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WO2024218197A1 - Oligosaccharides de lait humain - Google Patents

Oligosaccharides de lait humain Download PDF

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Publication number
WO2024218197A1
WO2024218197A1 PCT/EP2024/060503 EP2024060503W WO2024218197A1 WO 2024218197 A1 WO2024218197 A1 WO 2024218197A1 EP 2024060503 W EP2024060503 W EP 2024060503W WO 2024218197 A1 WO2024218197 A1 WO 2024218197A1
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Prior art keywords
cells
nutritional
composition
mix
cat
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Inventor
Stina JENSEN
Daniel VILLALBA
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Chr Hansen AS
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Chr Hansen AS
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Priority to CN202480026459.0A priority Critical patent/CN121057512A/zh
Publication of WO2024218197A1 publication Critical patent/WO2024218197A1/fr
Anticipated expiration legal-status Critical
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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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic 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

Definitions

  • the present disclosure relates, in part, to the use of a mix of human milk oligosaccharides for modulating immune responses in a 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 are thought to have various biological functions, such as preventing the attachment of pathogens to epithelial cells (Ruiz- Palacios, Cervantes, Ramos, Chavez-Munguia, & Newburg, 2003), modulating immune cell responses (Zhang et al., 2019) in vitro, modulating gut microbiota (Berger et al., 2020; Elison et al., 2016; Iribarren et al., 2020).
  • fucosylated HMOs (2'FL, 3FL and DFL
  • fucosylated HMOs has been seen in an in vitro anaerobic culture system using infant fecal microbiota (Yu et al., 2013).
  • 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 helpful bifidobacteria in support of overall gut health (Bode, Lars. Nutrition reviews suppl_2 (2009)), support microbial colonization and gut barrier function (Kong et. al., Mol. Nutr. Food Res. 2019, 63), and enhance mucus barrier function through direct modulation of intestinal goblet cells (Cheng et. al., Mol.
  • cytokines are indicative of different types of immune response. For example, IFN-y and IL-2 are associated with TH1 responses whereas IL-4, IL-5 and IL-13 are associated with TH2 responses.
  • compositions for balancing the immune system of a subject.
  • the present disclosure relates to compositions comprising 5 HMOs (2'-Fucosyllactose (2’-FL), 3-Fucosyllactose (3-FL), 3'-sialyllactose (3’-SL), 6'-sialyllactose (6’-SL) and lacto-N-tetraose (LNT)) for balancing the immune system of non-adult humans.
  • 5 HMOs (2'-Fucosyllactose (2’-FL), 3-Fucosyllactose (3-FL), 3'-sialyllactose (3’-SL), 6'-sialyllactose (6’-SL) and lacto-N-tetraose (LNT)
  • compositions for the modulation of immune activity such as allergies, and the like.
  • the 5 HMO mix balances the amount of cytokines that promote Tn1-related responses and the amount of cytokines that promote TH2- related responses. This balance helps avoid any one type of response predominating and allows for the immune system to respond appropriately to challenges. Moreover, the 5HMO mix also appears to enhance cytokines related to the TH17 immune response, which could be important for development of the immune system.
  • Figure 1 shows the effect of 5HMO mix on enhancing release of Th1 priming interleukin (IL)-12 from LPS-activated human dendritic cells (DC) and macrophages (M0).
  • IL-12 interleukin
  • naive CD4+ T cells differentiate towards Th1 rather than the Th2 direction.
  • Th1 cells produce large amounts of interferon (IFN)-y.
  • Figure 2 shows the effect of 5HMO mix on enhancing release of the Th2 priming IL-4 from LPS- activated human dendritic cells (DC) and macrophages (M0).
  • IL-4 naive CD4+ T cells favor differentiation into Th2 rather than the Th1 direction.
  • Th2 cells produce large amounts of IL-4, IL-5, and IL-13.
  • Figure 3 shows the effect of 5HMO mix on the Th2/Th1 ratio.
