WO2025190896A1 - Mélange de cinq oligosaccharides de lait humain pour réduire le risque d'infection dans le tractus gastro-intestinal provoquée par e. coli pathogène - Google Patents

Mélange de cinq oligosaccharides de lait humain pour réduire le risque d'infection dans le tractus gastro-intestinal provoquée par e. coli pathogène

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Publication number
WO2025190896A1
WO2025190896A1 PCT/EP2025/056528 EP2025056528W WO2025190896A1 WO 2025190896 A1 WO2025190896 A1 WO 2025190896A1 EP 2025056528 W EP2025056528 W EP 2025056528W WO 2025190896 A1 WO2025190896 A1 WO 2025190896A1
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composition
coli
weight
fucosyllactose
sialyllactose
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English (en)
Inventor
Erik Juncker BOLL
Stina JENSEN
Sara HESSING
Olivia COLBERG
Anja Wellejus
Gerben HERMES
Andrea Qvortrup HOLST
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Chr Hansen AS
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Chr Hansen AS
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Publication of WO2025190896A1 publication Critical patent/WO2025190896A1/fr
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates, in part, to the use of a mix of human milk oligosaccharides for reducing the risk of infections in the gastrointestinal tract, particularly diarrheal diseases, caused by Escherichia coli bacteria in a human subject. Further disclosed herein are methods, uses, processes, and the like.
  • HMOs Human milk oligosaccharides
  • HMOs can be structurally categorized as (a) fucosylated HMOs such as 2’- and 3-fucosyllactose (2’-FL and 3-FL), (b) neutral non- fucosylated HMOs such as lacto-N-tetraose (LNT) and (c) sialylated HMOs such as 3’- and 6’ sialyllactose (3’-SL and 6’-SL).
  • HMOs in human milk vary widely based on various influences such as genetics, lactation, and geographic location. While most HMO concentrations decrease over the course of lactation, at least two, 3’-SL and 3-FL, may increase. Different HMOs may work together in complementary ways to support the growth and development of infants.
  • HMOs can lower the risk of gut microbiome imbalance due to harmful bacteria (Weichert, Stefan, et al. Nutrition researchi 0 (2013): 831-838). Further, it has been suggested that HMOs can selectively stimulate growth and metabolic activity of helpful bifidobacteria in support of overall gut health (Bode, Lars. Nutrition reviews, 2009, Vol 67 suppl. 2,183.191). It has been shown that mixtures of HMOs increase the relative abundance of bifidobacteria in the microbiome of formula-fed infants towards that in the microbiome of breastfed infants (Holst A. et al., Nutrients, 2023, 15, 3087). E.
  • co!i is among the first colonizers and a common inhabitant of the infant gut microbiome. It persists due to e.g., the expression of fimbriae that enables it to attach to colonic epithelial cells (Nowrouzian, F. et al., 2003, Pediatric Research, 54(1), 8-14). It comprises a wide variety of strains ranging from commensals to severe pathogens depending on the presence of virulence factors that are frequently encoded on genetic elements (Evans DJ Jr. et al. in: Baron S, Medical Microbiology, 4th edition, Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 25 and Kaper et al.; 2004, Nature Reviews Microbiology, 2(2), 123-140.), thus transferable between strains.
  • E. coli strains are harmless and contained within the intestinal lumen but may cause infection given favorable circumstances such as breaches of the gastrointestinal barriers (Kai, A. et al., 2010, Nippon Rinsho. Japanese Journal of Clinical Medicine, 68, 6(1), 203-2075) or through acquired virulence factors (Makvana, S., & Krilov, L. R., 2015, Pediatrics in Review, 36(4), 167-171).
  • E. coli strains are classified into pathotypes that are associated with distinctive severe diarrheal diseases in infants such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) (Makvana, S., & Krilov, L. R., 2015, Pediatrics in Review, 36(4), 167-171).
  • EPEC enteropathogenic E. coli
  • ETEC enterotoxigenic E. coli
  • In vitro assays indicate that mixtures of human milk oligosaccharides concentrated from human milk have an inhibitory effect on the adhesion of EPEC serotype 0119 to epithelial cells (Coppa G. V. et al., 2006, Pedriatric Research, 59, 3, 2006, 377 - 382).
