Nutrients that stimulate the immune system

Abstract

The present invention discloses a nutritional composition comprising a combination of non-digestible oligosaccharides and a product obtained by incubation of an aqueous substrate with Bifidobacterium and optionally a product obtained by incubation of an aqueous substrate with Streptococcus thermophilus. The combination synergistically improves the immune system by stimulating Th1 response, and / or attenuating Th2 response, improving immunization response, improving resistance to infection and / or reducing allergic reactions.

Description

Technical Field

[0001] This invention relates to a nutrient for improving immune system response and a method for preparing the same. Background Technology

[0002] The immune system has different possible response modes. A decisive step in the type of immune response is the stimulation of different subsets of T cells. So-called Th1 cells primarily produce cytokines (IFN-γ, IL-12, IL-2) that stimulate cellular immune responses—such as against pathogens and vaccines. In contrast, Th2 cells primarily produce IL-4, IL-5, and IL-10. These cytokines amplify IgE-mediated allergic reactions and inflammation. Th1 and Th2-related cytokines act antagonistically, and Th1 and Th2 responses are in a well-controlled balance in normal physiological settings. Neither Th1 nor Th2 responses are dominant. If these responses are out of balance, then the dominance of either the Th1 or Th2 immune response plays a role in several pathological conditions.

[0003] WO 2005 / 039319 and WO 2006 / 091103 relate to an article containing a mixture of Bifidobacterium breve and non-digestible carbohydrates for use in infants who are not breastfed or partially breastfed, and its use for the treatment or prevention of immune disorders in infants who are not breastfed or partially breastfed. WO 2005 / 039597 relates to the use of acidic and neutral oligosaccharides for enhancing the immune system and for the treatment and / or prevention of immune system-related diseases. WO 01 / 642255 relates to a nutritional composition containing probiotics for enhancing immune responses. WO 2004 / 0938998 and WO 2004 / 0938999 relate to an immunomodulatory product derived from a culture of Bifidobacterium.

[0004] Significant efforts have been made to find other options for balancing and stimulating the immune system. Summary of the Invention

[0005] Surprisingly, the inventors have discovered that a combination of the following components synergistically stimulates the immune system: i) a product obtained by incubating an aqueous substrate—wherein the substrate is at least one selected from milk, milk protein, whey, whey protein hydrolysate, casein hydrolysate, and lactose—with Bifidobacteria, then inactivating the Bifidobacteria by heating the incubation mixture and / or removing the Bifidobacterial cells from the incubation mixture by centrifugation and / or filtration; and ii) at least two different non-digestible carbohydrates, wherein at least one, preferably two, is selected from fructo-oligosaccharide, galacto-oligosaccharide, and gluco-oligosaccharide. The ingredients include arabino-arabinose, mannan-oligosaccharide, xylo-oligosaccharide, fucose, arabinogalacto-oligosaccharide, glucomanno-oligosaccharide, glactomanno-oligosaccharide, raffinose, lactofrucose, sialic acid-containing oligosaccharides, and uronic acid oligosaccharides. It has been found that the articles of the present invention, i.e., those comprising the above-mentioned compounds, weaken the Th2 reaction and / or enhance the Th1 reaction. The observed effects are unexpectedly higher than the sum of the effects of the individual components.

[0006] Unexpectedly enhanced effects were observed in two different experimental animal models. In animals that ingested the product of this invention, a stronger delayed-type hypersensitivity response was observed after influenza vaccination, indicating an enhanced Th1 response, compared to animals that ingested a single component. A combination of the product obtained by incubating a substrate with Bifidobacterium and the product obtained by incubating a substrate with Streptococcus and two non-digestible carbohydrates resulted in an enhanced delayed-type hypersensitivity response.

[0007] Compared with animals that ingested a single component, a decrease in immediate hypersensitivity response was observed in animals that ingested the product of the present invention after pulmonary exposure to the allergen, indicating a weakened Th2 response.

[0008] The synergistic effect between the combination of components i) and ii) defined above is surprising. It cannot be explained by a symbiotic effect—whereby the non-digestible carbohydrate (ii) specifically stimulates the growth of beneficial microorganisms present in the same product—because no live Bifidobacterium cells are present in the product of the present invention. Removing and / or inactivating live Bifidobacterium cells has the advantage that the product can be pasteurized and / or sterilized, thereby reducing the possibility of contamination by harmful microorganisms. This is particularly advantageous in infants and young children due to their higher intestinal permeability. Furthermore, since the Bifidobacteria are removed or inactivated, they themselves cannot cause infection.

[0009] Another advantage is the ability to better control the dosage of the bioactive component received by each human subject. Equally advantageous is the ease and cost reduction in product preservation. Furthermore, it is advantageous that no post-acidification occurs in the preserved product, thus avoiding side effects and unpleasant taste associated with protein coagulation. Another advantage is that inactivated and / or removed Bifidobacteria are no longer able to break down and consume the non-digestible carbohydrates.

[0010] The present invention is particularly advantageous to the following populations: human subjects with a decreased Th1 response compared to healthy adults, especially newborns, elderly people with immunosenescence, people with AIDS or infected with human immunodeficiency virus, and cancer patients and cachectic cancer patients who are undergoing or have undergone chemotherapy or radiotherapy.

[0011] The products of this invention are particularly suitable for the treatment and / or prevention of diseases that can be prevented and / or treated by reducing Th2 response, especially allergic reactions, atopic dermatitis, asthma, food allergies, allergic rhinitis (e.g., hay fever), dust mite allergies, and other forms of hypersensitivity such as systemic anaphylaxis and acute urticaria.

[0012] The articles of this invention are suitable for treating and / or preventing infections, and / or for supporting vaccination responses before, during and / or after vaccination. Detailed Implementation

[0013] This invention relates to a method for manufacturing an article, comprising the following steps:

[0014] a: Incubating an aqueous substrate— wherein the substrate comprises at least one selected from milk, milk protein, whey, whey protein hydrolysate, casein hydrolysate and lactose—with Bifidobacterium to obtain an incubation mixture;

[0015] b: Inactivating the Bifidobacteria by heating the incubation mixture and / or removing the Bifidobacterial cells from the incubation mixture by centrifugation and / or filtration; and

[0016] c: Combining a composition comprising a mixture of the product obtained in step a or step b—preferably step b—with at least two different non-digestible carbohydrates, wherein at least one, preferably two, of the non-digestible carbohydrates are selected from fructooligosaccharides, galacto-oligosaccharides, glucose-oligosaccharides, arabino-oligosaccharides, manno-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides, glucomanno-oligosaccharides, galacto-manno-oligosaccharides, raffinose, lactulose-oligosaccharides, sialic acid-containing oligosaccharides, and uronic acid oligosaccharides.

[0017] In one aspect, the present invention relates to an article obtainable by the method of the present invention. In one embodiment, the present invention relates to a nutritional composition comprising or consisting of an article obtainable by the method of the present invention.

[0018] The present invention also relates to a method for treating and / or preventing diseases in mammals, the method comprising administering the article of the present invention to the mammal.

[0019] The present invention also relates to a method for providing nutrition to infants and young children, the method comprising administering to the infants and young children an article of the present invention or a nutritional composition comprising an article of the present invention.

[0020] This includes methods for incubating a substrate with Bifidobacteria.

[0021] This invention relates to an article obtainable or acquired by incubating an aqueous substrate with Bifidobacteria (hereinafter referred to as step (a)), wherein the aqueous substrate comprises at least one selected from milk, milk protein, whey, whey protein hydrolysate, casein hydrolysate, and lactose. Step (b) is performed on the incubation mixture obtained or acquireable in step (a), step (b) comprising an inactivation step by heat treatment and / or a step by centrifugation and / or filtration to remove Bifidobacterium cells. Step (b) is performed to reduce the amount of live Bifidobacteria in the article, preferably by at least 90%, more preferably by at least 99%.

[0022] In one embodiment, the incubation step includes a fermentation step and / or a biotransformation step. During fermentation, the aqueous substrate is fermented by the Bifidobacterium. During biotransformation, the aqueous substrate is biotransformed by the Bifidobacterium.

[0023] The product obtained or available (hereinafter, "obtained" also means "available") by the method of the present invention preferably comprises bacterial cell debris such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acid (LTA), lipoproteins, DNA, and / or capsular polysaccharides. These debris elicit an immune response. Advantageously, it is preferable to use an aqueous substrate containing at least one selected from milk, milk protein, whey, whey protein hydrolysate, casein hydrolysate, and lactose—incubated with Bifidobacteria, followed by inactivation and / or removal of the Bifidobacteria, as this results in a higher concentration of bacterial cell debris. Incubation of the aqueous substrate with the Bifidobacteria can form other bioactive compounds, such as organic acids, bioactive peptides, and / or oligosaccharides, which stimulate the immune system. When using commercial probiotic products, probiotic cells are typically washed and separated from aqueous growth media containing bacterial cell debris, thereby significantly reducing or even eliminating the supernatant containing the bacterial cell debris. This is not the case in the present invention. The presence of intact cells (living or dead cells) is not necessary for an immune response; after the incubation step with Bifidobacteria in this invention, the aqueous substrate itself already has a beneficial effect on the immune system.