  • DCs or M0 activated with LPS in the presence or absence of 5HMO mix were co-cultured with naive CD4 T cells, after which the amounts of Th1 signature cytokine IFN-y (see also Figure 5) and Th2 signature IL-13 (see also Figure 6) released was quantified and used to calculate the IL-13/IFN- y ratio.
  • FIG 4 shows the classification of macrophages into: 1) pro-inflammatory M1 macrophages, which are pro-inflammatory by nature and critical for host protection against viruses and intracellular bacteria; and 2) M2 macrophages, which are anti-inflammatory by nature and play an important role in tissue repair.
  • the macrophage experiments described herein are all performed using M1 macrophages.
  • Figure 5 shows the effect of 5HMO mix on IFN-y release in a co-culture system of human LPS- activated DCs and naive CD4+ T cells or LPS-activated M0 and naive CD4+ T cells.
  • the 5 HMO mix significantly boosts IFN-y release in a dose dependent manner in both co-culture systems compared to the respective LPS only groups.
  • Figure 6 shows the effect of 5HMO mix on IL-13 release in a co-culture system of human LPS- activated DCs and naive CD4+ T cells or LPS-activated M0 and T-lymphocytes.
  • the 5 HMO mix significantly decreases IL-13 release compared to the respective LPS only groups, which can be an effector of allergic responses if dominating.
  • Figure 7 shows the effect of 5HMO mix on release of TH17 priming cytokines IL-6 and IL-23 from LPS-activated DCs, as well as on release of TH17 signature effector cytokine IL-17 from naive CD4+ T cells co-cultured with LPS-activated DCs.
  • the 5HMO mix significantly increases DC release of IL23 and IL-6 as well as CD4 T cell release of IL-17 in a dose dependent manner.
  • Figure 8 shows the effect of 5HMO mix on release of the anti-inflammatory cytokine IL-10 from LPS-activated DCs and M0s.
  • 5HMO mix significantly increases IL-10 secretion from DCs and M0 compared to the respective LPS only groups, and thereby ensures a regulatory environment that adds to a balanced immune system.
  • Figure 9 shows the antibody response to Bordetella pertussis toxin (PT) in serum of mice administered a Diphtheria-Tetanus-Acellular Pertussis (DTaP) vaccine on Days 0, 14 and 28 and receiving oral gavage of 5HMO mix at different dosages (0.01 , 0.05 and 0.1% of bodyweight) starting 14 days prior to initial vaccination.
  • 5HMO mix supplementation enhances serum levels of PT-specific IgG on days 28 and 42/43 post initial vaccination.
  • Statistical significance was determined by a Kruskal-Wallis non-parametric test followed Dunn’s multiple comparisons test.
  • Figure 10 shows the cell-mediated immune response to PT of splenocytes isolated on the day of euthanization (day 42 or 43) from the mouse included in the DTaP vaccine study described in Figure 8.
  • 5HMO mix supplementation enhances the frequency of T-bet-expressing CD4+ T helper cells (Th1 cells) and tumor necrosis factor (TNF)-a expressing CD8+ cytotoxic T cells in splenocytes of DTaP-vaccinated mice stimulated ex vivo with 8pg/mL PT.
  • Stimulation with 12- myristate 13-acetate I ionomycin (PMA-IONO) was included as a positive control.
  • 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 animal 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 subsp. 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-neo- 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.
  • 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.
  • compositions comprising a mixture of 5 HMOs (2-FL, 3-FL, 3- SL, 6-SL and LNT) that promotes balancing the immune system.
  • balancing the immune system refers to the ratio of Th1 and Th2 related cytokines. While not wishing to be bound by theory, it is believed that newborns and infants with a more balanced immune system will be less susceptible to pathogens and show less propensity to develop allergic diseases. Modulation of the immune system is also supported by the 5HMO mix effect on mediating a TH17 immune response that supports protection to extracellular pathogens and fungi.
  • compositions comprising a mixture of 5 HMOs for modulating the secretion of pro-inflammatory cytokines (e.g. IL-12, IL-6, IL-23) and anti-inflammatory cytokines (e.g. IL-10 from human M0 and DCs) such that the ratio is more balanced.
