Nutritional composition for stimulating bifidobacteria

Abstract

A nutritional composition comprising galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of GOS:2'-FL of 1:9 to 9:1 for use in increasing the relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the digestive tract of a non-infant human subject.

Description

[Technical field]

[0001] The present invention relates to a nutritional composition for use in increasing the relative abundance of Bifidobacteria in the digestive tract of a non-infant human subject. [Background technology]

[0002] The nature of bacterial species in gut microbiota is largely determined by the food consumed. Various beneficial gut bacteria consume dietary fiber such as oligosaccharides and polysaccharides. Human breast milk contains various oligosaccharides (classified as human milk oligosaccharides (HMO)) that can reach the intestinal tract undigested and unchanged. As a result, they can be utilized as the main carbon and energy source by certain gut microbiota components, especially species of Bifidobacterium and Bacteroides. These species have been shown to have beneficial effects on infant health, more specifically on promoting bacterial colonization in the gut and defense against pathogens.

[0003] Although the amount and diversity of HMOs in human breast milk varies between women depending, for example, on geographic origin and genetic background, human breast milk can be said to contain three main HMO types: fucosylated HMOs (35%-50%), sialylated HMOs (12%-14%) and non-fucosylated neutral HMOs (42%-55%).Fucosylated HMOs include 2'-fucosyllactose (2'-FL), which is the most abundant HMO in human breast milk.

[0004] Bifidobacteria are common bacteria that reside in the human intestine throughout life. They are saccharolytic organisms with the ability to ferment glucose, galactose and fructose and are thought to play a central role in maintaining a healthy gastrointestinal tract.

[0005] Intestinal colonization with bifidobacteria begins at birth and this process proceeds continuously with the acquisition and loss of different species and strains over time. The transition from the infant microbiota to the adult microbiota is thought to begin after the transition to solid foods in the diet. One effect of this transition is a decrease in the relative abundance of bifidobacteria from approximately 60-70% in infants to approximately 2-14% in adults.

[0006] With age, the relative abundance of Bifidobacteria tends to decrease further, meaning that most elderly people, for example those over about 50, 60 or 70 years of age, have a significantly lower relative abundance of these species.

[0007] Colonization by bifidobacteria is believed to play an important role in maintaining human health. Various studies have shown that increased abundance of bifidobacteria in the digestive tract of non-infant human subjects is associated with the prevention or treatment of enteropathogenic infections, type 2 diabetes, intestinal barrier dysfunction, gut-brain disorders such as stress, anxiety and depression, allergies such as atopic dermatitis, asthma and inflammatory gut pathologies (WO 2018 / 157900; CB Wong et al., FEMS Microbiology Reviews 44(2020)369-385 and F. Turroni et al., Applied and Environmental Microbiology 75(2009)1534-1545).

[0008] More specifically, in elderly individuals, the abundance of bifidobacteria is reduced with a corresponding increase in the number of less desirable microbial species and pathogenic organisms, as described by S.C. Leahy et al., J. Appl. Microbiol. 98 (2005) 1303-1315. Furthermore, bifidobacteria, particularly B. longum, have been shown to reduce the incidence and duration of antibiotic-associated diarrhea and to exhibit inhibitory effects against many other pathogenic organisms, such as Salmonella typhimurium.

[0009] In addition, a positive effect on constipation and relief of severe premenstrual syndrome gastrointestinal symptoms has been reported.

[0010] Various studies have focused on the relationship between the gut microbiota and the development of irritable bowel syndrome (IBS), showing alterations in the microbiota and reduced levels of Bifidobacterium associated with IBS patients. In the gut microbiota of elderly people, a decreased number of Bifidobacteria was observed in elderly people suffering from diarrhea associated with Clostridium difficile compared to healthy controls. A decrease in the number of Bifidobacteria has also been observed in other diseases such as cystic fibrosis, hepatitis B and both types I and II diabetes (S. Arboleya et al., Frontiers in Microbiology, 7 (2016) 1204).

