 50 value.

[0161] In replicate 1, total HMOs, 2'-FL, and a mixture of 19 HMOs inhibited infection when pre-incubated with HEp-2 cells. This suggests that these HMOs act on cells prior to infection to prevent infection. While the GOS:FOS mixture showed some very limited antiviral activity (<50% inhibition) in the first replicate, 6'-SL showed no antiviral activity at the tested concentration.

[0162] In replicate 2, a higher infection percentage was achieved (94% in replicate 2 compared to 67% in replicate 1, data not shown), leading to the need for a higher dose of the positive control ribavirin to achieve EC. 50 Under these conditions of high infection percentage, only 19 HMO mixtures were found to show beneficial inhibition of infection when pre-incubated with HEp-2 cells. Notably, despite the high infection percentage in the second replicate, the 19 HMO mixtures still maintained a >50% inhibitory effect on viral infection (data not shown).

[0163] Table 2. Percentage of virus inhibition achieved by pre-incubating Hep-2 cells with different test compounds or mixtures.

[0164]

[0165] II. RSV-A2 pre-incubation with HMO before infecting HEp-2 cells Cell inoculation HEp-2 cells were seeded as described above and incubated at 37°C and 5% CO2 for 5 hours.

[0166] RSV-A2 processing As previously described, HMO and ribavirin were prepared at 10-fold test concentrations. RSV-A2 was diluted in assay medium and added to round-bottom 96-well plates [Corning 3788]. The ability of the following compounds to inhibit RSV-A2 viral infection was evaluated: -2'-FL - A mixture of GOS:FOS at a weight ratio of 9:1 -5 HMO mixtures -19 HMO mixtures -6'-SL - Ribavirin (positive control for viral inhibition) Transfer the test compound to a plate containing the virus and add additional assay medium to ensure the test concentration range is 0.13–16 mg / mL. Incubate the plate at 37°C and 5% CO2 for 1 hour.

[0167] HEp-2 cell infection The assay medium was removed from the HEp-2 cell plates and replaced with HMO-treated RSV-A2 to achieve an MOI of 0.5. The plates were then incubated at 37°C and 5% CO2 for another 72 hours.

[0168] Assay for viral inhibition Viral ToxGlo™ [Promega G8943] was added to the plates to evaluate cell metabolism following RSV-A2 infection with either an untreated RSV-A2 control or HMO pretreatment. After 20 minutes of incubation, luminescence was measured on each plate using a spectrophotometer. Data were analyzed using GraphPad Prism software to generate EC50 values ​​for ribavirin and HMO. 50 Value (in relevant cases).

[0169] result The experiments were conducted in duplicate. The percentage of virus inhibition for each experimental condition is shown in Table 3. For some test compounds, the evaluated concentration range was too low to accurately determine the EC50. 50 The values ​​show the EC values ​​of a mixture of GOS:FOS and 19 HMOs. 50 value.

[0170] GOS:FOS and a mixture of 19 HMOs showed antiviral activity upon pre-incubation with RSV-A2 before infection. This was observed in both replicates, with similar infection percentages (94%–95%) achieved in the RSV-A2 control group. 6'-SL showed no antiviral activity at the tested concentrations, and the mixture of 5 HMOs showed only low levels of viral inhibition.

[0171] Overall, the 19-HMO mixture appeared to have broad effects on both viral neutralization and inhibition of cell infection, similar to the effects of total HMO, but superior to those observed with other oligosaccharides. Despite a higher percentage of infection in the second replicate, only the 19-HMO mixture maintained >50% inhibition of viral infection after Hep2 cell pre-incubation.

[0172] Table 3. Percentage of virus inhibition achieved by pre-incubating RSV-A2 with different test compounds or mixtures.

[0173]

[0174] Example 5: The effect of HMOs on the growth of pathogenic bacteria The effects of total HMO mixtures, mixtures of 19 HMOs, mixtures of 5 HMOs, and mixtures of total HMOs and lactose on inhibiting the growth of Gram-negative enterobacteria such as Escherichia coli at concentrations ranging from 0.5 mg / ml to 16 mg / ml were assessed by growth percentage assays (Table 4).