  • pro-inflammatory cytokines e.g. IL-12, IL-6, IL-23
  • anti-inflammatory cytokines e.g. IL-10 from human M0 and DCs
  • LPS+5HMO Mix-primed DCs led to a significant down-regulation in IL-13 production.
  • 5HMO mix supplementation balanced the TH1/TH2 ratio immunity and support the maturation of other immune phenotypes including TH17 (IL-6, IL-23 and IL-17).
  • 5-HMO mix supplementation was tested to see if it could improve humoral and adaptive immune responses against bortedella Pertussis (PT) in a specific DTaP vaccination mice model. It was observed that dietary 5-HMO mix enhanced serum levels of anti-PT IgG.
  • the present compositions may be used to improve the efficacy of a vaccine.
  • the present compositions may be administered to a subject who has recently received, or is about to receive a vaccine, in order to enhance the immune response to said vaccine.
  • the subject may, for example, be a newborn.
  • the subject may, for example, be an infant.
  • the subject may, for example, be a toddler.
  • the subject may, for example, be a child.
  • the present compositions comprise five Human Milk Oligosaccharide (HMO) namely, 2'- fucosyllactose, 3-fu cosy I lactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.
  • HMO Human Milk Oligosaccharide
  • the present compositions may comprise other HMOs such as, for example, lacto-N-neotetraose.
  • PSD particle size distribution
  • 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 .
  • 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%.
  • Percent through mesh #45 (355 pm) - greater than about 95%, greater than about 92%, greater than or equal to about 90%.
  • Percent through mesh #20 (850 pm) - 100%.
  • 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 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 be and typically are shelf stable.
  • the nutritional emulsions typically contain up to about 95% by weight of water, including from about 50% to about 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions.
  • the nutritional emulsions may have a variety of product densities, but most typically have a density greater than about 1 g/mL, including greater than about 1 .05 g/mL, including greater than about 1 .055 g/mL to about 1.12 g/mL, and also including from about 1 .085 g/mL to about 1 .10 g/mL.
  • 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 solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions, that may optionally be compressed into tablets.
  • Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dry-blended powder compositions.
  • the compositions can easily be scooped and measured with a spoon or similar other device, and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use.
  • "immediate" use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution.
  • 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 balancing the immune system in newborns, infants, toddlers, children.
  • the present compositions may be useful in newborns.
  • the present compositions may be useful in infants.
  • 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 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).
  • 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.
  • compositions of the present disclosure may further comprise other optional components that may modify the physical, chemical, aesthetic or processing characteristics of the composition or to serve as pharmaceutical or additional nutritional components.
  • optional ingredients include preservatives, emulsifying agents, buffers, pharmaceutical actives, nutrients, colorants, flavors, thickening agents and stabilizers, flowing agents, minerals, emulsifying agents, lubricants, sweetening agents, and the like.
  • a flowing agent or anti-caking agent may be included in the present compositions to retard clumping or caking of the powder overtime 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.
  • the 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 dentium, Bifidobacterium longum subsp.
  • infantis Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum), or any strain from the genera of Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family Coriobacteriaceae, as well as suitable combinations of the foregoing.
  • 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.
  • Human monocytes were isolated from PBMC using Easysep Human Monocyte Enrichment Kit following manufacturer’s protocol.
  • 1 .5 x 10 6 of purified monocytes were cultured in flat-bottomed 6-well plates (Cat: Nunc) during 6 days in 3 ml FBS medium ((RPMI-1640 medium (Cat: R5886, Sigma Aldrich) with 1 % streptomycin, 1 % penicillin, 1 % L-glutamine, and 10 % heat-inactivated and endotoxin-free fetal bovine serum (FBS) (Cat: 10082-147, Gibco) in the presence of GM-CSF (50 ng/ml, Cat: AF-HDC, Peprotech).