[0011] It is therefore generally desirable to achieve an increased abundance of Bifidobacteria in the gastrointestinal tract of non-infant human subjects, more particularly adults, and more particularly the elderly.

[0012] A non-infant human subject is defined herein as a human subject that is at least 3 years of age.

[0013] Not only does the number of bifidobacteria decrease with age, but the diversity of bifidobacterial species also changes: B. longum subspecies longum, B. longum subspecies infantis, B. breve and B. bifidum are generally predominant in infants, while B. catenulatum, B. adolescentis and B. longum subspecies longum are more predominant in adults (S. Arboleya et al., Frontiers in Microbiology, 7 (2016) 1204).

[0014] These different species show different abilities to ingest various HMOs. Infantile bifidobacteria are adapted to utilize HMOs, whereas the majority of adult bifidobacteria are unable to utilize HMOs. B. longum subsp. infantis actively ingests almost all types of HMOs, including fucosylated and sialylated molecules, whereas the ability of B. longum subsp. Longum strains to digest HMOs is limited. The majority of B. longum subsp. Longum strains were found to ingest only LNT and LNB, leaving other HMOs unchanged (CBWong et al., FEMS Microbiology Reviews, 44, 2020, 369-385).

[0015] Meanwhile, WO 2017 / 144062 and WO 2017 / 46711 disclose that administration of neutral HMOs such as 2'-FL increases the relative abundance of Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum in the gastrointestinal microbiota of non-infant humans, and WO 2018 / 127900 discloses administration of neutral HMOs such as 2'-FL for at least 14 days or less to increase the relative abundance of Bifidobacterium longum and / or Bifidobacterium bifidum in non-infant humans.

[0016] In other words, there are conflicting studies on the effect of 2′-FL-containing HMOs on bifidobacteria, particularly on the abundance of bifidobacteria, in the non-infant microbiota. Summary of the Invention [Means for solving the problem]

[0017] In our studies on the effect of oligosaccharides on bifidobacteria in the non-infant intestine, we surprisingly found that the combined administration of both GOS and 2'FL to non-infant infants, and more specifically to (older) adults, synergistically increased the relative abundance of bifidobacteria, particularly Bifidobacterium adolescentis and Bifidobacterium longum, above the levels achieved by administration of these compounds individually.

[0018] Although a synergistic effect of these compounds on the infant microbiota has been suggested (WO 2011 / 008086), their synergistic effect on the non-infant microbiota, especially on bifidobacterial species, and even more specifically on the species Bifidobacterium adolescentis and Bifidobacterium longum in older adults, is surprising. As explained above, the adult gut microbiota is much more diverse compared to the infant gut microbiota, containing much fewer bifidobacteria and consisting of different subspecies that are generally less responsive to 2'-FL.

[0019] The present invention therefore relates to the use of galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of 1:9 to 9:1 GOS:2'-FL for increasing the relative abundance of Bifidobacteria, in particular Bifidobacterium adolescentis and / or Bifidobacterium longum, in the gastrointestinal tract of a non-infant human subject, preferably an adult human, both therapeutically and non-therapeutically.

[0020] According to one embodiment, the present invention relates to a nutritional composition comprising galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of GOS:2'-FL of 1:9 to 9:1 for use in increasing the relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the gastrointestinal tract of a non-infant human subject in a method for treating or preventing the onset of a disease associated with a low relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum.

[0021] Examples of such diseases are enteropathogenic infections, intestinal barrier dysfunction, type 2 diabetes, gut-brain disorders such as stress, anxiety and depression, allergies such as atopic dermatitis, asthma and / or inflammatory bowel conditions such as inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC).

[0022] In relation to a given disease or disorder, the term "treatment" includes, but is not limited to, preventing the disease or disorder (e.g., arresting the onset of the disease or disorder), alleviating the disease or disorder (e.g., causing regression of the disease or disorder); and / or alleviating a pathology caused by or resulting from the disease or disorder (e.g., alleviating, preventing, or treating a symptom of the disease or disorder).