[0175] Escherichia coli was inoculated into 5 ml of YCFA broth (YCFAG) supplemented with glucose as the sole energy source and incubated anaerobically at 37°C for 24 h. Subsequently, the strain was multiplied overnight in 5 ml of YCFAG broth as a pre-culture; then, different strains were added in duplicate at a ratio of 1:100 to tubes supplemented with different carbon sources of YCFA broth at 37°C. OD600 was measured using an Ultraspec 10 cell density meter (Amersham Biosciences GmbH, Germany) from 0-h to 72-h, and growth in the cultures was monitored spectrophotometrically every 4 h.

[0176] Growth percentage was determined by assessing the total HMO fraction using lactose as a negative control using the following four parameters: i) µMAX (A), ii) time to reach 50% OD600, iii) maximum OD, and iv) area under the curve. Therefore, a higher growth percentage indicates less or no growth inhibition, and vice versa.

[0177] Overall, 19 HMO mixtures were found to effectively inhibit the growth of bacteria and a variety of viruses, thus it is considered a multifunctional mixture that provides broad-spectrum protection against pathogenic infections.

[0178] Example 6 - Infant formula with a mixture of 19 HMOs This infant formula is intended for infants aged 0-6 months and contains per 100 ml (obtained by reconstituted 13.7 g of powder with water): -67 kcal - Digestible carbohydrates (mainly lactose): 7.3 g - Protein (whey protein, casein): 1.3 g -Lipids: 3.4 g - 0.9 g of non-digestible oligosaccharides consisting of the following: - 0.7 g GOS / lcFOS, wt / wt ratio 7.6 : 1 - 0.2 g of a mixture of 19 HMOs according to Example 1, comprising: 28.3 wt% 2'-FL, 21.2 wt% 3-FL, 2.1 wt% 3'-SL, 4.1 wt% 6'-SL, 3.4 wt% DFL, 7.5 wt% LNT, 0.9 wt% LNnT, 3.3 wt% LNFP I, 4.1 wt% LNFPII, 3.3 wt% LNFP III, 0.3 wt% LNFP V, 1.2 wt% LSTa, 1 wt% LSTb, 4.5 wt% LSTc, 4.6 wt% LNDH I, 0.001 wt% LNnDFH I, 1 wt% LNDFH II and LNnDFHII, 9.3 wt% DSLNT, - Micronutrients as specified in the Infant Formula Directive Example 7 - Infant formula with a mixture of 19 HMOs This infant formula is intended for infants aged 0-6 months and contains per 100 ml (obtained by reconstituted 13.7 g of powder with water): -67 kcal - Digestible carbohydrates (mainly lactose): 7.3 g - Protein (whey protein, casein): 1.3 g -Lipids: 3.4 g - 0.9 g of non-digestible oligosaccharides consisting of the following: - 0.32 g GOS / lcFOS, wt / wt ratio of 2.6 : 1 - 0.58 g of a mixture of 19 HMOs according to Example 1, comprising: 28.3 wt% 2'-FL, 21.2 wt% 3-FL, 2.1 wt% 3'-SL, 4.1 wt% 6'-SL, 3.4 wt% DFL, 7.5 wt% LNT, 0.9 wt% LNnT, 3.3 wt% LNFP I, 4.1 wt% LNFPII, 3.3 wt% LNFP III, 0.3 wt% LNFP V, 1.2 wt% LSTa, 1 wt% LSTb, 4.5 wt% LSTc, 4.6 wt% LNDH I, 0.001 wt% LNnDFH I, 1 wt% LNDFH II and LNnDFHII, 9.3 wt% DSLNT. - Micronutrients as specified in the Infant Formula Directive Example 8: Infant formula containing a mixture of 19 HMOs This infant formula is intended for infants aged 0-6 months and contains per 100 ml (obtained by reconstituted 13.7 g of powder with water): -66 kcal - Digestible carbohydrates (mainly lactose): 7.2 g - Protein (whey protein, casein): 1.3 g -Lipids: 3.4 g - 0.872 g of non-digestible oligosaccharides consisting of the following: - 0.7 g GOS / lcFOS, wt / wt ratio of 9:1 - 0.2 g of a combination of 19 HMOs consisting of: 28.3 wt% 2'-FL, 21.2 wt% 3-FL, 2.1 wt% 3'-SL, 4.1 wt% 6'-SL, 3.4 wt% DFL, 7.5 wt% LNT, 0.9 wt% LNnT, 3.3 wt% LNFP I, 4.1 wt% LNFPII, 3.3 wt% LNFP III, 0.3 wt% LNFP V, 1.2 wt% LSTa, 1 wt% LSTb, 4.5 wt% LSTc, 4.6 wt% LNDH I, 0.001 wt% LNnDFH I, 1 wt% LNDFH II and LNnDFH II, 9.3 wt% DSLNT - Micronutrients as specified in the Infant Formula Directive.