  • FBS fetal bovine serum
  • DCs dendritic cells
  • 1 .5 x 10 6 of purified monocytes were cultured in flat-bottomed 6- well plates (Cat: Nunc) during 6 days in 3 ml FBS medium ((RPMI-1640 medium (Cat: R5886, Sigma Aldrich) with 1 % streptomycin, 1 % penicillin, 1 % L-glutamine, and 10 % heat-inactivated and endotoxin-free fetal bovine serum (FBS) (Cat: 10082-147, Gibco) in the presence of GM- CSF and IL-4 (50 ng/ml, Cat: AF-HDC, Peprotech). After 3 days, FBS medium and cytokines were replenished.
  • FBS medium (RPMI-1640 medium (Cat: R5886, Sigma Aldrich) with 1 % streptomycin, 1 % penicillin, 1 % L-glutamine, and 10 % heat-inactivated and endotoxin-free fetal bovine serum (
  • naive M0 were stimulated with LPS (50 ng/ml) (Cat: 5568, Sigma-Aldrich) and IFNy (50 ng/ml) (Cat: 285-IF-100/CF, R&D Systems), and naive DCs were activated with LPS (50 ng/ml) (Cat: 5568, Sigma-Aldrich) in the presence of 5-HMO Mix increasing concentrations (1-5 mg/ml) for 24 hours.
  • M0 and DCs were collected, washed with PBS and resuspended in X-VIVO 15 medium. Subsequently, 1 x 10 5 of M0 and DCs were co-cultured with 1 x 10 6 naive CD4 + T cells in flat-bottomed, 24-well culture plates (Cat: Nunc) for 5 days. Hereafter, supernatants were collected for cytokines analysis.
  • PBMCs Peripheral blood mononuclear cells
  • Lymphoprep 11 14547, Axis-Shield, Oslo, Norway
  • naive CD4 + T cells were purified by negative selection using Easysep Human Naive CD4 + T cell Enrichment Kit (Cat:19155 Stemcell Technologies) according to manufacturer’s protocol.
  • naive CD4 + T cells were counted by optical microscopy.
  • naive CD4 + T cells were cultured with allogeneic macrophages either allogeneic DCs in a 1 :10 ratio (M0:TC) (DC:TC) in serum-free X-VIVO 15 medium (Cat: BE02-060F, Lonza, Verviers, Belgium) in flat-bottomed 24 well culture plates (Cat: 142475, Nunc) for 5 days.
  • the concentration of IL-4, IL-6, IL-10, IL-12 and IL-23 in mono-cultures (DCs or M0), and IFN-y, IL-13 and IL-17 in co-cultures (DC-CD4+ T-cells or M0-CD4+ T cells) were measured by ELISA according to the manufacturer's instruction (InVitroGen, IFNG Cat: 88-7316-88 and IL-13 Cat: 88-7439-88).
  • supernatants were isolated and stored at -80C.
  • Standard and positive control are pool of mouse sera obtained from mice vaccinated with DTaP.
  • the negative control used was naive mouse serum.
  • the standard curve (Nexelis, Lot. 09Nov2022) starting dilution was 1/200. Serum samples starting dilution were either 1/50 or 1/1000. Positive (Nexelis, Lot. 09Nov2022) and negative control serum starting dilution were 1/50 and 1/50, respectively.
  • Samples, standard, and controls were serially diluted 2-fold in subsequent rows using pre-dispensed blocking buffer. Then plates were incubated for 1 hr at RT on an orbital shaker set at 350 rpm.
  • the EC50 values of standard curves generated during the anti-PT specific IgG ELISA development experiments were compiled.
  • the geometric mean concentration of the EC50 values were calculated and an arbitrary concentration of 2704 ELU/ml was determined for the standard.
  • the positive control was determined by obtaining the reference value ⁇ 2SD.