  • E. coli Enteropathogenic E. coli
  • EHEC Enterohemorrhagic E. coli
  • Enterohemorrhagic E. coli is a pathotype of E. coli associated with foodborne outbreaks worldwide. Clinical manifestations of EHEC infection range from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. Infants and children are the main affected patients (Gomes TAT et al, 2016; 47; 3-30). EHEC requires binding to the intestinal epithelium by virtue of adhesins to establish infection (McWilliams BD, Torres AG, Microbiol Spectr. 2014; 2(3)). Thus, reduction of this binding may lower the risk of colonization and ensuing diarrheal disease caused by EHEC and other E. coli pathotypes.
  • E. coli is the leading cause of infantile diarrhea, which is the second most common cause of death among children under the age of five worldwide (Johansson, E. W. et al., 2009, in The United Nations Children’s Fund (UNICEF)ZWorld Health Organization (WHO), Vol. 44, 11 , 1- 68).
  • UNICEF Children’s Fund
  • WHO World Health Organization
  • Figure 1 B shows the reduction of adhesion of a strain of enteropathogenic E. coli (EPEC) serotype 0127 to monolayers of human colon cancer cells (Caco-2) in the presence of galactooligosaccharides (GOS) at a dosage of 30 mg/ml and 5HMO-mix at 5 mg/mL and 30 mg/mL.
  • EPEC enteropathogenic E. coli
  • GOS galactooligosaccharides
  • Figure 3 shows the relative abundance of E. coli in an infant in vitro colonic model, l-TIM-2 and the comparisons within donors (INF5-7) between samples of the donor fecal inocula and l-TIM-2 samples with the feed SIIEM (without HMOs) and SIIEM-HMO (with the 5HMO-mix) in independent experiments with individual donors (INF5-7).
  • A EPEC levels in the NSC and HMO study arms, quantified by qPCR and expressed as copies/mL. Statistical differences between NSC and the individual treatments are visualised via * (0.1 ⁇ Padjusted ⁇ 0.2), ** (0.05 ⁇ padjusted ⁇ 0.1) or *** (padjusted ⁇ 0.05).
  • B The impact of HMO on EPEC levels, expressed as log2Fold change vs NSC, as quantified via qPCR. The different symbols represent the 8 individual donors. Overall, lower levels of Enteropathogenic E.
  • E.coli of serotype 0127 can be observed in the presence of 5HMOmix (right) compared to a nonsubstrate control (NSC) without HMOs (left) in an infant ex vivo 20hour fermentation model and subsequently a 24 hour E.coli infection model.
  • NSC nonsubstrate control
  • Figure 5 shows the impact of 5HMO mix on (A) pH, (B) gas production, (C) total SCFA, at 20h and 44h.
  • Statistical differences between NSC and the individual treatments are visualised via * (0.1 ⁇ p adjusted ⁇ 0.2), ** (0.05 ⁇ P adjusted ⁇ 0.1) or *** (p adjusted ⁇ 0.05).
  • the different symbols represent the 8 individual donors.
  • modulation of the metabolic activity of the infant microbiota is indicated by decreased pH, increased gas production, increased production of short-chain fatty acids in the presence of 5HMO mix compared to non-substrate control without HMOs in an infant ex vivo 20hour fermentation model and subsequently a 24 hour E.coli infection model .
  • any and all examples, or exemplary language (e.g., "such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
  • the term “and/or” is intended to mean the combined (“and”) and the exclusive (“or”) use, i.e. “A and/or B” is intended to mean “A alone, or B alone, or A and B together”.
  • the terms "effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of a material sufficient to improve the overall health of the subject and confer benefits similar to the ones demonstrated in the examples.
  • the actual effective dosage in absolute numbers depends on factors including the state of health of the subject in question, and other ingredients present.
  • the "effective amount”, “effective concentration”, or “effective dosage” of the material may be determined by routine assays known to those skilled in the art.
  • isolated means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
  • a bacterial “strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied and that originates from a single isolate or pure culture. Probiotics are classified by their genus (e.g. Bifidobacterium), species and subspecies (e.g. animalis subs p. lactis), and strains (e.g. DSM 15954 and/or BB-12®). FAO/WHO has stated that probiotic effects are strain specific and that most probiotic characteristics of a particular strain cannot therefore be extrapolated to other strains of the same species.
  • probiotic refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects the host (Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic).
  • human milk oligosaccharide refers generally to a number of complex carbohydrates found in human breast milk that can be in acidic or neutral form, and to precursors thereof.