[0179] Example 9: Follow-up infant formula containing a mixture of 19 HMOs This follow-up formula is intended for infants aged 6-12 months and contains per 100 ml (obtained by reconstituted 14.4 g of powder with water): -68 kcal - Digestible carbohydrates (mainly lactose): 8.2 g - Protein (whey protein, casein): 1.4 g -Lipids: 3.2 g - 0.786 g of non-digestible oligosaccharides composed of the following: - 0.7 g GOS / lcFOS, wt / wt ratio of 9:1 - 85 mg consists of a combination of 19 HMOs: 28.3 wt% 2'-FL, 21.2 wt% 3-FL, 2.1 wt% 3'-SL, 4.1 wt% 6'-SL, 3.4 wt% DFL, 7.5 wt% LNT, 0.9 wt% LNnT, 3.3 wt% LNFP I, 4.1 wt% LNFPII, 3.3 wt% LNFP III, 0.3 wt% LNFP V, 1.2 wt% LSTa, 1 wt% LSTb, 4.5 wt% LSTc, 4.6 wt% LNDH I, 0.001 wt% LNnDFH I, 1 wt% LNDFH II and LNnDFH II, 9.3 wt% DSLNT - Micronutrients as specified in the Guidelines for Formula for Older Infants Example 10: Infant formula containing a mixture of 19 HMOs This infant formula is intended for infants aged 12 to 47 months and contains per 100 ml (obtained by reconstituted 14.4 g of powder with water): -65 kcal - Digestible carbohydrates (mainly lactose): 8.3 g - Protein (whey protein, casein): 1.3 g -Lipids: 2.7 g - Non-digestible oligosaccharides: 1.243 g, composed of the following: - 1.2 g GOS / lcFOS, wt / wt ratio of 9:1 - 43 mg consists of a combination of 19 HMOs: 28.3 wt% 2'-FL, 21.2 wt% 3-FL, 2.1 wt% 3'-SL, 4.1 wt% 6'-SL, 3.4 wt% DFL, 7.5 wt% LNT, 0.9 wt% LNnT, 3.3 wt% LNFP I, 4.1 wt% LNFPII, 3.3 wt% LNFP III, 0.3 wt% LNFP V, 1.2 wt% LSTa, 1 wt% LSTb, 4.5 wt% LSTc, 4.6 wt% LNDH I, 0.001 wt% LNnDFH I, 1 wt% LNDFH II and LNnDFH II, 9.3 wt% DSLNT - Micronutrients as specified in the Follow-up Infant Formula Directive.

[0180] Example 11 - Effects of HMO mixtures on Clostridium difficile toxins To assess the potential of 19 HMO mixtures to provide anti-infective effects against Clostridium difficile infection (a Gram-positive bacterium), a 3D intestinal model was used to evaluate the role of HMOs in the intestinal barrier.

[0181] Caco-2 cell tubular model (organoplate) ® ） In the method of this invention, an organoplate (organoplate 3, channel 40, Mimetas, Leiden, Netherlands) was used as a three-dimensional tubular model. Caco-2 cells were introduced into the chip and maintained in Duchenne Modified Igor Medium (DMEM) supplemented with high glucose (4.5 g / L), GlutaMAX, and 10% FCS (Gibco). After three days, the tubular structures formed. The experimental procedure began on the fourth day after cell introduction.

[0182] Human milk oligosaccharides The five HMO mixtures (2'-FL, 3-FL, LNT, 3'-SL, 6'-SL) and 19 HMO mixtures according to the present invention were tested at the same total concentration of 9 g / L.

[0183] Intestinal barrier attack To simulate the transition of the intestinal barrier from tight to leaky during Clostridium difficile infection, a tubular model was challenged with toxin A (TcdA) derived from Clostridium difficile (SML1154-2UG, Sigma-Aldrich). The toxin was applied at a concentration of 10 ng / mL in FCS-free medium. This exposure was sustained for 16 hours in both the presence and absence of human lactose oligosaccharides (HMOs). Cell culture was performed using Organoter in a cell incubator. ® (Mimitas) monitors transepithelial resistance (TEER). The percentage of TEER that evolves over time is determined as a quantitative marker of barrier integrity.