[0023] In the context of a disease or disorder, the term "prevention" refers to preventing the onset of disease development when none has yet occurred, preventing a disease or disorder in a subject who may be predisposed to the disorder or disorder but has not yet been diagnosed as having the disorder or disorder, and / or preventing further development of the disease / disorder when the disease / disorder is already present.

[0024] In one embodiment the nutritional composition is administered to a non-infant human subject having a below average abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the gastrointestinal tract and / or suffering from or at risk of developing an enteropathogenic infection, intestinal barrier dysfunction, type 2 diabetes, gut-brain disorders such as stress, anxiety and depression, allergies such as atopic dermatitis, asthma and / or inflammatory gut conditions such as inflammatory bowel disease (IBD).

[0025] According to another embodiment, the present invention relates to a non-therapeutic use of a nutritional composition comprising galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of GOS:2'-FL of 1:9 to 9:1 to increase the relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the gastrointestinal tract of a non-infant human subject.

[0026] As used herein, an increase in the relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the digestive tract of a subject refers to a higher number of these microorganisms relative to the total number of bacteria in the digestive tract of a subject after the intervention of the nutritional composition compared to before supplementation. The increase is preferably at least 3%, more preferably at least 8%, such as at least 10%, even more preferably at least 15%. The degree of increase may depend on the daily dosage of the nutritional composition. The relative abundance of these microorganisms may be determined as described below in the Examples.

[0027] As used herein, the lower than average relative abundance of these microorganisms in the digestive tract refers to the number of these microorganisms relative to the total number of bacteria in the digestive tract of a subject being lower than the average number of these microorganisms in the intestines of a group of 10 healthy subjects of the same age group.A healthy subject is one who has not been diagnosed with a disease and does not suffer from any problems related to the digestive tract.

[0028] The nutritional composition may be administered to any non-infant human subject, but is preferably administered to an adult human subject over 18 years of age, preferably over 40 years of age, more preferably over 50 years of age, even more preferably over 60 years of age, more preferably over 65 years of age, and most preferably at least 70 years of age.

[0029] The basic structure of galactooligosaccharides (GOS) contains a lactose core at the reducing end extended with up to about seven galactose residues (degree of polymerization 8; DP8). Commercial GOS preparations are generally produced by transgalactosylation reaction by enzymatic treatment of lactose with the enzyme β-galactosidase (EC.3.2.1.23), resulting in a mixture of approximately 100 different types of oligomers with various DPs and linkages. Beta-galactosidase is produced by many microorganisms, including Bacillus circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, and Papiliotrema terrestris (Cryptococcus Papiliotrema terrestris). Beta-galactosidases differ in their three-dimensional structure, which results in stereo- and regioselective formation of glycosidic bonds.

[0030] After the enzymatic reaction, the GOS is isolated and purified using conventional methods such as nanofiltration and / or continuous simulated moving bed (SSMB). The resulting product is a GOS-containing syrup, which can be optionally dried (e.g., by spray drying, freeze drying, or spray chilling) to a powder.

[0031] A particularly preferred type of GOS is Biotis TMGOS, such as GOS prepared by beta-galactosidase enzyme from Bacillus circulans, has particularly strong transgalactosylation activity and has been commercialized worldwide.

[0032] The nutritional composition contains GOS and 2'-FL in a weight ratio of GOS:2'-FL in the range of 1:9 to 9:1, preferably 1:8 to 8:1, more preferably 1:7 to 7:1, even more preferably 1:6 to 6:1, even more preferably 1:5 to 5:1, more preferably 1:4 to 4:1, more preferably 1:3 to 4:1, more preferably 1:2 to 4:1, and most preferably 1:1 to 4:1.