[0024] Bifidobacteria

[0025] Bifidobacteria used in the methods of the present invention are preferably provided as single or mixed cultures. Bifidobacteria are Gram-positive, anaerobic, rod-shaped lactic acid-producing bacteria. The Bifidobacteria species of the present invention preferably have at least 95% 16S rRNA sequence identity, more preferably at least 97%, as defined in manuals on this subject, such as Sambrook, J., Fritsch, EF, and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (NY) Laboratory Press. The preferred Bifidobacterium species used are also described in Scardovi, V. Genus Bifidobacterium, pp. 1418-1434. In: Bergey's manual of systematic Bacteriology, Vol. 2. Sneath, PHA, NSMair, MESharpe and JGHolt (ed.). Baltimore: Williams & Wilkins, 1986, p. 635. Preferably, the Bifidobacterium used in producing the articles of the present invention is at least one selected from the following: *Bifidobacterium breve*, *Bifidobacterium infantis*, *Bifidobacterium bifidum*, *Bifidobacterium catenulatum*, *Bifidobacterium adolescentis*, *Bifidobacterium thermophilum*, *Bifidobacterium gallicum*, *Bifidobacterium animalis*, or *Bifidobacterium lactis*, *Bifidobacterium angulatum*, *Bifidobacterium pseudodocatenulatum*, *Bifidobacterium thermophilum*, and *Bifidobacterium longum*, more preferably *Bifidobacterium breve*, *Bifidobacterium infantis*, *Bifidobacterium bifidum*, *Bifidobacterium catenulatum*, and *Bifidobacterium thermophilum*, more preferably *Bifidobacterium breve*, *Bifidobacterium infantis*, *Bifidobacterium bifidum*, *Bifidobacterium catenulatum*, and *Bifidobacterium longum*, more preferably *Bifidobacterium longum* and *Bifidobacterium breve*, even more preferably *Bifidobacterium breve*, and most preferably *Bifidobacterium breve* deposited at CNCM in Paris, France. I-2219.Bifidobacterium breve CNCM I-2219 was deposited by Compagnie Gervais Danone on May 31, 1999, at the Collection Nationale de Cultures de Microorganisms van Institute Pasteur in Paris, France, pursuant to the Budapest Treaty. This strain was disclosed in WO 2004 / 093899.

[0026] Preferably, the composition further comprises a product obtained by incubating a streptococcus with an aqueous substrate comprising at least one selected from milk, milk protein, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate, and lactose, preferably at least one selected from whey and lactose, and preferably subsequently inactivating and / or removing the streptococcus. Streptococcus is a Gram-positive, anaerobic, cocci-forming lactic acid-producing bacterium. The streptococcal strains of the present invention preferably have at least 95% 16S rRNA sequence identity, more preferably at least 97%, as defined in manuals on this subject, such as Sambrook, J., Fritsch, EF, and Maniatis, T. (1989), *Molecular Cloning*, A Laboratory Manual, 2nd ed., Cold Spring Harbor (NY) Laboratory Press. Preferably, the production process of the product obtained by incubating a streptococcus with an aqueous substrate and then inactivating the streptococcus is carried out using streptococcus species selected from the following genera: *Streptococcus salivarius* and *Streptococcus thermophilus*, more preferably *Streptococcus thermophilus*, even more preferably *Streptococcus thermophilus* strain CNCM I-1620 or strain CNCM I-1470, and most preferably strain CNCM I-1620. *Streptococcus thermophilus* strains CNCM I-1620 and I-1470 advantageously produce large amounts of β-galactosidase. *Streptococcus thermophilus* CNCM I-1620 was deposited by Compagnie Gervais Danone on August 23, 1995, in accordance with the Treaty of Budapest at Collection Nationale de Cultures de Microorganisms van Institute Pasteur, Paris, France. Streptococcus thermophilus CNCM I-1470 was deposited by Compagnie Gervais Danone on August 25, 1994, at Collection Nationale de Cultures de Microorganisms van Institute Pasteur in Paris, France, under the Treaty of Budapest. These strains are disclosed in EP 778885.

[0027] Method steps a) Incubation of aqueous substrates

[0028] Step (a) is preferably performed in the following manner:

[0029] a1 will use Bifidobacterium at 1×102 Up to 1×10 11 A concentration of CFU / ml of Bifidobacterium was inoculated into an aqueous substrate with a pH of 4 to 8, and the substrate contained at least one selected from milk, whey, whey protein, whey protein hydrolysate, casein hydrolysate, and lactose.

[0030] a2 The Bifidobacterium is incubated in the aqueous culture medium for at least 2 hours under aerobic or anaerobic conditions and at a temperature of 20°C to 50°C.

[0031] The aqueous substrate to be incubated with Bifidobacteria comprises at least one selected from milk, whey, whey protein, whey protein hydrolysate, casein hydrolysate, and lactose, more preferably at least two. The substrate preferably does not contain intact casein. It has been found that when the aqueous substrate contains a large amount of intact casein, less immunostimulatory substances are formed. Therefore, the aqueous substrate preferably contains less than 25 g / L of casein, more preferably less than 15 g / L, even more preferably less than 5 g / L, and most preferably less than 1 g / L of intact casein. The aqueous substrate therefore even more preferably contains whey and / or whey protein and / or whey protein hydrolysate.

[0032] The milk can be whole milk, semi-skimmed milk, and / or skimmed milk. Skimmed milk is preferred. The whey can be sweet whey, acid whey, or whey or whey permeate obtained by, for example, filtration to remove casein. The whey is preferably present at a concentration of 3 to 80 g dry weight per liter (L) of aqueous substrate, more preferably 40 to 60 g / L. Preferably, whey protein concentrate is used. Preferably, whey protein hydrolysate is used and is present at an amount of 2 to 80 g dry weight per liter (L) of aqueous substrate, more preferably 5 to 15 g / L. Lactose is preferably present at an amount of 5 to 50 g dry weight per liter (L) of aqueous substrate, more preferably 1 to 30 g / L. The aqueous substrate preferably contains a buffer salt to maintain the pH within the desired range. Preferably, sodium dihydrogen phosphate or potassium dihydrogen phosphate is used as a buffer salt, preferably in an amount of 0.5 to 5 g / L, more preferably 1.5 to 3 g / L. The aqueous substrate preferably contains 0.1 to 0.5 g / L of aqueous substrate, more preferably 0.2 to 0.4 g / L of cysteine. The presence of cysteine ​​results in a lower redox potential of the substrate, which is beneficial to the activity of lactic acid-producing bacteria, particularly Bifidobacteria. The aqueous substrate preferably contains 0.5 to 5 g / L of aqueous substrate, more preferably 1.5 to 3 g / L of yeast extract. Yeast extract is a rich source of enzyme cofactors and growth factors for lactic acid-producing bacteria. The presence of yeast extract will enhance the biotransformation and / or fermentation carried out by Bifidobacteria.

[0033] The aqueous substrate to be incubated preferably contains a high concentration of solids, preferably more than 20% by weight by volume, and more preferably more than 40% by weight. This high concentration is advantageous for subsequent processing steps—such as spray drying, centrifugation, or filtration.

[0034] To remove unwanted live bacteria, the aqueous substrate may be pasteurized appropriately before the incubation step. To inactivate enzymes, the product may be pasteurized appropriately after incubation. Suitable enzyme inactivation is performed at 75°C for 1 minute. Suitable enzyme inactivation is performed at 75°C for 3 minutes. The aqueous substrate may be homogenized appropriately before and / or after the incubation step. Homogenization results in a more stable product, especially in the presence of fats (lipids).

[0035] The incubation density is preferably 1×10⁻⁶. 2 Up to 1×10 11 CFU / ml aqueous substrate, preferably 1×10⁻⁶. 4 Up to 1×10 10 CFU / ml aqueous substrate, preferably 1×10 7 Up to 1×10 9 CFU / ml aqueous substrate. Methods for obtaining a concentrated starting culture of Bifidobacteria to be incubated in an aqueous substrate are known in the art. The final bacterial density of Bifidobacteria after incubation is preferably 1 × 10⁻⁶. 3 Up to 1×10 11 cfu / ml aqueous substrate, more preferably 1×10 4 Up to 1×10 9 cfu / ml aqueous substrate.

[0036] Incubation with Bifidobacteria is preferably carried out at a temperature of about 20°C to 50°C, more preferably 30°C to 45°C, and even more preferably about 37°C to 42°C. The optimal temperature for the growth and / or activity of Bifidobacteria is 37°C to 42°C.

[0037] Incubation with Bifidobacteria is preferably carried out under anaerobic conditions because the growth of Bifidobacteria and the enzymatic activity of many Bifidobacterial enzymes are inhibited under aerobic conditions. However, acidification is not always necessary. Therefore, in one embodiment, the incubation step is suitably carried out under aerobic conditions.

[0038] Incubation with Bifidobacteria is preferably carried out at pH 4 to 8, more preferably pH 5.6 to 7.5, and even more preferably pH 6 to 7.5. Such pH values ​​do not induce protein precipitation and / or unpleasant flavors, while allowing Bifidobacteria to interact with the aqueous substrate.

[0039] The incubation time is preferably at least 2 hours, more preferably 4 to 48 hours, more preferably 6 to 24 hours, and even more preferably 6 to 15 hours. A sufficiently long time allows for a greater degree of interaction between the Bifidobacterium and the substrate and / or a greater degree of generation of cell debris such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acid (LTA), lipoproteins, DNA, and / or capsular polysaccharides, while for economic reasons, the incubation time need not be too long.

[0040] Methods for inactivating and / or physically removing live Bifidobacterium cells

[0041] In step (b) of the method of the present invention, after incubation in step a), it is preferable to remove substantially all live Bifidobacterium cells, for example, by inactivation and / or physical removal through heat treatment. The cells are preferably inactivated by heat treatment. The Bifidobacteria are preferably killed by heat after incubation step a). Preferred methods of heat killing include pasteurization, sterilization, ultra-high temperature treatment, spray cooking at a temperature where Bifidobacteria cannot survive, and / or spray drying. The heat treatment is preferably performed at at least 50°C, more preferably at least 65°C. The heat treatment is preferably performed for at least 5 minutes, more preferably at least 10 minutes. The heat treatment is preferably performed at at least 50°C for at least 5 minutes, more preferably at at least 65°C for at least 10 minutes. The heat treatment is preferably performed at at least 75°C for at least 1 minute, more preferably at at least 75°C for at least 3 minutes.