[0184] Data Analysis In three separate experiments, each mixture was applied to five chips (reproduced). Statistical analysis was performed using SPSS, and an estimated marginal mean model was used to explain the differences between experiments. A post-experimental LSD test was then conducted.

[0185] result The mean TEER of the untreated tubular models was 77.6%, which significantly decreased after exposure to TcdA (Table 4). Exposure of the tubular models to TcdA in combination with a mixture of five HMOs improved TEER compared to exposure to toxins without HMOs. The TEER of the tubular models exposed to both TcdA and a mixture of 19 HMOs was even further (and significantly) restored.

[0186] Table 4. Estimated marginal mean of the effect of different HMO mixtures on TEER evolution.

[0187]

[0188] The results showed that exposure to a mixture of 19 HMOs had a preventive or protective effect against Clostridium difficile toxin-induced intestinal barrier disruption.

[0189] Example 12 - Effect of HMO mixtures on synthesized dsRNA virus substitutes To evaluate the effects of 19 HMO mixtures on the inflammatory response to double-stranded RNA virus infection, an in vitro model was used in which HT-29 cells were pre-incubated with the test compounds and then challenged with Lyovec-complexed polyI:C as a synthetic substitute for dsRNA infection.

[0190] Materials and methods Cell culture HT-29 cell lines (HTB-38™, ATCC) were seeded (65,000 cells / mL; 100 µl / well) in 96-well flat-bottomed plates (Falcon) using McCoy 5A medium (Thermo Fisher Scientific, 26600080) supplemented with 10% heat-inactivated FCS (Gibco, A5256701) and 1% penicillin / streptomycin (Thermo Fisher Scientific, 15140130). Cells were incubated at 37°C and 5% CO2, and the medium was replaced on day 3.

[0191] Pre-incubation with test compound On day 7, when the cells reached confluence, they were washed once with PBS (Thermo Fisher Scientific, 14190144). Then, the cells were exposed to culture medium (McCoy 5A supplemented with 2% heat-inactivated FCS and 1% penicillin / streptomycin) or 1% (w / v) 2'FL, 5 HMOs or 19 HMOs for 24 hours.

[0192] Stimulation with poly(I:C)After 24 h of incubation, the culture medium was removed, and the cells were re-exposed to fresh culture medium (McCoy 5A supplemented with 2% heat-inactivated FCS and 1% penicillin / streptomycin) or 2 µg / mL Lyovec complex poly-I:C (Ingenie, tlrl-picwlv) for 24 h.

[0193] Assay of cell viability and IL-8 secretion After incubation, the supernatant was collected and stored at -20°C for further analysis. Cell viability was also measured according to the manufacturer's protocol using LDH-Glo™ (Promega, J2381) and WST-1 (Merck, 11644807001). Results showed no viability issues after stimulation. IL-8 secretion in the conditioned supernatant was measured using the Human IL-8 / CXCL8 DuoSet ELISA Kit (R&D Systems, DY208). Therefore, the supernatant was diluted (25-fold) with reagent diluent according to the manufacturer's protocol.

[0194] result The response of IL-8 secretion to challenge with poly-I:C (an alternative to dsRNA viral infection) was assessed (Table 5). Exposure to poly-I:C resulted in a significant increase in mean IL-8 secretion, while exposure to a mixture of 19 HMOs resulted in a significant decrease in mean IL-8 secretion; this effect was not observed in 2'-FL or 5 HMOs. The results suggest a beneficial prevention of inflammatory responses induced by viral infection.

[0195] Table 5. Mean IL-8 secretion (pg / ml) of cells exposed to control conditions and cells challenged with poly-I:C and exposed to cellobiose, lactoferrin, a mixture of 5 HMOs or a mixture of 19 HMOs.

[0196]

[0197] Example 13 - Effects of 19 HMO mixtures on the growth inhibition of pathogenic bacteria The effects of total HMO mixtures, mixtures of 19 HMOs, mixtures of 5 HMOs, and 2'-FL at concentrations ranging from 1 mg / ml to 16 mg / ml on the growth inhibition of different bacterial strains (Escherichia coli, Salmonella typhimurium, Proteus mirabilis, and Staphylococcus aureus) were assessed by growth percentage assays and K factor inhibition assays.