[0033] The nutritional composition is preferably administered at a daily dosage of 0.5-30 g, preferably 0.5-20 g, more preferably 1-10 g, even more preferably 1-8 g, most preferably 2-6 g of GOS+2'-FL.

[0034] In this respect, it should be noted that GOS is a complex mixture of carbohydrates differing in chain length, bond type and degree of branching. It preferably contains 40-100% by weight, more preferably 50-90% by weight, most preferably 60-80% by weight of oligosaccharides (DP≧3) on a dry weight basis, and may further contain monosaccharides such as glucose and galactose, and disaccharides such as lactose, lactulose and allolactose. The lactose content is generally in the range of 0-60% by weight, preferably 0-40% by weight, most preferably 0-30% by weight on a dry weight basis, and the monosaccharide content is generally in the range of 0-10% by weight.

[0035] All weight percentages of GOS herein are calculated based on the weight of di- and oligosaccharides (i.e., DP > 2) present in said GOS, excluding lactose, except lactose and monosaccharides such as glucose and galactose, which are not included in the calculation.

[0036] The same applies to the weight ratios described herein, in which when calculating them, monosaccharides and lactose are not included, but all longer sugars (i.e. DP≧2) present in GOS are taken into account.

[0037] In the nutritional composition according to the invention, GOS and 2'-FL are preferably present in a total amount of at least 10% by weight, such as at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight or at least 90% by weight, for example up to 91% by weight, up to 92% by weight, up to 93% by weight or up to 94% by weight, relative to the total weight of the nutritional composition.

[0038] The nutritional composition may have the form of a food product, a drink or a dietary supplement. Furthermore, it may be administered as a prebiotic formulation, i.e. without bacteria, or as a probiotic formulation, i.e. with desirable (probiotic) bacteria such as bifidobacteria and / or lactobacilli.

[0039] The nutritional composition may have liquid, semi-liquid or solid components.

[0040] The food product is not mammalian milk, such as milk from humans, goats, sheep, cows, camels or ruminants. The food product or nutritional composition is a synthetic composition. The term "synthetic composition" refers to an artificially prepared composition, preferably a composition containing at least one compound produced ex vivo chemically and / or biologically, for example by chemical reaction, enzymatic reaction or recombinantly.

[0041] Examples of suitable food products and beverages are dairy products such as milk, milkshakes, chocolate milk, yogurt, cream, cheese, pudding and ice cream; bars such as nutritional bars, energy bars, snack bars, cereal bars and bars for diabetics; liquid products such as energy drinks, diet drinks, liquid complete meals, sports drinks and other nutritional beverages; savory snacks such as chips, tortillas, puffed baked snacks, crackers, pretzels and savory biscuits; bakery products such as muffins, cakes and biscuits; confectioneries such as gummies and candies; and pasta such as spaghetti.

[0042] Food supplements can be in the form of pills, capsules or dry powders. Dietary supplements can be ready to consume or need to be dissolved in a liquid such as water. Products in dry powder form can come with a device such as a spoon for measuring the desired amount of powder (e.g., daily dose or unit dose).

[0043] The nutritional composition may be provided in a jar, bottle, sachet, carton, wrapping, or the like.

[0044] In a preferred embodiment, the nutritional composition is provided in the form of a single serving. Each serving may optionally be individually packaged. A single serving preferably contains 0.5-30 g, preferably 0.5-20 g, more preferably 1-10 g, even more preferably 1-8 g, and most preferably 2-6 g of 2'-FL and GOS.

[0045] It is preferred that a unit dose of the nutritional composition is administered at least once a week, preferably at least once every three days, more preferably at least once every two days, and most preferably at least once a day.

[0046] The treatment preferably continues for a period of at least 2 weeks, for example at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months or at least 6 months. In order to maintain the health benefits provided by the administration of the nutritional composition, the composition is preferably administered at least once a day. The nutritional composition may be taken with a meal, such as at breakfast, or at the end of the day, for example 0-120 minutes, more preferably 0-60 minutes, most preferably 0-30 minutes before going to bed. Alternatively, the nutritional composition may be administered twice a day, preferably in the morning and in the evening, for example at breakfast and dinner or at breakfast and before going to bed. Administration with a meal is convenient and reduces the risk that the consumer will forget to take the nutritional composition.