[0042] The intact cells of Bifidobacterium are preferably removed from the incubation product by physical removal methods such as filtration and / or centrifugation (e.g., centrifugation at 3000g for 1 hour), with the intact cells remaining in the flaky precipitate or retentate, while the product obtained by incubating milk and / or milk-derived substrate with Bifidobacterium and subsequently inactivating Bifidobacterium cell debris is retained in the supernatant and / or filtrate.

[0043] Heat inactivation and / or physical removal of live cells result in a quantity of live Bifidobacteria below the detection limit known in the art for conventional plate culture techniques. The detection limit is less than 10 per gram of dry weight of the composition. 4 CFU Bifidobacterium live cells, preferably less than 10 3 cfu / g. The heat inactivation and / or removal steps preferably remove at least 90%, more preferably at least 99%, of the cells present in the incubation mixture after step a).

[0044] The requirement to inactivate live cells has the advantage that the final nutritional composition can be pasteurized and / or sterilized after production, thereby reducing the chance of harmful microbial contamination. Therefore, this invention enables ready-to-use liquid formulations to be prepared and stored at room temperature. Furthermore, the dosage of the bioactive component received by each human subject can be more easily controlled because no further growth occurs in the liquid product or in the human subject's gut. The latter is a variable factor dependent on the individual gut environment and can therefore lead to differences in the degree of beneficial effects among individual subjects. Another advantage is that the inactivated and / or removed Bifidobacteria and Streptococci are no longer able to degrade and consume the non-digestible carbohydrates.

[0045] Another advantage is that the nutritional composition is easier to store and less expensive because no special precautions are required to maintain the viability of Bifidobacteria at an acceptable level. This is especially true for products with a water activity greater than 0.3. Post-acidification does not occur in stored high water activity products and / or during the period after reconstitution of the powdered nutritional composition with water and before consumption. Side effects and unpleasant taste associated with protein coagulation are also avoided in this way.

[0046] The addition of other components and other optional method steps

[0047] Optionally, one or more of the following steps may follow step b) of the above method:

[0048] i) The incubated product is ultrafiltered through a membrane with a cut-off threshold of 100 to 300 kDa to obtain a concentrated permeate. The membrane is preferably a polyethersulfone membrane, and filtration is preferably carried out at a temperature below 60°C.

[0049] ii) Wash the concentrated residue with water.

[0050] iii) Dehydrate the concentrated permeate, preferably by freeze drying.

[0051] iv) Dissolve the dehydrated effluent in a buffer solution, preferably a Tris buffer solution with a pH of 6-8.

[0052] v) Using a column with an exclusion threshold of 600 kDa—preferably a Dextran or agarose column such as Superdex. At 200°C, the permeate solution was subjected to gel size exclusion chromatography.

[0053] vi) At the end of chromatography, recover the filtered fraction or exclusion fraction.

[0054] vii) The product was desalted using a membrane with a retention limit of 10 kDa. The size exclusion fraction was recovered after chromatography.

[0055] These steps are preferably performed under aseptic conditions. After step a), preferably just before or after step b), other ingredients that facilitate obtaining the desired final nutritional composition may be added. These ingredients are preferably added after step b). For an infant formula, some ingredients known in the art may be added, such as skim milk, whey, lactose, vegetable fats, minerals, and vitamins.

[0056] Preferably, an aqueous substrate containing whey, whey protein, and / or whey protein hydrolysate is pasteurized, cooled, and incubated with one or more Bifidobacterium strains, preferably the short Bifidobacterium strain CNCM I-2219. The incubation product is then heat-treated and stored. Optionally, the incubation product is mixed with other components constituting the nutritional composition. A fat component may or may not be included, but it is preferred not to add a fat component at this stage. Preferably, the mixture is preheated, followed by the sequential (in-line) addition of fat (also referred to herein as "lipids"), homogenization, heat treatment, and drying.

[0057] Another preferred method for preparing the incubation product of the present invention is disclosed in WO 01 / 01785, more specifically in Examples 1 and 2. Another preferred method for preparing the incubation product of the present invention is described in WO 2004 / 093899, more specifically in Example 1.

[0058] On a dry weight basis, the final nutritional composition preferably contains 5 to 100% by weight of the product obtained by step b, more preferably 5 to 99.5% by weight, even more preferably 5 to 95% by weight, even more preferably 5 to 80% by weight, even more preferably 5 to 40% by weight, and most preferably 10 to 40% by weight. The final nutritional composition preferably contains 0.5 to 20% by weight of the product obtained by step b per 100 ml, more preferably 0.5 to 14% by weight per 100 ml, even more preferably 1 to 10% by weight, and even more preferably 1 to 5% by weight.

[0059] Based on the dry weight of the final composition, the final nutritional composition of the present invention preferably contains more than 1 × 10⁻⁶ per g. 3 cfu, preferably more than 1×10 4 cfu, or even more than 1×10 5 Inactivated Bifidobacteria and / or Bifidobacterium-derived bacterial fragments obtained from CFU of Bifidobacteria. Based on the dry weight of the final composition, the inactivated Bifidobacteria and / or Bifidobacterium-derived fragments are preferably from less than 1 × 10⁻⁶ per g. 11cfu, preferably less than 1×10 10 cfu, or even less than 1×10 9 These values ​​are obtained from Bifidobacteria in CFU. These values ​​can be calculated by determining the amount of Bifidobacteria in the mixture after incubation in step a) and before step b), and subsequently determining how many grams of the article of the present invention are present in the final composition (on a dry weight basis).

[0060] Other ingredients that may be beneficial in obtaining the desired final nutritional composition may be added after method step a) or b). These ingredients are preferably added after step b). For an infant formula, some ingredients known in the art may be added, such as skim milk, whey, lactose, vegetable fats, minerals, and vitamins.

[0061] The method preferably includes the following additional steps:

[0062] d: Incubate a substrate with Streptococcus thermophilus, preferably strain CNCM I-1620 or strain CNCM I-1470, to obtain an incubation mixture, wherein the substrate is selected from milk, milk protein, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate, and lactose.

[0063] e: The thermophilic streptococci are inactivated by heating the incubation mixture of step e and / or thermophilic streptococcal cells are removed from the incubation mixture of step e by centrifugation and / or filtration. Steps e and b are preferably performed simultaneously. Intact streptococcal cells are preferably removed from the incubation product by physical removal methods such as filtration and / or centrifugation (e.g., centrifugation at 3000g for 1 hour), with the intact cells remaining in the flaky precipitate or exudate, while the product obtained by incubating milk and / or milk-derived substrate with streptococci and subsequently inactivating streptococcal cell debris is retained in the supernatant and / or filtrate.

[0064] Heat inactivation and / or physical removal of live cells result in a level of live streptococci below the detection limit known in the art for conventional plate culture techniques. The detection limit is less than 10 per gram of dry weight of the composition. 4 CFU streptococcal live cells, preferably less than 10 3 Therefore, in one embodiment of the invention, the article after step e) preferably contains less than 10 cfu / g. 3 CFU of live streptococci / g of the product dry weight. The heat inactivation and / or removal steps preferably remove at least 90%, more preferably at least 99%, of the cells present in the incubation mixture after step d).

[0065] Another preferred method for preparing incubation products from the thermophilic Streptococcus strain of the present invention is disclosed in EP 0778885, and more specifically in Examples 5 and 6. Another preferred method for preparing incubation products from the thermophilic Streptococcus strain of the present invention is disclosed in Examples 2 to 6 of FR2723960.

[0066] Preferably, an aqueous substrate containing whey, whey protein, and / or whey protein hydrolysate is pasteurized, cooled, and incubated with one or more Bifidobacterium strains, preferably the short Bifidobacterium strain CNCM I-2219. The incubation product is then heat-treated and stored. Preferably, a second aqueous substrate containing whey and / or lactose is incubated with Streptococcus thermophilus, preferably strain CNCM I-1620 or strain CNCM I-1470. Subsequently, the two incubation products are preferably mixed together and combined with other components constituting the nutritional composition. A fat component may or may not be included, but it is preferred not to add a fat component at this stage. Preferably, the mixture is preheated, followed by the sequential (in-line) addition of fat (also referred to herein as "lipids"), homogenization, heat treatment, and drying.

[0067] Incubation step d can be performed simultaneously with the incubation step with Bifidobacterium in step a. Preferably, incubation with Streptococcus thermophilus and incubation with Bifidobacterium are performed in separate method steps. Separate incubation allows for optimal conditions for each different bacterium and / or prevents adverse interference from different bacteria and releases immunostimulatory components. Preferably, the incubation mixture obtained after incubation of the substrate with Streptococcus thermophilus is added to the mixture obtained in step a), step b, or step c, more preferably after step a). When the composition of the present invention further comprises the mixture obtained after incubation with Streptococcus thermophilus, an improvement effect on delayed-type hypersensitivity is observed. Therefore, in one embodiment, the method of the present invention includes another step:

[0068] f: Combine the incubation mixture obtained in step d or e—preferably step d—with the incubation mixture obtained in step a, b, or c—preferably step a. In one embodiment, the incubation mixture obtained in step d is combined with the incubation mixture obtained in step a, and steps b and e are performed simultaneously.

[0069] On a dry weight basis, the final nutritional composition preferably contains 2 to 94.5% of the product obtained by step d, more preferably 5 to 80% by weight, and even more preferably 5 to 40% by weight. The final nutritional composition preferably contains 0.2 to 20% by weight of the product obtained by step d per 100 ml, more preferably 0.5 to 14% by weight per 100 ml, more preferably 1 to 10% by weight per 100 ml, and even more preferably 1 to 5% by weight per 100 ml.