[0198] Bacteria were inoculated into 5 ml of YCFA broth (YCFAG) supplemented with glucose as the sole energy source and anaerobically incubated at 37°C for 24 h. Subsequently, the strains were multiplied overnight in 5 ml of YCFAG broth culture as a pre-culture. Then, different strains were added in duplicate at a ratio of 1:50 to tubes supplemented with different carbon sources in YCFA broth at 37°C and pH 5.5. OD600 was measured using an Ultraspec 10 cell density meter (Amasia Biosciences, Germany) from 0 h to 24 h, and growth in the cultures was monitored spectrophotometrically.

[0199] Calculation of the area under the curve (AUC) The area under the curve (AUC) was calculated using samples collected up to the time point corresponding to the reference line. The reference line was determined as the average of measurements taken during the early stationary phase of the growth curve (N = 10). All calculations were performed using GraphPad Prism software. Therefore, a higher AUC indicates less or no growth inhibition, and vice versa.

[0200] Growth factor (k-factor) calculation Growth factors, denoted as (k) factors, were determined using a nonlinear fit to the logistic growth model. The k factor represents the growth rate of the bacterial population. This analysis was also performed using GraphPad Prism software. The logistic growth model used is defined by the following equation: Y=(YM-Y0) exp(-k x)+Y0YM Y0. Here, (Y0) is the initial population, (YM) is the largest population, (k) is the rate constant (the reciprocal of (X)), and (\frac{1}{k}) is the (X) coordinate of the first inflection point, indicating the time when the growth rate begins to slow down.

[0201] Escherichia coli E374 The effect of HMO mixtures on the growth inhibition of Escherichia coli E374 (ETEC, ATCC 35401) was evaluated, and the AUC and k-factor were calculated based on the growth curves (Tables 6 and 7, respectively).

[0202] Table 6. Percentage of growth inhibition of Escherichia coli 374 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL based on AUC.

[0203]

[0204] Table 7. Percentage of growth inhibition of Escherichia coli 374 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL, expressed as K factor.

[0205]

[0206] Starting at low doses, a dose-dependent inhibition of Escherichia coli growth was observed in the mixture of 19 HMOs, with both the percentage inhibition and the inhibition of the K factor being higher than those observed against the total HMOs. These results demonstrate the anti-infective potential of the 19 HMO mixtures according to the present invention.

[0207] Staphylococcus aureus S17 The effect of HMO mixtures on the growth inhibition of Staphylococcus aureus S17 (ATCC 29213) was evaluated, and the AUC, percentage of growth inhibition, and k-factor were calculated based on the growth curves (Tables 8 and 9, respectively).

[0208] Table 8. Percentage of growth inhibition of Staphylococcus aureus S17 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL based on AUC.

[0209]

[0210] Table 9. Percentage of growth inhibition of Staphylococcus aureus S17 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL, expressed as K factor.

[0211]

[0212] Both the mixture of 19 HMOs and total HMOs were observed to inhibit the growth of Staphylococcus aureus S17 in a dose-dependent manner, showing significant inhibition at 8 mg / ml and 16 mg / ml. The percentage inhibition and inhibition K factor of the 19 HMOs were similar to those observed with total HMOs. The results support the broad anti-infective potential of the mixture of 19 HMOs according to the present invention.

[0213] Salmonella Typhimurium S19 The effect of HMO mixtures on growth inhibition of Salmonella Typhimurium S19 (ATCC 14028) was evaluated, and AUC, percentage of growth inhibition and k factor were calculated based on growth curves (Tables 10 and 11, respectively).

[0214] Table 10. Percentage of growth inhibition of Salmonella Typhimurium S19 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL based on AUC.

[0215]

[0216] Table 11. Percentage of growth inhibition of Salmonella Typhimurium S19 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL, expressed as K factor.

[0217]

[0218] Starting at low doses, a dose-dependent inhibition of Salmonella Typhimurium S19 growth was observed in a mixture of 19 HMOs, with both the percentage inhibition and the inhibition factor K of the 19 HMOs being higher than the inhibition observed against the total HMOs. These results demonstrate that the 19 HMO mixtures according to the present invention possess broad and potent anti-infective potential.