  • exemplary non-limiting human milk oligosaccharides include 3'- sialyllactose, 6'-sialyllactose, 3-fucosyllactose, 2'-fucosyllactose, and lacto-N-tetraose.
  • treat or “treating” should not be taken to imply that an individual is treated until total recovery. Accordingly, these terms broadly include amelioration and/or prevention of the onset of the symptoms or severity of a particular condition.
  • lowering the risk of an infection include prevention of the onset of the symptoms or reduction of the severity of the symptoms. The risk of an infection is reduced or lowered compared to the situation in absence of the claimed composition.
  • shelf stable refers to a nutritional product that remains commercially stable after being packaged and then stored at 18-24°C for at least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months.
  • nutritional formulation or “nutritional composition” as used herein, are used interchangeably and, unless otherwise specified, refer to nutritional liquids, nutritional powders, nutritional supplements, and any other nutritional food product as known in the art.
  • the nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human.
  • nutritional powder refers to nutritional products in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spray-dried and dry-mixed dry-blended powders.
  • infant as used herein, unless otherwise specified, refers to a person 12 months or younger.
  • preterm refers to a baby born prior to 36 weeks of gestation.
  • toddler refers to a person greater than one year of age up to three years of age.
  • child refers to a person greater than three years of age up to twelve years of age.
  • formula refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by a human.
  • human milk fortifier refers to liquid and solid nutritional products suitable for mixing with breast milk or formula for consumption by a preterm or term infant.
  • the purity can be determined by any suitable method known to the person skilled in the art.
  • One suitable method is HPLC (high-performance liquid chromatography). In the obtained chromatogram, the ratio of the area underneath the peak(s) representing the amount of HMO(s) to the sum of areas underneath the peaks representing the HMO(s) and all other compounds than said HMO(s) in the chromatogram is calculated.
  • SIIEM simulated infant ileal efflux medium
  • 5HMO-mix 2’- Fucosyllactose, 3-Fucosyllactose, Lacto-N-Tetraose, 3’-Sialyllactose and 6’-Sialyllactose
  • HMO supplementation maintained the relative abundance of HMO-utilizing bacteria, while the relative abundance of E. coli was decreased ( Figure 2, Figure 3). While not wishing to be bound by theory, it is believed that the decrease in the relative abundance of E. coli may lower the risk of an infection of the gastrointestinal tract.
  • an EPEC (E.coli 0127) infection was simulated with fecal samples from formula-fed infants in an ex vivo system using the SIFR® technology (Van den Abbeele, P. et al. Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology. Frontiers Microbiol 14, (2023)).
  • This technology provides insights into compositional changes in the gut microbiota. The technology has been validated with clinical data. It was found that the 5HMO-mix significantly lowered EPEC 0127 levels compared to the situation without the 5HMO-mix.
  • the composition comprising the 5HMO-mix is used to reduce the risk of an infection in the gastrointestinal tract by inhibiting the adhesion of E. coli to intestinal epithelial cells. It is known that one step in the mechanism of infection is the attachment to the epithelial cells.
  • the composition is a nutritional composition such as a formula or a dietary supplement.
  • a preferred composition herein is a nutritional composition such as a formula.
  • the nutritional compositions may be in any product form comprising the ingredients described herein, and which is safe and effective for oral administration.
  • the nutritional compositions may be formulated with optional ingredients such as those described herein.
  • the nutritional compositions of the present disclosure are preferably formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof.
  • the nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific diseases or conditions or with a targeted nutritional benefit as described below.
  • Specific non-limiting examples of product forms suitable for use as HMO-containing compositions as disclosed herein include, for example, liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid, and powdered formula.
  • Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure.
  • Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates. These emulsions are generally flowable or drinkable liquids at from about 1 °C to about 25°C and are typically in the form of oil- in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.
  • the nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter.
  • the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter.
  • the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.
  • the nutritional emulsion may have a pH ranging from about 3.5 to about 8, from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2.
  • the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 mL, or even at least 10 mL, or even at least 25 mL, including ranges from about 1 mL to about 300 mL, including from about 4 mL to about 250 mL, and including from about 10 mL to about 240 mL.
  • the nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to form compositions comprising generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter.
  • the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter.
  • the reconstituted powder may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.
  • the present compositions may be useful in newborns, infants, toddlers, or children.
  • the present compositions may be useful in newborns.