[0070] Non-digestible carbohydrates

[0071] The article obtained by the method of the present invention comprises at least two different non-digestible carbohydrates. These non-digestible carbohydrates are added in step c) of the method. The non-digestible carbohydrates can advantageously stimulate the immune system. This stimulation can be achieved through the improvement of the intestinal microbiota and / or a direct effect on the immune system. The presence of the two different non-digestible carbohydrates synergistically improves the intestinal microbiota and / or synergistically stimulates the immune system.

[0072] The presence of two non-digestible carbohydrates and a product obtained by incubating an aqueous substrate with Bifidobacteria, followed by inactivation and / or removal of the Bifidobacteria, synergistically enhances the immune response. The conjugate unexpectedly and synergistically results in a higher Th1 response, a lower Th2 response, and / or a higher Th1 / Th2 response ratio. The conjugate synergistically enhances the inoculation response, synergistically improves allergic reactions, and / or enhances resistance to infection. This synergistic effect between the two compounds is unexpected and cannot be explained by a symbiotic effect—whereby the non-digestible carbohydrates specifically stimulate the growth of beneficial microorganisms present in the same product—because no live Bifidobacteria are present in the incubated milk and / or milk-derived product.

[0073] As used herein, the term "oligosaccharide" refers to a carbohydrate with a degree of polymerization (DP) of 2-250, preferably 2-100, more preferably 2-60, and even more preferably 2-10. If the articles of the present invention include oligosaccharides with a DP of 2-100, this includes compositions containing oligosaccharides with DPs of 2-5, 50-70, and 7-60. As used herein, the term "non-digestible carbohydrate" refers to carbohydrates that are not digested in the intestine by acids or digestive enzymes in the human upper digestive tract (small intestine and stomach), but are preferably fermented by the human gut microbiota. For example, sucrose, lactose, maltose, and maltodextrin are considered digestible.

[0074] Preferably, the non-digestible carbohydrates of the present invention are soluble. When referring to carbohydrates, the term "soluble" as used herein means that the carbohydrate is soluble according to the method described in L. Prosky et al., J. Assoc. Off. Anal. Chem. 71, 1017-1023 (1988).

[0075] In this invention, the different non-digestible carbohydrates refer to non-digestible carbohydrates with different monosaccharide unit compositions, different degrees of polymerization (DP), or both. Two non-digestible carbohydrates are considered different in monosaccharide composition when there is a difference of at least 30 mol% in monosaccharide composition, more preferably at least 50 mol%, based on total molar monosaccharide units. For example, the difference between galacto-oligosaccharides with an average composition of Glu-Gal3 and fructooligosaccharides with an average composition of Glu-Fru3 is 75 mol%. If the difference in average DP between two non-digestible carbohydrates exceeds 5 monosaccharide units, preferably more than 10 units, and even more preferably more than 15 units, then the two carbohydrates are considered different in DP. For example, the difference in DP between hydrolyzed inulin with an average DP of 4 and long-chain inulin with an average DP of 25 is 21 units.

[0076] The non-digestible carbohydrate is selected from at least one, more preferably at least two, of the following: fructooligosaccharides, galacto-oligosaccharides, glucose-oligosaccharides, arabino-oligosaccharides, mannan-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides, glucomannan-oligosaccharides, galacto-manno-oligosaccharides, sialic acid-containing oligosaccharides, and uronic acid oligosaccharides. Preferably, the article of the present invention comprises fructooligosaccharides, galacto-oligosaccharides, and / or galacturonic acid oligosaccharides, more preferably galacto-oligosaccharides, and most preferably β-galacto-oligosaccharides. Fructooligosaccharides include inulin; galacto-oligosaccharides include trans-galacto-oligosaccharides or β-galacto-oligosaccharides; oligodextroses include gentio-oligosaccharide, nigero-oligosaccharide, cyclodextrin-oligosaccharide, and polydextrose; arabinogalacto-oligosaccharides include gum arabic; and galactomannan-oligosaccharides include partially hydrolyzed guar gum.

[0077] To further improve the quality, the non-digestible carbohydrates of the present invention preferably have a high content of short-chain oligosaccharides, as these short-chain oligosaccharides strongly promote the growth of Bifidobacteria. Therefore, preferably, at least 10% by weight of the non-digestible carbohydrates in the articles of the present invention have a DP of 2 to 5 (i.e., 2, 3, 4, and / or 5), and at least 5% by weight of the non-digestible carbohydrates have a DP of 10 to 60. Preferably, at least 50% by weight, more preferably at least 75% by weight of the non-digestible carbohydrates have a DP of 2-9 (i.e., 2, 3, 4, 5, 6, 7, 8, and / or 9).

[0078] The articles obtained by the method of the present invention more preferably contain galactooligosaccharides. Preferably, the galactooligosaccharides are selected from β-galactooligosaccharides, lacto-N-tetrasaccharides (LNT), lacto-N-neo-tetrasaccharides (neo-LNT), fucoidyllactose, fucoidylated LNT, and fucoidylated neo-LNT. In a particularly preferred embodiment, the articles of the present invention contain β-galactooligosaccharides. The β-galactooligosaccharides used in the present invention refer to oligosaccharides with a degree of polymerization (DP) of 2-20 and composed of more than 50%, preferably more than 65%, of galactose units based on monomer subunits, wherein at least 50%, more preferably at least 75%, more preferably at least 90% of the galactose units are linked together by β-glycosidic bonds (preferably β-1,4-glycosidic bonds). β-bonds are also predominant in human milk oligosaccharides. The average DP is preferably in the range of 3 to 6. A glucose unit may be present at the reducing end of the galactose unit chain. β-galactooligosaccharides are sometimes also called trans-galactooligosaccharides (TOS). A suitable source of β-galactooligosaccharides is Vivinal. GOS (commercially available from Borculo Domo Ingredients, Zwolle, Netherlands). Other suitable sources are Oligomate (Yakult), Cupoligo (Nissin), and Bi2muno (Classado). β-galactooligosaccharides have been found to promote the growth of lactic acid-producing bacteria, particularly Bifidobacteria.

[0079] The article obtained by the method of the present invention more preferably contains fructooligosaccharides. The fructooligosaccharides used in the present invention refer to carbohydrates composed of more than 50%, preferably more than 65%, of fructose units (based on monomer subunits), wherein at least 50%, more preferably at least 75%, and even more preferably at least 90% of the fructose units are linked together by β-glycosidic bonds (preferably β-2,1-glycosidic bonds). A glucose unit may be present at the reducing end of the fructose unit chain. The fructooligosaccharides preferably have a degree of polymerization (DP) of 2 to 250, more preferably 2 to 100, and even more preferably 10 to 60 or an average DP. Fructooligosaccharides include fructans, hydrolyzed fructans, inulin, hydrolyzed inulin, and synthetic fructooligosaccharides. Preferably, the article contains short-chain fructooligosaccharides with an average degree of polymerization (DP) of 3-6, more preferably hydrolyzed inulin or synthetic fructooligosaccharides. The article preferably contains long-chain fructooligosaccharides with an average DP greater than 20. Preferably, the article contains both short-chain and long-chain fructooligosaccharides. The fructooligosaccharides suitable for the method of this invention are also readily available, such as Raftiline. HP (Orafti).

[0080] The articles obtained by the method of the present invention are more preferably combinations comprising galactooligosaccharides and fructooligosaccharides (more preferably long-chain fructooligosaccharides). Such mixtures synergistically stimulate the growth of healthy gut microbiota, particularly Bifidobacteria.

[0081] The product obtained by the method of the present invention preferably comprises uronic acid oligosaccharides, more preferably mannonuric acid oligosaccharides and / or galacturonic acid oligosaccharides, and even more preferably galacturonic acid. The term uronic acid oligosaccharide as used in the present invention refers to an oligosaccharide in which at least 50% of the monosaccharide units present are uronic acids. The term galacturonic acid oligosaccharide as used in the present invention refers to an oligosaccharide in which at least 50% of the monosaccharide units present are galacturonic acids. The galacturonic acid oligosaccharides used in the present invention are preferably prepared by degradation of pectin, pectate, and / or polygalacturonic acid. Preferably, the degraded pectin is prepared by hydrolysis and / or β-elimination of fruit and / or vegetable pectin, more preferably apple, citrus, and / or beet pectin, and even more preferably apple, citrus, and / or beet pectin degraded by at least one lysin. In a preferred embodiment, at least one terminal galacturonic acid unit of the galacturonic acid oligosaccharide has a double bond. This double bond effectively protects intestinal epithelial cells from adhesion by pathogenic bacteria. Preferably, one of the terminal galacturonic acid units contains a C4-C5 double bond. The galacturonic acid oligosaccharide can be derivatized. The galacturonic acid oligosaccharide can be methoxylated and / or amidated. Preferably, the galacturonic acid oligosaccharide is characterized by a methoxylation degree greater than 20%, more preferably greater than 50%, and more preferably greater than 70%. The galacturonic acid oligosaccharide can advantageously reduce the adhesion of pathogenic microorganisms to intestinal epithelial cells. Furthermore, the galacturonic acid oligosaccharide can stimulate the immune system by enhancing the Th1 response.

[0082] Therefore, in one embodiment, the article obtained by the method of the present invention and used in the present invention preferably contains at least β-galactooligosaccharides. In one embodiment, the article obtained by the method of the present invention and used in the present invention preferably contains at least short-chain fructooligosaccharides and / or long-chain fructooligosaccharides, preferably long-chain fructooligosaccharides. In one embodiment, the article obtained by the method of the present invention and used in the present invention preferably contains at least uronic acid oligosaccharides. In one embodiment, the article used in the present invention preferably contains at least β-galactooligosaccharides and at least short-chain fructooligosaccharides and / or long-chain fructooligosaccharides. In one embodiment, the article used in the present invention preferably contains at least β-galactooligosaccharides and at least uronic acid oligosaccharides. In one embodiment, the article used in the present invention preferably contains at least short-chain fructooligosaccharides and uronic acid oligosaccharides, or at least long-chain fructooligosaccharides and uronic acid oligosaccharides. In one embodiment, the article according to the present invention preferably contains at least β-galactooligosaccharides, short-chain fructooligosaccharides and uronic acid oligosaccharides, or at least β-galactooligosaccharides, long-chain fructooligosaccharides and uronic acid oligosaccharides. Conjugates of uronic acid oligosaccharides with galacto-oligosaccharides and / or fructooligosaccharides result in higher Th1 responses compared to uronic acid oligosaccharides alone or galacto-oligosaccharides and / or fructooligosaccharides alone.