[0219] Proteus mirabilis P80 The effect of HMO mixtures on growth inhibition of Proteus mirabilis P80 was evaluated, and AUC, percentage of growth inhibition, and k-factor were calculated based on growth curves (Tables 12 and 13, respectively).

[0220] Table 12. Percentage of growth inhibition of Proteus mirabilis P80 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL based on AUC.

[0221]

[0222] Table 13. Percentage of growth inhibition of Salmonella Typhimurium S19 exposed to different concentrations of total HMO, 19 HMOs, 5 HMOs and 2'-FL, expressed as K factor.

[0223]

[0224] Both the mixture of 19 HMOs and the total HMOs were observed to inhibit the growth of *Proteus mirabilis* P80 in a dose-dependent manner, showing inhibition starting from 2 mg / ml. The percentage of inhibition and the inhibition factor K of the 19 HMOs were similar to those observed with the total HMOs.

[0225] The results support the anti-infective potential of the 19 HMO mixtures according to the invention, which show broad anti-infective potential against Gram-negative pathogenic bacteria Escherichia coli, Salmonella typhimurium and Proteus mirabilis, as well as Gram-positive bacteria Staphylococcus aureus.

Claims

1. A synthetic HMO mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO, and wherein the mixture comprises at least 19 HMOs.

2. The synthesized HMO mixture according to claim 1, wherein the acidic HMOs comprise at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:

2.

3. The synthesized HMO mixture according to any one of claims 1 and 2, comprising 3'SL, 6'SL, LSTA, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFHI, LNnDFH I, LNDFH II, and LNnDFH II.

4. The synthesized HMO mixture according to any one of claims 1 to 3, wherein the HMO comprises, preferably substantially, the following based on the total dry weight of the synthesized HMO mixture: (i) 1.4 wt% to 2.6 wt%, more preferably 1.6 wt% to 2.4 wt%, even more preferably 1.8 wt% to 2.2 wt% 3'-SL; (ii) 3.5 wt% to 4.7 wt%, more preferably 3.7 wt% to 4.5 wt%, even more preferably 3.9 wt% to 4.3 wt% 6'SL; (iii) 0.8 wt% to 1.6 wt%, more preferably 0.9 wt% to 1.5 wt%, and even more preferably 1.0 wt% to 1.4 wt% LSTa; (iv) 0.7 wt% to 1.4 wt%, more preferably 0.8 wt% to 1.3 wt%, and even more preferably 0.9 wt% to 1.2 wt% LSTb; (v) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, and even more preferably 4.3 wt% to 4.6 wt% LSTc; (vi) 7.8 wt% to 11.2 wt%, more preferably 8.0 wt% to 11.0 wt%, and even more preferably 8.5 wt% to 10.5 wt% DSLNT; (vii) 25 wt% to 32 wt%, more preferably 26 wt% to 31 wt%, even more preferably 27 wt% to 30 wt% 2'FL; (viii) 19 wt% to 25 wt%, more preferably 20 wt% to 24 wt%, even more preferably 20.5 wt% to 23 wt% 3FL; (ix) 4.1 wt% to 4.8 wt%, more preferably 4.2 wt% to 4.7 wt%, and even more preferably 4.3 wt% to 4.6 wt% DFL; (x) 5 wt% to 10 wt%, more preferably 6 wt% to 9 wt%, even more preferably 7 wt% to 8 wt% LNT; (xi) 0.5 wt% to 1.1 wt%, more preferably 0.6 wt% to 1 wt%, even more preferably 0.7 wt% to 0.9 wt% LNnT; (xii) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP I; (xiii) 3.8 wt% to 4.4 wt%, more preferably 3.9 wt% to 4.3 wt%, and even more preferably 4 wt% to 4.2 wt% LNFP II; (xiv) 2.9 wt% to 3.6 wt%, more preferably 3 wt% to 3.5 wt%, even more preferably 3.1 wt% to 3.4 wt% LNFP III; (xv) 0.2 wt% to 0.5 wt%, more preferably 0.25 wt% to 0.45 wt%, even more preferably 0.3 wt% to 0.4 wt% LNFP V; (xvi) 4.2 wt% to 5 wt%, more preferably 4.4 wt% to 4.8 wt%, even more preferably 4.5 wt% to 4.75 wt% LNDFH I; (xvii) 0.00001 wt% to 0.02 wt%, more preferably 0.0001 wt% to 0.015 wt%, even more preferably 0.001 wt% to 0.01 wt% LNnDFH I; and (xviii) 0.7 wt% to 1.3 wt%, more preferably 0.8 wt% to 1.2 wt%, and even more preferably 0.9 wt% to 1.15 wt% of a combination of LNDFH II and LNnDFH II.