  • the Positive control acceptance range was: 236-308 ELU/ml
  • Spleen cell suspensions were prepared by gently crushing spleens through 100pm cell strainers placed on a tube containing 48mL of plain RPMI-1640 (Lonza, Cat. 12-167Q) with plunger part of a 3-5mL syringe piston. Cell suspensions were centrifuged and washed with plain RPMI. After transfer of cell suspension to 15ml falcon tubes, cells were centrifuged at 400g for 10min at RT and cell pellet resuspend in 10mL of complete RPMI (RPMI-1640 (Lonza, Cat. 12-167Q), containing 10% FBS (Gibco, Cat. 12483-020), 100U/mL Penicillin-100pg/mL Streptomycin (Gibco, Cat.
  • Brefeldin A GolgiPlug (BD, Cat 51-2301 KZ) and Monensin (GolgiStop BD, Cat 51-2092KZ), intracellular protein transport blocking agents, were added 2hrs after the initiation of the stimulation to inhibit cytokine transport and secretion. Following an overnight incubation (17hr ⁇ 1 hr) at 37°C under 5% CO2, cells were harvested, washed with D-PBS and stained with Fixable Viability Stain 780. After 15 mins at RT protected from light, cells were washed with staining buffer and stained with surface markers (For panel 1 : CD4, CD8, CD69 and for panel 2: CD4, CD8, CD62L, CD25, CD44).
  • Cells were then stained with intracellular marker antibodies (for panel 1 : CD3, IFN- y, TNF-a, IL-2, IL-13 and for panel 2: CD3, KI-67, T-bet, Foxp3) 30 mins at RT. After intracellular staining, cells were washed with 1X Perm/Wash buffer, fixed with 1% PFA (EMS, Cat. 15735- 20S) fixation solution and kept at 4°C protected from light until acquisition on flow cytometer.
  • intracellular marker antibodies for panel 1 : CD3, IFN- y, TNF-a, IL-2, IL-13 and for panel 2: CD3, KI-67, T-bet, Foxp3
  • FMO fluorescence minus one
  • Stimulated T cells by allogeneic 5-HMO Mix-conditioned DCs support Th1 responses whilst regulate Th2 immunity

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Abstract

La présente invention concerne une composition destinée à être utilisée pour équilibrer le système immunitaire d'un nouveau-né ou d'un nourrisson, ladite composition comprenant une quantité efficace de 2'-fucosyllactose, de 3-fucosyllactose, de 3'-sialyllactose, de 6'-sialyllactose et de lacto-N-tétraose.
PCT/EP2024/060503 2023-04-19 2024-04-18 Oligosaccharides de lait humain Pending WO2024218197A1 (fr)

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WO2021186258A1 (fr) * 2020-03-20 2021-09-23 Glycom A/S Procédés d'obtention d'un produit hmo solide, et produits hmo solides obtenus par ceux-ci
WO2022034078A1 (fr) * 2020-08-10 2022-02-17 Inbiose N.V. Procédé de production d'un mélange purifié d'oligosaccharides différents produits par culture cellulaire ou fermentation microbienne
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WO2022078859A1 (fr) * 2020-10-16 2022-04-21 Société des Produits Nestlé S.A. Composition nutritionnelle comprenant des oligosaccharides de lait humain
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EP4129302A1 (fr) * 2020-04-03 2023-02-08 Inner Mongolia Yili Industrial Group Co., Ltd. Oligosaccharides du lait maternel pour l'amélioration de la résistance d'un organisme contre une infection au staphylococcus aureus

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US10435427B2 (en) * 2013-10-04 2019-10-08 Jennewein Biotechnologie Gmbh Process for purification of neutral human milk oligosaccharide using simulated moving bed chromatography
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WO2021186258A1 (fr) * 2020-03-20 2021-09-23 Glycom A/S Procédés d'obtention d'un produit hmo solide, et produits hmo solides obtenus par ceux-ci
EP4129302A1 (fr) * 2020-04-03 2023-02-08 Inner Mongolia Yili Industrial Group Co., Ltd. Oligosaccharides du lait maternel pour l'amélioration de la résistance d'un organisme contre une infection au staphylococcus aureus
WO2022034078A1 (fr) * 2020-08-10 2022-02-17 Inbiose N.V. Procédé de production d'un mélange purifié d'oligosaccharides différents produits par culture cellulaire ou fermentation microbienne
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