[0083] Preferably, the weight ratio between the mixture of two different non-digestible carbohydrates (preferably β-galactooligosaccharides and fructooligosaccharides) is 0.05-20, more preferably 1-20. β-galactooligosaccharides are more similar to human milk oligosaccharides. Preferably, the articles of the present invention comprise β-galactooligosaccharides with a DP of 2-10 and / or fructooligosaccharides with a DP of 2-60. This combination has been found to synergistically increase Bifidobacteria and Lactobacilli. The presence of these three non-digestible oligosaccharides further stimulates Bifidobacteria. The weight ratio of trans-galactooligosaccharides:fructooligosaccharides:uronic acid oligosaccharides is preferably (20-2):1:(1-20), more preferably (12-7):1:(1-3).

[0084] Preferably, the final nutritional composition comprising or containing the article obtained by the method of the present invention contains 80 mg to 2 g, more preferably 150 mg to 1.50 g, and even more preferably 300 mg to 1 g of non-digestible carbohydrates per 100 ml. Based on dry weight, the nutritional composition preferably contains 0.25-20% by weight, more preferably 0.5-10% by weight, and even more preferably 1.5-7.5% by weight of non-digestible carbohydrates. Small amounts of non-digestible carbohydrates are less effective in stimulating the immune system and / or beneficial bacteria in the microbiome, while excessive amounts can cause side effects such as bloating and abdominal discomfort.

[0085] The two different non-digestible carbohydrates are added after step a), preferably before or after step b) (i.e., step c), preferably after step b), preferably after step e), i.e. after inactivation by heat treatment and / or after removal of Bifidobacteria and optional Streptococcus thermophilus.

[0086] The product obtained by the method of the present invention preferably comprises an aqueous substrate incubated with Bifidobacterium breve, more preferably strain CNCM I-2219, and at least one, preferably two, non-digestible carbohydrates. The aqueous substrate comprises at least one selected from milk, milk protein, whey, whey protein hydrolysate, and lactose. The non-digestible carbohydrates are selected from galacto-oligosaccharides and fructooligosaccharides.

[0087] In one aspect, the present invention relates to the method of the invention, wherein only one non-digestible carbohydrate is added in step c. This is particularly advantageous when the only non-digestible carbohydrate is fructooligosaccharide.

[0088] In one aspect, the present invention relates to an article obtainable by the method of the present invention described above. In one embodiment, a final nutritional composition comprising or containing an article obtained by the method of the present invention comprises 0.5 to 10 g of non-digestible carbohydrates as defined above per 100 g of the dry weight of the composition. In one embodiment, the final nutritional composition comprising or containing an article obtained by the method of the present invention is subjected to a process at 20°C and for 100 seconds. -1 The viscosity at the shear rate is 1 to 60 mPa·s.

[0089] The above method preferably includes a drying step. Drying steps are well known in the art. A suitable drying step is spray drying. The drying step is preferably carried out in such a manner that the dried product is a powder containing less than 10% by weight water, more preferably less than 5% by weight water. The drying step is preferably carried out after step c. Alternatively, the drying step may be carried out after step b and / or step e, after which the non-digestible oligosaccharide is dry-mixed into the product.

[0090] Nutrients

[0091] The articles of the present invention have been found to be advantageously applicable to food, such as infant formula and clinical food. The articles of the present invention, or compositions containing the articles of the present invention, are preferably administered enterally, more preferably orally. The compositions are preferably complete nutrients.

[0092] The nutrients are preferably suitable for infants and young children. More preferably, the nutritional composition of the present invention is an infant formula or follow-on formula. The composition of the present invention can be advantageously used as a complete nutrient for infants and young children.

[0093] The composition of the present invention is preferably an infant nutrition product, comprising, based on the dry weight of the infant nutrition product:

[0094] i) A total of 0.5 to 10% by weight of galactooligosaccharides and fructooligosaccharides, and

[0095] ii) 5 to 99.5% by weight of the article obtained after step b of the method according to the invention, wherein the Bifidobacterium in step a belongs to the species Bifidobacterium breve, preferably the Bifidobacterium breve strain CNCM I-2219.

[0096] iii) and optionally 2 to 94.5% by weight of the article obtained after step e.

[0097] Such nutrients preferably contain lipids, proteins, and carbohydrates, and are preferably administered in liquid form. As used herein, the term "liquid food" includes dry foods (such as powders) accompanied by instructions on mixing said dry food mixture with a suitable liquid (such as water).

[0098] Therefore, the nutritional composition of the present invention preferably comprises 5% to 60% lipids, 5% to 60% protein, and 15% to 90% digestible carbohydrates, based on total calories. When intended for adult subjects, the nutritional composition of the present invention preferably comprises 5% to 30% lipids, 15% to 40% protein, and 25% to 75% digestible carbohydrates, based on total calories. When intended for infants and young children, the nutritional composition of the present invention preferably comprises 30% to 60% lipids, 5% to 15% protein, and 25% to 75% digestible carbohydrates, based on total calories; more preferably, 35% to 50% lipids, 7.5% to 12.5% ​​protein, and 40% to 55% digestible carbohydrates, based on total calories. To calculate the protein percentage based on total calories, the total calories provided by protein, peptides, and amino acids need to be considered.

[0099] The lipid preferably comprises a vegetable oil. The vegetable lipid is preferably at least one oil selected from: soybean oil, palm oil, coconut oil, safflower oil, sunflower seed oil, corn oil, rapeseed oil, and lecithin. Preferably, a conjugate of the vegetable lipid with at least one oil selected from fish oil and ω-3-containing vegetable oils, algal oils, or bacterial oils is used. In a preferred embodiment, the method of the invention further includes administering long-chain polyunsaturated fatty acids (LC-PUFAs). Since these are believed to act on the immune system through a mechanism different from that of non-digestible carbohydrates and different from that of products obtained by incubating milk and / or milk-derived substrates with Bifidobacteria and subsequently inactivating said Bifidobacteria, the conjugate of the article of the invention with LC-PUFAs is considered to act synergistically.

[0100] When intended for adults, the nutritional composition of the present invention preferably contains 5% to 60% lipids, more preferably 5% to 30% lipids, based on total calories; when intended for infants and young children, the nutritional composition of the present invention preferably contains 30% to 60% lipids, more preferably 35% to 50% lipids, based on total calories.

[0101] The proteins used in the nutritional products are preferably selected from non-human animal proteins (e.g., milk proteins, meat proteins, and egg proteins), plant proteins (e.g., soy proteins, wheat proteins, rice proteins, and pea proteins), hydrolysates of the above proteins, free amino acids, and mixtures of proteins, hydrolysates, and free amino acids. Bovine milk proteins, such as casein and whey proteins, are particularly preferred. Since the compositions of the present invention are suitable for reducing allergic reactions, especially in infants and young children, the proteins are preferably selected from hydrolyzed milk proteins. Preferably, the compositions of the present invention comprise hydrolyzed casein and / or hydrolyzed whey protein, hydrolyzed plant proteins, and / or free amino acids, most preferably hydrolyzed whey protein. The use of these proteins further reduces allergic reactions. The use of these hydrolyzed proteins can advantageously improve the absorption of dietary protein components. This is particularly advantageous for infants and young children and sick subjects.

[0102] When intended for use in adult subjects, the nutritional composition of the present invention preferably contains 5% to 60% protein, more preferably 15% to 40% protein, based on total calories; and when intended for use in infants and young children, the nutritional composition of the present invention preferably contains 5% to 15% protein, more preferably 7.5% to 12.5% ​​protein, based on total calories. To calculate the percentage of protein based on total calories, the total calories provided by protein, peptides, and amino acids need to be taken into account.

[0103] A digestible carbohydrate source can be added to the nutritional formulation. Any suitable digestible carbohydrate source can be used, such as sucrose, lactose, glucose, fructose, solid corn syrup, and maltodextrin, as well as mixtures of the above. Therefore, the nutritional composition of the present invention preferably contains 15% to 90% carbohydrates based on total calories, more preferably 25% to 75% carbohydrates based on total calories, and even more preferably 40% to 55% carbohydrates based on total calories.

[0104] The nutritional composition of the present invention is preferably in liquid form. It preferably has a defined viscosity. It has been found that the method of the present invention can provide liquid nutrients with sufficiently low viscosity, so that it can be used, for example, as liquid infant food and liquid patient food, which can be fed through a nipple, tube, or straw while maintaining low viscosity. In a preferred embodiment, the composition of the present invention is heated at 20°C and for 100 seconds... -1The viscosity at the shear rate is below 600 mPa·s, preferably below 250 mPa·s, more preferably below 60 mPa·s, even more preferably below 35 mPa·s, and most preferably below 6 mPa·s. When the term viscosity is used herein, it refers to a physical parameter determined by means of a Carri-Med CSL rheometer. The geometry used is conical (6 cm 2-degree acrylic cone), and the gap between the plate and the geometry is set to 55 μm. The viscosity is measured from 0 to 150 s. -1 The linear continuous ramp shear rate was applied for 20 seconds. It should be noted that the invention also covers compositions in powder form, with instructions for preparing aqueous solutions—for example, by adding water in a certain proportion and then obtaining a specified viscosity.