5. A nutritional composition comprising a synthetic HMO mixture according to any one of claims 1 to 4, wherein the nutritional composition is selected from infant formula, follow-up formula and toddler milk.

6. A synthetic human lactose oligosaccharide (HMO) mixture comprising 10 wt% - 30 wt% acidic HMO and 70 wt% - 90 wt% neutral HMO, wherein the neutral HMO comprises 75 wt% - 95 wt% fucosylated HMO based on the weight of the neutral HMO, for use in the treatment and / or prevention of infection, preferably pathogenic infection, wherein the mixture preferably comprises at least 19 HMOs.

7. The synthetic HMO mixture for use according to claim 6, wherein the acidic HMOs comprise at least 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), and wherein the weight ratio of 3'SL to 6'SL is between 0.5:3 and 1:1, preferably between 1:3 and 1:2, and even more preferably about 1:

2.

8. The synthetic HMO mixture according to claims 6 and 7, used for the treatment and / or prevention of pathogenic infections, preferably caused by viruses selected from double-stranded RNA viruses, single-stranded positive and antisense RNA viruses, and DNA viruses, and / or bacteria selected from Gram-positive and Gram-negative bacteria.

9. The synthetic HMO mixture for use according to any one of claims 6 to 8, wherein it is used for the treatment and / or prevention of infection, preferably pathogenic infection, which is against a virus selected from rotavirus, respiratory syncytial virus (RSV) and SARS-CoV-2 and / or Listeria monocytogenes (RSV). L. monocytogenes ), Escherichia coli ( E. coli Staphylococcus aureus ( S. aureus Salmonella typhimurium ( S. typhimurium Listeria, Enterococcus faecalis E. faecium ), Enterococcus faecalis ( E. faecalis ), Proteus mirabilis ( P. mirabilis ) and Klebsiella pneumoniae ( K. pneumoniae (i) infection caused by bacteria, preferably Escherichia coli.

10. The synthetic HMO mixture for use according to any one of claims 6 to 9, wherein the treatment and / or prevention of infection, preferably pathogenic infection, is more similar to the treatment and / or prevention of infection observed in breastfed infants, and / or is improved compared to the treatment and / or prevention of infection in infants given a composition not containing the HMO mixture.

11. The synthetic HMO mixture for use according to any one of claims 6 to 10, wherein the neutral HMOs comprise at least LNT and LNnT, and wherein the weight ratio of LNT to LNnT is between 12:1 and 1:1, preferably between 10:1 and 3:1, and even more preferably between 9:1 and 6:

1.

12. The synthetic HMO mixture for use according to any one of claims 6 to 11, wherein the HMO is selected from 3'SL, 6'SL, LSTa, LSTb, LSTc, DSLNT, 2'FL, 3'FL, DFL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNnDFH I, LNDFH II and LNnDFH II.

13. The synthetic HMO mixture for use according to any one of claims 6 to 12, wherein the HMO mixture comprises a nutritional composition selected from infant formula, follow-up formula and toddler formula.

14. A nutritional composition comprising a synthetic HMO mixture for use according to any one of claims 6 to 13, wherein the nutritional composition further comprises a Class II non-digestible sugar selected from the group consisting of: fructooligosaccharides, non-digestible dextrins, galacto-oligosaccharides (e.g., β-galacto-oligosaccharides), xylooligosaccharides, arabinose oligosaccharides, arabinose-galacto-oligosaccharides, glucosamine oligosaccharides, gentian oligosaccharides, glucomannan oligosaccharides, galacto-mannino oligosaccharides, mannan oligosaccharides, isomaltooligosaccharides, Aspergillus niger oligosaccharides, chitosan oligosaccharides, soybean oligosaccharides, uronic acid oligosaccharides, and mixtures thereof, and wherein the total weight ratio of the HMO mixture and the short-chain oligosaccharides with a DP between 3 and 6 (DP 3-6) and the long-chain oligosaccharides with a DP of 7 and above in these Class II non-digestible sugars is 5:1 to 12:1, preferably 8:1 to 10:1, and even more preferably about 9:1.

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