  • the present compositions may be useful in infants. It has been shown that the 5HMO-mix reduces the relative abundance of E. coli in the infant TIM-2 model, thus indicating that the mixture may have a beneficial effect for infants. Also, E. coli is the leading cause of infantile diarrhea, which indicates that infants may particularly benefit from a composition comprising the 5HMO-mix.
  • the present composition may comprise an effective amount of 2'-fucosyllactose, 3- fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.
  • the present composition may comprise at least 0.01%, by weight, of 2'-fucosyllactose; at least 0.01%, by weight, 3-fucosyllactose; at least 0.01%, by weight, 3'-sialyllactose; at least 0.01%, by weight, 6'-sialyllactose; and at least 0.01%, by weight, lacto-N-tetraose.
  • compositions may comprise individual HMOs in any suitable amount, such as, for example, at least 0.001 mg/mL, including from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 10 mg/mL, including from about 0.01 mg/mL to about 5 mg/mL (mg of particular HMO per mL of composition).
  • the present composition may comprise the 5 HMOs in specific ratios, wherein the ratios of 2'- fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose in the composition are: 45% to 60%, by weight, of 2’-fucosyllactose; 8% to 18%, by weight, of 3- fucosyllactose; 2% to 10%, by weight, of 3’-sialyllactose; at least 2% to 10%, by weight, of 6’- sialyllactose; and 20% to 31 %, by weight, of lacto-N-tetraose.
  • the concentration of individual HMOs in the nutritional powder is preferably from about 0.001% to about 5%, including from about 0.01% to about 1% (by weight of the nutritional powder).
  • the concentration of individual HMOs is preferably from about 0.001% to about 0.50%, including from about 0.001% to about 0.15%), including from about 0.01% to about 0.10%, and further including from about 0.01%) to about 0.03% (by weight of the ready-to-feed nutritional liquid).
  • the concentration of individual HMOs is preferably from about 0.002% to about 0.6%, including from about 0.002% to about 0.3%, including from about 0.02% to about 0.20% (by weight of the concentrated nutritional liquid).
  • the present composition may be in the form of a powder.
  • Formulating compositions with HMOs can be somewhat problematic. It has been found that a more reproducible and consistent composition can be achieved through controlling the particle size distribution (PSD) of the HMO. While not wishing to be bound by theory, it is believed that having a somewhat narrow PSD improves the flowability of the HMO enabling a more effective mixing with the other ingredients. In addition, it is believed that a PSD within a certain range provides a better solubility profile.
  • Particle size of an HMO may be determined using a standard method, such as using a sieve tower, which separates the powder into the different fractions after a defined time with a predefined amplitude. The sieves used in such a method may be sieves which comply with DIN ISO 3310-1.
  • HMOs in particular 2’FL and 3FL, used in the present compositions have the following particle size characteristics:
  • Percent through mesh #100 150 pm - greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.
  • the human milk oligosaccharides when in form of a powder have a water activity a w between 0.10 and 0.3, preferably between 0.10 and 0.25.
  • the water activity may be determined using known hygrometers; preferably the water activity is determined according to ISO 18787:2017. The water activity ensures the microbial stability of the powder and prevents contamination with undesired microorganisms.
  • the present composition preferably comprises HMOs of synthetic origin, such as HMOs produced by microbial fermentation, or alternatively HMOs produced by biocatalysis or chemical synthesis.
  • HMOs produced by microbial fermentation, or alternatively HMOs produced by biocatalysis or chemical synthesis.
  • microbial fermentation allows production on an industrial scale in high purities, which may be used in nutritional compositions.
  • HMOs are used that have a purity of more than 85%, preferably a purity of more than 90%, more preferably a purity of more than 95%.
  • compositions of the present disclosure may optionally include anti-inflammatories such as long-chain polyunsaturated fatty acids (LCPUFAs) and/or antioxidants such as carotenoids.
  • LCPUFAs may be included in the compositions to provide nutritional support and to enhance growth and functional development of the intestinal epithelium and associated immune cell populations.
  • Exemplary LCPUFAs for use in the present compositions include, for example, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils.
  • the present compositions preferably comprise total concentrations of LCPUFA of from about 0.01 mM to about 10 mM and including from about 0.01 mM to about 1 mM.
  • the compositions comprise total concentrations of LCPUFA of from about 0.001 g/L to about 1 g L.
  • antioxidants such as carotenoids, and particularly, combinations of the carotenoids, lutein, lycopene, zeaxanthin and/or beta-carotene may be included in the present compositions.