[0105] Irregular bowel movements (e.g., hard stools, insufficient stool volume, diarrhea) are a major problem in many infants and young children, as well as in sick subjects receiving liquid foods. It has been found that bowel problems can be alleviated by administering the present invention in liquid food form with a molar osmolality of 50-500 mOsm / kg, more preferably 100-400 mOsm / kg.

[0106] For the reasons stated above, it is equally important that the liquid food does not have an excessively high calorie density, yet still provides sufficient calories for the subject. Therefore, the liquid food preferably has a calorie density of 0.1-2.5 kcal / ml, and even more preferably 0.5-1.5 kcal / ml. When used as an infant formula, the calorie density is most preferably 0.6-0.8 kcal / ml.

[0107] application

[0108] The articles of this invention obtained by the method of this invention have been found to synergistically stimulate the immune system. Specifically, they weaken the Th2 response and / or enhance the Th1 response. The effect of the combination of these two components is greater than the sum of the effects of the individual components.

[0109] The articles of the present invention can be advantageously used for the treatment and / or prevention of diseases, and therefore the present invention relates to a method for treating and / or preventing diseases in mammals, the method comprising administering the articles of the present invention to the mammal. In other words, the present invention also relates to the use of the articles of the present invention for the preparation of a composition, preferably a nutritional composition, for the treatment and / or prevention of diseases. In other words, the present invention relates to an article or a nutritional composition containing the articles of the present invention for the treatment and / or prevention of diseases. The mammal is preferably a human, and even more preferably a human infant. Therefore, the present invention also relates to the use of the articles of the present invention for the preparation of a composition, preferably a nutritional article, for the treatment and / or prevention of diseases in infants and young children. Or in other words, the present invention relates to an article or a nutritional composition containing the articles of the present invention for the treatment and / or prevention of diseases in infants and young children.

[0110] In the context of this invention, the age of the infant is 0 to 6 years, preferably 0 to 4 years, more preferably 0 to 2 years, and more preferably 0 to 1 year.

[0111] This invention also relates to a method for providing nutrition to infants and young children, the method comprising administering the article of the invention or a nutritional composition thereof to the infant or young child. In other words, this invention also relates to the use of the article of the invention in preparing a nutritional composition for providing nutrition to infants and young children. In other words, this invention relates to an article or a nutritional composition containing the article of the invention for providing nutrition to infants and young children.

[0112] The articles of the present invention can advantageously be used to enhance the Th1 response, weaken the Th2 response, repair the Th1 / Th2 response imbalance, maintain a favorable Th1 / Th2 balance, and / or for the treatment and prevention of diseases associated with Th1 / Th2 imbalance. Therefore, compositions proposed for, for example, promoting immune system maturation, enhancing resistance to pathogens by strengthening the immune system, and / or supporting the immune system are part of this invention. In another aspect, the present invention provides a method for treating and / or preventing immune system-related diseases, the method comprising administering to the mammal a composition containing a therapeutically effective amount of the article of the present invention. In yet another aspect, the present invention provides a method for enhancing the immune response of a mammal, the method comprising administering to the mammal the article of the present invention.

[0113] The immune system of human newborn infants is characterized by an excessive Th2 response. During immune system maturation, the Th1 response is enhanced and the Th1 / Th2 balance changes toward measured values ​​in healthy adults. Therefore, the articles of the present invention are particularly beneficial to human infants and young children. The present invention supports the maturation of the immune system in infants and young children. In another embodiment, the method of the present invention involves administering the articles of the present invention to a person aged 0-6 years, preferably 0-4 years, preferably 0-2 years, more preferably 0-1 years. In a preferred embodiment, the method of the present invention involves promoting the maturation of the immune system in human subjects aged 0-6 years, preferably 0-4 years, preferably 0-2 years, more preferably 0-1 years.

[0114] An excessive Th2 response leads to extreme sensitivity to foreign components that should not elicit any immune response, such as allergic reactions and related diseases like atopic dermatitis, asthma, food allergies, allergic rhinitis (e.g., hay fever), house dust mite allergies, and other forms of hypersensitivity such as systemic anaphylaxis and acute urticaria. Therefore, the articles of the present invention are particularly advantageous for the treatment and / or prevention of diseases selected from allergic reactions, food allergies, allergic rhinitis, asthma, atopic dermatitis, house dust mite allergies, and acute urticaria. The present invention attenuates the Th2 response.

[0115] An enhanced Th1 response leads to an enhanced response to pathogenic bacteria and / or viruses. Therefore, the products of the present invention are suitable for the treatment and / or prevention of infections. The products of the present invention can be advantageously used for the treatment and / or prevention of intestinal infections, systemic infections, and / or respiratory infections.

[0116] The articles of the present invention have also been found to be suitable for supporting vaccination methods, such as enhancing the effectiveness of vaccination methods. The articles of the present invention are suitable for supporting vaccination responses before, during, and / or after vaccination. Specifically, they can suitably enhance the effectiveness of vaccines against diphtheria-tetanus, pertussis, polio, measles / mumps / rubella, pneumococcal conjugate vaccine, Haemophilus influenzae conjugate vaccine, hepatitis B, hepatitis A, varicella, and influenza. Therefore, the articles of the present invention can be advantageously used for the treatment and / or prevention of infections, and / or for enhancing vaccination responses.

[0117] In the context of this invention, “prevention” of a disease or condition also refers to the treatment of a person at risk of developing said disease or condition.

[0118] Therefore, the present invention is beneficial to human subjects with immunodeficiency, especially elderly people with immunosenescence, people with AIDS or infected with human immunodeficiency virus, as well as cancer patients, especially cancer patients undergoing or having undergone chemotherapy, radiotherapy and cachectic cancer patients, patients with chronic obstructive pulmonary disease and / or patients with diabetes.

[0119] Example

[0120] Example 1: Preparation of incubation mixture using Bifidobacterium breve

[0121] Pasteurized skim milk was concentrated to 43% by weight based on the weight of the skim milk by evaporation. The concentrate was cooled to 37°C and then concentrated with 3 × 10⁻⁶ ppm per ml. 9 Inoculate with a 10% (v / w) culture of *Bifidobacterium breve* CNCM I-2219 using CFU. The preparation of this inoculum is described below. The initial pH was 6–6.1. Incubate at 37°C for 8 hours in a water bath with stirring for 10 minutes every 2 hours. Afterward, maintain the pH at 6–6.1 and the *Bifidobacterium breve* count at 10⁻⁶. 6 1000 bacteria / ml (step (a)). This concentrate was pasteurized (step b) and then spray-dried, and named BbC50cf.

[0122] The inoculum was prepared as follows: a concentrated, precipitated starting culture was added to pasteurized skim milk; and incubated anaerobically at 37°C for 8 hours. During this process, the number of *Bifidobacterium breve* cells increased to approximately 3 × 10⁻⁶. 9 The concentration is cfu / ml, and the acidity changes from approximately pH 6.7 to pH 4.5-5.0. Suitably, 0.1-0.5 g / L of cysteine ​​and / or 0.5-5 g / L of yeast extract of the aqueous substrate are present.

[0123] Example 2: Preparation of incubation mixture using Streptococcus thermophilus

[0124] Preheated inoculum was prepared from Streptococcus thermophilus CNCM I-1620 culture by maintaining frozen inoculum at approximately 40°C for approximately 7 hours. A pasteurized lactose solution (350-450 g / L) was cooled to approximately 45-55°C, and then inoculated with approximately 3 × 10⁻⁶ g / L of lactose solution. 9 Inoculate with approximately 10% (v / w) of preheated Streptococcus thermophilus CNCM I-1620 inoculum (cfu / mL). The initial pH is approximately pH 6. Incubate in a tank at approximately 50°C for approximately 7 hours, stirring for 10 minutes every 2 hours. Afterward, maintain the pH at 6-8 and the Streptococcus thermophilus count at approximately 10⁻⁶. 6 1000 bacteria / ml (step (d)). This concentrate was pasteurized (step e) and then spray-dried, and named St065cf.

[0125] Example 3: Enhanced Th1 response in incubation mixtures and non-digestible carbohydrates Synergistic effect

[0126] method:

[0127] The effects of a diet containing the following components were tested in a mouse model: (a) BbC50cf prepared according to Example 1; (b) a conjugate of galacto-oligosaccharides (Elixor) and fructooligosaccharides (Raftilin HP); and (c) a conjugate of (a) and (b) (i.e., comprising step c) of the method of the present invention), wherein the response to the antigen was measured by a delayed-type hypersensitivity (DTH) reaction. This ear DTH response following local stimulation with an antigen present in the vaccine is a measure of Th1 cell proliferation. During the response to infection and / or vaccination, Th1 cells proliferate in response to stimulation with the antigen. Upon subsequent stimulation of the ear with the antigen, these Th1 cells infiltrate the ear and cause swelling. The infiltration of Th1 cells into the ear takes approximately 24 hours, therefore the swelling is delayed. The more Th1 cells proliferate during the initial vaccination and / or infection, the stronger the DTH observed after stimulation with the antigen.

[0128] BbC50cf was lyophilized and administered to mouse diets at a final concentration of 3% by weight. The weight ratio of the non-digestible oligosaccharide mixture (GF) containing trans-galacto-oligosaccharide (GOS) (derived from Vivinal-GOS (Borculo Domo Ingredients, Netherlands)) and fructooligosaccharide (FPS) (derived from Raftiline HP, Orafti, Tiense, Belgium) was 9:1. Diets containing 1% by weight of GF based on total mouse weight were tested. The effect of the combination of non-digestible oligosaccharides and Bifidobacterium breve incubation mixture (GF+BbC50cf) was tested using a diet containing 1% by weight of GF and 3% by weight of BbC50cf based on total dietary weight.