[0047] In a preferred embodiment, the nutritional composition further comprises other prebiotic ingredients such as fructooligosaccharides (FOS), inulin and other HMOs.

[0048] The nutritional composition may further contain additional nutritional agents such as lipids, digestible carbohydrates, proteins and / or vitamins, minerals and / or biologically active peptides.

[0049] Examples of proteins are milk proteins (eg, casein and / or whey proteins), plant proteins (eg, soybean proteins and / or rice proteins), hydrolysates thereof and mixtures thereof.

[0050] Examples of digestible carbohydrates are sucrose, lactose, glucose, fructose, corn syrup solids, starch and maltodextrin.

[0051] Examples of vitamins and minerals are iron, magnesium, zinc, vitamin B3 and vitamin B6, vitamin D, vitamin C, vitamin E and / or beta carotene.

[0052] The nutritional composition may further contain flavouring agents, preservatives and / or colouring agents. [Brief description of the drawings]

[0053] [Figure 1] The relative abundance of bifidobacteria with respect to the total bacterial content obtained with different weight ratios of GOS and 2'-FL is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Fecal fermentation simulating the distal colon of elderly people As described in MMO'Donnell et al., Front. Microbiol., vol. 9, August 10, 2018, article 1844, microMatrix TM Fecal fermentation was performed in 24-well plates using Biotin (Applikon Biotechnology). Oligosaccharide GOS (Biotis TM GOS ex-FrieslandCampina), 2'-FL(Aequival TM The wells were filled with medium (composition: see Table 1) to which was added 2'-FL, ex-FrieslandCampina) or a mixture thereof at a final concentration of 0 (blank) or 0.5% by weight (each condition was repeated twice). TM The plates were placed in a hood for approximately 20 minutes to allow the dissolved oxygen concentration to fall below 5% and the temperature of the medium to increase to 37°C. After this 20 minutes, the plates were inoculated with a pool of fecal material from healthy 70+ human subjects to initiate fermentation. During fermentation, pH was controlled at pH 6.8 with a dead zone of 0.1 using ammonium gas and CO2. The plates were orbitally shaken at 300 rpm. A gas mixture (CO2 / H2 / N2: 10 / 5 / 85) was used to keep the culture anaerobic during fermentation. After 7 hours of fermentation, a 1 ml sample was taken.

[0055] [Table 1]

[0056] Fecal pool preparation Fecal samples from eight healthy elderly individuals were included in the pool.

[0057] Inclusion criteria were: age >70 years; no intake of antibiotics, prebiotics, probiotics and / or laxatives in the 3 months prior to donation; no symptoms of infection such as fever or diarrhea in the 3 days prior to donation; BMI 22.5–27.5.

[0058] Individual fecal samples were diluted 10-fold by adding phosphate buffer (50 mM pH 7.2 ± 0.1, 0.05% cysteine-HCl) under anaerobic conditions and homogenized in a stomacher. 20% glycerol was then added to obtain a fecal slurry with a final fecal concentration of 8%. The fecal slurry was aliquoted and stored in a freezer (-80°C). Immediately before inoculating the plates, fecal samples from each of the eight donors were thawed and mixed in the anaerobic chamber. Each reaction well (working volume 5 ml) was inoculated with 625 μl of the fecal pool (1% feces).

[0059] Sample preparation and analysis Immediately after inoculation, a 1 ml sample was taken from the wells without oligosaccharides. Additionally, a 1 ml sample was taken during fermentation at t=7 (in the anaerobic chamber). The sample was centrifuged at 13200 rpm for 5 min at 4°C. The bacterial pellet was used to determine the microbiota composition by shotgun metagenomics sequencing (BaseClear) after DNA isolation (QIAamp PowerFecal Pro DNA kit). The supernatant was diluted 10 / 100 times with 30% acetonitrile and used for analysis of oligosaccharides by HPLC.