  • a flowing agent or anti-caking agent may be included in the present compositions to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container.
  • Non-limiting examples include tricalcium phosphate, silicates, and combinations thereof.
  • the concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.
  • compositions of the present disclosure may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids or powders and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.
  • compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support of the subject. It will be appreciated that the exact amounts and rates of administration will depend on a number of factors such as the age, body weight, general health, sex, and dietary requirements of the subject. Based on the teaching herein those skilled in the art can, by routine trial and experimentation, determine suitable dosage regimes on a case-by-case basis.
  • compositions may comprise at least one probiotic strain, for example, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lactobacillus easel subsp. casei, Lactobacillus delbrueckii subsp.
  • probiotic strain for example, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lactobacillus easel subsp. casei, Lactobacillus delbrueckii subsp.
  • Bifidobacterium including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dent!
  • Bifidobacterium longum subsp. infantis Bifidobacterium longum subsp. longum
  • Bifidobacterium magnum Bifidobacterium pseudocatenulatum
  • compositions may comprise at least one strain of a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241 , Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012 and suitable combinations thereof.
  • a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241 , Lactobacillus rhamnosus
  • compositions preferably comprise an effective amount of probiotic.
  • the probiotic has a concentration ranging from 0.05 x 10 9 CFU/g to 30 x 10 9 CFU/g, preferably from 0.5 x 10 9 CFU/g to 25 x 10 9 CFU/g.
  • EHEC 0157 DSM 17076 was grown agitated overnight in Luria-Bertani (LB) broth at 37°C, then washed twice using Hanks balanced salt solution (HBSS, Gibco), and resuspended and ODeoonm-norrnalized to 0.5 in DMEM.
  • the EHEC strain was then diluted 1 :20 in DMEM to approximately 2x10 6 colony forming units (CFU)Zml and pre-incubated or not with 5-HMO mix at different dosages (range 1 to 30mg/ml) or GOS (30mg/ml) with agitation at room temperature.
  • CFU colony forming units
  • the cell culture media was removed from the Caco-2 cell monolayers, which were then gently washed twice using HBSS.
  • the EHEC strain suspensions (with and without 5-HMO mix or GOS) were then added the Caco-2 cell monolayers, and after 1 hr of incubation, the media containing non-adhering EHEC was removed.
  • the Caco-2 cell monolayers were gently washed trice in HBSS and following 3 washing steps, 0.1% Triton X-100 in HBSS was added to wells to release the adhering EHEC.
  • the number of EHEC adhering to the intestinal cell monolayers was then quantified by serial plating on tryptic soy broth (plates) and counting of CFU) following overnight incubation at 37°C.
  • the human epithelial intestinal cancer cell line Caco-2 (ACC 169, DSMZ, passages 5-20) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX Supplement (Gibco) supplemented with 1% non-essential amino acids (Merck Life Science), 1% penicillinstreptomycin (10,000 U/mL) (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in a 5% CO2 atmosphere.
  • DMEM Modified Eagle Medium
  • Gibco Modified Eagle Medium
  • the cells were seeded at a density of 8x10 4 cells/well in 24-well culture plates, and the media was replaced every 3-4 days until cells were ready for use in the experiment after 14 days.
  • the cell culture media was removed from the Caco-2 cell monolayers, which were then gently washed twice using HBSS.
  • the EPEC strain suspensions (with and without 5-HMO mix or GOS) were then added to the Caco-2 cell monolayers, and after 1 hr of incubation, the media containing non-adhering EPEC was removed.
  • the Caco-2 cell monolayers were gently washed trice in HBSS and following 3 washing steps, 0.1% Triton X-100 in HBSS was added to wells to release the adhering EPEC.
  • the number of EPEC adhering to the intestinal cell monolayers was then quantified by serial plating on tryptic soy broth (plates) and counting of CFU) following overnight incubation at 37°C.

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Abstract

La présente divulgation concerne une composition destinée à être utilisée pour réduire le risque d'infection du tractus gastro-intestinal provoquée par E. coli pathogène chez un sujet humain, ladite composition comprenant une quantité efficace de 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose et lacto-N-tétraose.
PCT/EP2025/056528 2024-03-11 2025-03-11 Mélange de cinq oligosaccharides de lait humain pour réduire le risque d'infection dans le tractus gastro-intestinal provoquée par e. coli pathogène Pending WO2025190896A1 (fr)

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