[0129] Female, 6-week-old C57B1 / 6 mice (Harlan Nederland BV, Horst, the Netherlands) were housed in groups under regular 12-hour light / dark conditions. Each group consisted of 10 animals, with 3 animals in the negative control group. These animals were fed a semi-synthetic diet (Research Diet Services, Wijk bij Duurstede, the Netherlands). The diet was controlled according to AIN93G standards (Reeves et al. (1993) J Nutrition 123(11): 1923-31), and the oligosaccharide-supplemented diet was also based on these standards.

[0130] Vaccination began 20 days after the mice acclimatized to their new habitat and diet. Blood samples were collected on day 0, prior to vaccination. The first vaccination was administered subcutaneously on day 1. A booster vaccination was given three weeks later (day 22). Nine days after the booster (day 31), baseline ear thickness was measured using a digital outside micrometer (Mitutoyo, Veenendaal, the Netherlands), and a delayed-type hypersensitivity (DTH) response was induced by intradermal (ic) injection of the antigen solution into the mouse auricle. The DTH response was measured 24 hours later (day 32), blood samples were collected, and the mice were euthanized. This is the ear thickness 24 hours later minus the ear thickness at t=0.

[0131] The vaccination consisted of 100 μl of an intradermal (ic) injection containing a 1:1 mixture of antigen solution and Stimune adjuvant (Specol, Cedi-diagnostics BV, Lelystad, the Netherlands). The antigen solution was a 1:100 dilution of Influvac 2002 / 2003 (Solvay Pharmaceuticals, Weesp, the Netherlands) in PBS. Influvac is a trivalent protein vaccine containing 3 x 30 μg / ml hemagglutinins from three different influenza strains. For DTH response, 25 μl of dialyzed Influvac was administered intradermally to both ears of mice as a DTH stimulant.

[0132] result:

[0133] Diets containing 1% wt% GF or 3% wt% BbC50cf both resulted in statistically insignificant, minor enhancements to the DTH response. A combination of 1% wt% GF and 3% wt% BbC50cf resulted in a statistically significant 89% enhancement to the DTH response (see Table 1). Since these effects were significantly higher than the DTH response of diets containing only oligosaccharides, and much higher than the additive effect of GF and BbC50cf—which could be calculated as a 49% enhancement of DTH—these results suggest a synergistic effect of administering non-digestible oligosaccharides and BbC50cf to enhance the Th1 response. The measured effects indicate the advantageous use of combinations of the following components in the application of this invention: at least two non-digestible carbohydrates, and a product obtained by incubating an aqueous substrate with Bifidobacteria, followed by heating the incubation mixture and / or removing the Bifidobacterium cells by centrifugation and / or filtration.

[0134] Table 1: DTH Reaction

[0135] Group: Average DTH μm(SE) ΔDTHμm relative to DTH fake injection -1.7(4.6) 0 0 placebo 67.6(14.9) 69.3 1.00 GF 73.55(8.6) 75.25 1.09 BbC50cf 95.55(5.5) 97.25 1.40 GF+BbC50cf 129.4*(17.1) 131.1 1.89 Theoretical value of GF+BbC50cf 101.50 103.2 1.49

[0136] * Significantly different from the control (P < 0.01)

[0137] The results of this experiment indicate that the present invention can be advantageously used to support vaccination responses. The results also indicate that it can be advantageously used in subjects with low Th1 responses, particularly infants and young children. Furthermore, the results indicate that it can be advantageously used in subjects with low Th1 responses, particularly the elderly with immunosenescence or at risk of immunosenescence, HIV patients, AIDS patients and / or cancer patients undergoing or having undergone chemotherapy and / or radiation therapy, cachectic cancer patients, patients with COPD, and / or patients with diabetes. This model indicates underlying immunological changes that can be beneficial in all conditions of immune system dysfunction. It is known that the immune systems of infants, the elderly, HIV-infected individuals, cancer patients, COPD patients, and / or diabetic patients are not fully functional. For all of the above, the method of the present invention is another potentially beneficial aid.

[0138] Example 4: Incubation mixture obtained from Bifidobacterium breve and Streptococcus thermophilus with non-sterilized bacteria Enhancement effect of chemochemical carbohydrates on Th1 responses

[0139] method:

[0140] Using the same mouse model as in Example 3, the effects of diets containing the following components were tested in independent experiments: (a) BbC50cf (3 wt%) prepared according to Example 1 and a combination of galacto-oligosaccharide (Elixor) and fructooligosaccharide (Raftilin HP); (b) BbC50cf (3 wt%) prepared according to Example 1 and St065cf (3 wt%) prepared according to Example 2; and (c) a combination of BbC50cf (3 wt%), St065cf (3 wt%), and galacto-oligosaccharide and fructooligosaccharide.

[0141] Galacto-oligosaccharides (Elixor) and fructooligosaccharides (Raftilin HP) (GF) are present in a weight ratio of 9:1 and in an amount of 1% by weight of total diet.

[0142] result:

[0143] Diets containing a conjugate of 1% by weight GF and 3% by weight BbC50cf resulted in a statistically significant 100% enhancement of the DTH response (see Table 2).

[0144] The combination of BbC50cf and St065cf also caused a statistically significant 112% enhancement of the DTH reaction.

[0145] Surprisingly, the combination of all three components showed a maximum response enhancement of 192%.

[0146] These results indicate that the addition of BbC50cf and St065cf to the non-digestible oligosaccharide provides a further enhanced effect on the Th1 response. The experimental results demonstrate the advantageous use of combinations of the following components in the application of this invention: i) at least two different non-digestible carbohydrates, ii) products obtained by incubating an aqueous substrate with Bifidobacteria, followed by inactivation of the incubation mixture by heating and / or removal of the Bifidobacterium cells by centrifugation and / or filtration, and iii) products obtained by incubating an aqueous substrate with Streptococcus, followed by inactivation of the incubation mixture by heating and / or removal of the Streptococcus cells by centrifugation and / or filtration.

[0147] Table 2: DTH Reaction

[0148] Group: Average DTH μm(SE) ΔDTHμm relative to DTH fake injection 24.3(0.6) 0 0 placebo 66.5(0.3) 42.2 1.00 BbC50cf+GF 108.9(0.3) * 84.6 2.00 BbC50cf+St065cf 113.6(0.3) * 89.3 2.12 BbC50cf+GF+St065cf 147.6(0.2) *a 123.2 2.92

[0149] * Compared with placebo, p < 0.05

[0150] a Compared with Bbc50f+GF and Bbc50f+St065cf, p < 0.05

[0151] Example 5: Synergistic effect of incubation products and non-digestible carbohydrates on the weakening of Th2 response Same effect

[0152] method

[0153] The effects of a diet containing the following components were tested in a mouse model: (a) BbC50cf prepared according to Example 1; (b) a conjugate of non-digestible carbohydrates (GF) containing galacto-oligosaccharides (Elixor) and fructooligosaccharides (Raftilin HP); and (c) a conjugate of (a) and (b) (i.e., including step c) of the method of the present invention), wherein the response to the allergen was measured by an immediate-type hypersensitivity (ITH) reaction. This ear ITH response following allergen stimulation in the lungs is a measure of enhanced Th2 response. During the response to the allergen in the lungs, mast cells throughout the body, including in the ears, undergo almost immediate degranulation. These responses all involve IgE, which in turn requires Th2 response during helper T cell development. Therefore, enhanced ITH indicates increased IgE and thus enhanced Th2 response.

[0154] Male BALB / c mice free of specific pathogens were obtained from Charles River (Maastricht, the Netherlands). Food and water were provided freely, and the mice were given these medications at 6–9 weeks of age. Ovalbumin (Grade V) and acetyl-β-methylcholine chloride (acetylcholine) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aluminum hydroxide (AlumImject) was purchased from Pierce (Rockford, IL, USA).

[0155] Mice were tested with the same diet as in Example 2 for 14 days, followed by ovalbumin (OVA) sensitization until the end of the experiment. Mice were sensitized by two intraperitoneal injections on days 0 and 7, either in saline containing 10 μg of ovalbumin adsorbed onto 2.25 mg of aluminum hydroxide or saline alone. Mice were challenged on days 35, 38, and 41 by 20-minute inhalation of an ovalbumin aerosol in an plexiglass contact chamber. The aerosol was generated by nebulizing a saline solution of ovalbumin (10 mg / ml) using a Pari LC Star nebulizer (Pari Respiratory Equipment, Richmond, VA, USA). ITH was measured 1 hour after challenge with the allergen ovalbumin.

[0156] result

[0157] The obtained ITH results are shown in Table 3:

[0158] Table 3: ITH Reaction

[0159] Group: Average ITH μm (SE) ΔITH μm Compared to ITH Comparison 104.3(3.8) 0 0 placebo 184.7(8.7) 80.4 1 GF 159.8(17.9) * 55.5 0.69 BbC50cf 173.1(20.3) 68.8 0.86 GF+BbC50cf 136.6(14.4) ** 32.3 0.40 Theoretical value of GF+BbC50cf 151.7 47.4 0.59

[0160] * Significantly different from the control ( * P < 0.05 ** P < 0.01).

[0161] Diets containing 1% wt% GF or 3% wt% BbC50cf both resulted in a slight attenuation of the ITH response. A conjugate of 1% wt% GF and 3% wt% BbC50cf resulted in a statistically significant attenuation of the ITH response of approximately 60% (see Table 3). Since these effects were significantly higher than the ITH response of diets containing only oligosaccharides, and much higher than the additive effect of GF and BbC50cf—which could be calculated as an attenuation of approximately 40% of the ITH response—these results suggest a synergistic effect in attenuating the Th2 response by administering non-digestible oligosaccharides and incubation products.