[0060] result As shown in FIG. 1, the relative abundance of both B. adolescentis and B. longum and their total abundance were increased by the addition of 2'-FL and GOS individually compared to blanc.

[0061] Moreover, it can be observed that GOS has a greater effect than 2'-FL, however, surprisingly, replacing a portion of GOS with 2'FL further increases the relative abundance, thereby demonstrating a synergistic effect of 2'-FL and GOS.

Claims

1. In a method for treating or preventing the onset of a disease associated with a low relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, the method comprises: A nutritional composition comprising galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of GOS:2'-FL of 1:9 to 9:1 for use in increasing the relative abundance of Lactobacillus longum.

2. 2. The nutritional composition of claim 1, wherein said diseases associated with a low relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, are selected from the group consisting of enteropathogenic infections, intestinal barrier dysfunction, type 2 diabetes, gut-brain disorders such as stress, anxiety and depression, allergies such as atopic dermatitis, asthma and inflammatory bowel conditions such as inflammatory bowel disease (IBD).

3. 3. The nutritional composition for use according to claim 1 or 2, wherein said non-infant human subject has a below-average abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the digestive tract.

4. 3. The nutritional composition for use according to claim 1 or 2, wherein the non-infant human subject suffers from or is at risk of developing enteropathogenic infections, intestinal barrier dysfunction, type 2 diabetes, gut-brain disorders such as stress, anxiety and depression, allergies such as atopic dermatitis, asthma and / or inflammatory bowel conditions such as inflammatory bowel disease (IBD).

5. 3. The nutritional composition for use according to claim 1 or 2, wherein said non-infant human subject is an adult human subject over 18 years of age, preferably over 40 years of age, more preferably over 50 years of age, even more preferably over 60 years of age, more preferably over 65 years of age, most preferably at least 70 years of age.

6. 3. A nutritional composition for use according to claim 1 or 2, wherein the weight ratio of GOS:2'-FL is in the range of 1:8 to 8:1, preferably 1:7 to 7:1, more preferably 1:6 to 6:1, even more preferably 1:5 to 5:1, more preferably 1:4 to 4:1, more preferably 1:3 to 4:1, more preferably 1:2 to 4:1 and most preferably 1:1 to 4:

1.

7. The nutritional composition for use according to claim 1 or 2, wherein the daily dosage of GOS+2'-FL administered to said non-infant human subject is in the range of 0.5 to 30 g, preferably 0.5 to 20 g, more preferably 1 to 10 g, even more preferably 1 to 8 g, most preferably 2 to 6 g.

8. 3. A nutritional composition for use according to claim 1 or 2, having the form of a drink, ice cream, bar, snack, bakery product, confectionery, candy, pasta or dietary supplement.

9. 1. Non-therapeutic use of a nutritional composition comprising galactooligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) in a weight ratio of GOS:2'-FL of 1:9 to 9:1 to increase the relative abundance of Bifidobacteria, preferably Bifidobacterium adolescentis and / or Bifidobacterium longum, in the digestive tract of a non-infant human subject.

10. 10. The non-therapeutic use according to claim 9, wherein the weight ratio of GOS:2'-FL is in the range of 1:8 to 8:1, preferably 1:7 to 7:1, more preferably 1:6 to 6:1, even more preferably 1:5 to 5:1, more preferably 1:4 to 4:1, more preferably 1:3 to 4:1, more preferably 1:2 to 4:1, most preferably 1:1 to 4:

1.

11. 11. The non-therapeutic use according to claim 9 or 10, wherein said non-infant human subject is an adult over 18 years of age, preferably over 40 years of age, more preferably over 50 years of age, even more preferably over 60 years of age, more preferably over 65 years of age, most preferably at least 70 years of age.

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