[0162] Experimental results demonstrate the advantageous use of the following components in the application of this invention: non-digestible carbohydrates, and products obtained by incubating the substrate with Bifidobacteria, followed by inactivation and / or removal of the Bifidobacteria. The results of this experiment suggest that the invention can be advantageously used to support the prevention and / or treatment of, specifically, asthma, allergic reactions, atopic dermatitis, allergic conjunctivitis, dust mite allergies, urticaria, and allergic rhinitis.

[0163] Example 6: Infant formula

[0164] An infant formula is prepared by mixing the following ingredients: demineralized whey, vegetable fat, lactose, skim milk, non-digestible carbohydrates, whey protein concentrate, fish oil, minerals, and vitamins.

[0165] The skim milk was pre-incubated using the method described in Example 1.

[0166] The concentration of the product obtained by incubation with Bifidobacterium breve in steps a) and b) was 15.6% by weight based on the dry weight of the infant formula.

[0167] No live Bifidobacteria were detected in the final product.

[0168] The final composition of each 100ml of the infant formula comprises:

[0169] -66kcal

[0170] -1.3g of milk protein (casein and whey protein)

[0171] -7.3g of digestible carbohydrates (mainly lactose)

[0172] -3.5g of fat (mainly plant-based fats)

[0173] -0.8g trans-galactooligosaccharides (from Vivinal GOS) and fructooligosaccharides (from raftilin HP)

[0174] - Trace elements, minerals, vitamins and other micronutrients known in the art.

[0175] The infant formula claims to enhance the immune system and / or reduce the incidence of atopic eczema, and / or reduce the incidence of infections, and / or reduce the incidence of allergic reactions.

[0176] Example 7: Infant formula

[0177] Pasteurized skim milk was concentrated to approximately 43% by weight based on the weight of the skim milk by evaporation. The concentrate was cooled to approximately 37°C and then... 9Inoculate with approximately 10% (v / w) *Bifidobacterium breve* CNCM I-2219 culture at cfu / ml. The inoculum was prepared according to methods known in the art. The initial pH was 6-7.1. Incubate in a water bath at 37°C for 8 hours, stirring periodically for 10 minutes every 2 hours. Afterward, maintain the pH at 6-7.1 and the *Bifidobacterium breve* count at approximately 10... 6 bacteria / ml (step (a)).

[0178] Preheated inoculum was prepared from Streptococcus thermophilus CNCM I-1620 culture by maintaining frozen inoculum at approximately 40°C for approximately 7 hours. A pasteurized lactose solution (350-450 g / L) was cooled to approximately 45-55°C, and then inoculated with approximately 3 × 10⁻⁶ g / L of lactose solution. 9 Inoculate with approximately 10% (v / w) of preheated Streptococcus thermophilus CNCM I-1620 inoculum (cfu / ml). The initial pH is approximately pH 6. Incubate in a tank at approximately 50°C for approximately 7 hours, stirring for 10 minutes every 2 hours. Afterward, maintain a constant pH of 6-8 and a Streptococcus thermophilus count of approximately 10⁻⁶. 6 bacteria / ml (step (d)).

[0179] The incubated product, skim milk, vegetable fat, maltodextrin, trans-galacto-oligosaccharides and fructooligosaccharides, as well as other ingredients known for use in infant formula (e.g., vitamins, minerals, trace elements) are mixed together (steps c and f).

[0180] The mixture is pasteurized (steps b and e) and then spray-dried.

[0181] The final composition of each 100ml of the infant formula comprises:

[0182] -68kcal

[0183] -1.45g milk protein (derived from casein and whey protein in milk; partially hydrolyzed)

[0184] -8.6g of digestible carbohydrates (mainly lactose and maltodextrin)

[0185] -3.1g of fat (mainly plant-based fats)

[0186] -0.8g trans-galactooligosaccharides (from Vivinal GOS) and fructooligosaccharides (from raftilin HP)

[0187] - Trace elements, minerals, vitamins and other micronutrients known in the art (taurine, choline, inositol, nucleotides, carnitine).

Claims

1. A method for manufacturing an article of articles, comprising the following steps: -a) Incubating an aqueous substrate— wherein the substrate comprises at least one selected from milk, milk protein, whey, whey protein hydrolysate, casein hydrolysate and lactose—with Bifidobacterium to obtain an incubation mixture; -b) Inactivate the Bifidobacteria by heating the incubation mixture and / or remove the Bifidobacterium cells from the incubation mixture by centrifugation and / or filtration; -c) Combining a composition comprising the mixture obtained in step b) with two different non-digestible carbohydrates, wherein the non-digestible carbohydrates are selected from fructooligosaccharides and galacto-oligosaccharides.

2. The method of claim 1, wherein the Bifidobacterium belongs to the species Bifidobacterium breve.

3. The method of claim 2, wherein the Bifidobacterium breve belongs to the Bifidobacterium breve strain CNCM I-2219.

4. The method of any one of claims 1-3, further comprising the following steps: -d) Incubating a substrate with *Streptococcus thermophilus* to obtain an incubation mixture, wherein the substrate is selected from milk, milk protein, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate, and lactose. -e) Inactivate the thermophilic streptococci by heating the incubation mixture of step d) and / or remove thermophilic streptococcal cells from the incubation mixture of step d) by centrifugation and / or filtration. -f) Combine the incubation mixture obtained in step d) or e) with the incubation mixture obtained in step c).

5. The method of any one of claims 1-3, further comprising the following steps: -d) Incubating a substrate with Streptococcus thermophilus to obtain an incubation mixture, wherein the substrate is selected from milk, milk protein, whey, whey protein hydrolysate, casein, casein hydrolysate and lactose; and - Combine the incubation mixture obtained in step d) with the incubation mixture obtained in step a); - Inactivate the Bifidobacteria and Streptococcus thermophilus by heating the incubation mixture, and / or remove the Bifidobacteria cells and Streptococcus thermophilus from the incubation mixture by centrifugation and / or filtration; - The composition containing the mixture is combined with two different non-digestible carbohydrates, wherein the non-digestible carbohydrates are selected from fructooligosaccharides and galacto-oligosaccharides.

6. The method of claim 4, wherein the thermophilic streptococcus belongs to the thermophilic streptococcus strain CNCM I-1620.

7. The method of claim 5, wherein the thermophilic streptococcus belongs to the thermophilic streptococcus strain CNCM I-1620.

8. The method of any one of claims 1-3, wherein the article after step b) comprises less than 10 3 Live Bifidobacteria CFU / g of the product dry weight.

9. The method of any one of claims 1-3, wherein in step a), the substrate is treated with Bifidobacterium at a concentration of 1 × 10⁻⁶. 2 Up to 1×10 11 Inoculation with a concentration of cfu / mL of substrate, wherein the pH of the substrate is 4-8, and wherein incubation is carried out at a temperature of 20°C-50°C for at least 2 hours.

10. The method of any one of claims 1-3, wherein step b) comprises removing Bifidobacteria by centrifugation and / or filtration.

11. The method of any one of claims 1-3, wherein step b) comprises inactivation by heating at a temperature above 50°C for at least 5 minutes.

12. The method of any one of claims 1-3, further comprising a drying step.

13. The method of any one of claims 1-3, wherein in step a), the substrate is simultaneously incubated with Streptococcus thermophilus.

14. Articles obtainable by the method of any one of the preceding claims.

15. A nutritional composition comprising the article of claim 14 or thereof.

16. The composition of claim 15, wherein the composition comprises 0.5-10 g of non-digestible carbohydrates per 100 g dry weight of the composition.

17. The composition of claim 15 or 16, at 20°C and 100s -1 The viscosity at the shear rate is 1-60 mPa·s.

18. Infant nutrients, comprising, based on the dry weight of the infant nutrients: i) A total of 0.5 to 10% by weight of galactooligosaccharides and fructooligosaccharides, and ii) 5 to 99.5% by weight of the article obtained after step b) of claim 1, wherein the Bifidobacterium in step a) belongs to the species Bifidobacterium breve.

19. Infant nutrients, comprising, based on the dry weight of the infant nutrients: i) A total of 0.5 to 10% by weight of galactooligosaccharides and fructooligosaccharides, and ii) 5 to 99.5% by weight of the article obtained after step b) of claim 5, wherein the Bifidobacterium in step a) belongs to the species Bifidobacterium breve. iii) and 2-94.5% by weight of the article obtained after step e) of claim 4.

20. The infant nutrition of claim 18 or 19, wherein the Bifidobacterium breve belongs to the Bifidobacterium breve strain CNCM I-2219.

21. Use of the composition of any one of claims 15-17 or the infant nutrient of claim 18 or 19 in the preparation of a composition for providing nutrition to infants.

22. Use of the composition of any one of claims 15-17 or the infant nutrition of claim 18 or 19 in the preparation of a composition for the treatment and / or prevention of immune diseases selected from asthma and allergic reactions.

23. The use of claim 22, wherein the allergic reaction is atopic dermatitis, allergic conjunctivitis, dust mite allergy, urticaria, or allergic rhinitis.

24. The use of claim 22 or 23, wherein the immune disease is treated and / or prevented in infants and young children.

25. Use of the composition of any one of claims 15-17 or the infant nutrition of claim 18 or 19 in the preparation of a composition for the treatment and / or prevention of infection, and / or for enhancing the response to vaccination.

26. The use of claim 25, wherein the treatment and / or prevention of infection, and / or enhancement of vaccination response, is in infants and young children.

27. Use of the composition of any one of claims 15-17 or the infant nutrient of claim 18 or 19 in the preparation of a composition for use in: - Improves the immune system, the improvement being selected from enhancing the Th1 response, increasing the Th1 / Th2 balance, and weakening the Th2 response. - Treatment and / or prevention of immune aging in older adults.

28. The use of claim 27, wherein the improvement of the immune system is in infants and young children.