Preconditioning with L. reuteri

Abstract

The present invention relates to probiotic Lactobacillus reuteri strains obtained by growing the bacteria in a medium containing galactooligosaccharides (GOS), a process known as preconditioning. The preconditioning method enhances the effects of the probiotic L. reuteri strains, for example, improving mineral absorption in the gastrointestinal tract. The present invention also includes methods for producing and culturing probiotic Lactobacillus reuteri strains, as well as products and primary and secondary medical uses of such strains.

Description

[Technical field]

[0001] The present invention relates generally to enhancing the viability, persistence and / or probiotic effect of probiotic Lactobacillus reuteri strains in mammals or birds. The invention includes methods for producing and culturing probiotic L. reuteri strains, as well as products containing such strains. More specifically, the invention relates to probiotic L. reuteri strains obtained by growing the bacteria in a growth medium containing galactooligosaccharides (GOS), thereby enhancing the viability and persistence and / or enhancing the beneficial probiotic effects of the probiotic L. reuteri strains, such as improved mineral absorption, improved protective effect of epithelial integrity in the gastrointestinal tract, and improved bone formation and mineralization. Furthermore, the invention relates to preparations containing substrate components specifically selected to enhance the viability, persistence and / or probiotic effect of such probiotic L. reuteri strains in certain circumstances. [Background technology]

[0002] Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host". This is a scientific definition that is widely accepted worldwide (see, for example, Hill et al. 2014; Nat. Rev. Gastro. & Hepathology, 11:506-514). Probiotic products, which are usually dietary supplements or foods, can be recommended for various conditions or symptoms experienced by individuals. Lactic acid-producing bacteria, such as lactobacilli, are commonly used as probiotics in various types of foods, e.g., yogurt. The growth and colonization of harmful microorganisms can be prevented by such lactic acid-producing bacteria, by their own colonization in the intestinal system, by competition for available nutrients, and / or by the production of certain substances, e.g., hydrogen peroxide, bacteriocins, and / or organic acids, including lactic acid and acetic acid, that lower the pH of the intestine. It is well established that the interaction between the host and the intestinal microorganisms is important for the health and well-being of the host. The gut microbiota provides nutrients to the host, but also produces metabolic products that may be involved in immune responses and the regulation and development of the host's immune system, as well as in reducing inflammation and preventing allergic responses.

[0003] Prebiotics are compounds that induce the growth or activity of beneficial microorganisms, such as bacteria and fungi, by selectively stimulating their growth and / or activity. Prebiotics are substrates that are selectively utilized by such host microorganisms to confer health benefits (see, for example, Gibson et al. 2017; Nat. Rev. Gastro. & Hepathology, 14:491-502). Thus, in the gastrointestinal tract, prebiotics can change the composition of the gut microbiome. Dietary prebiotics are non-digestible fiber compounds that typically pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of beneficial bacteria that colonize the large intestine by acting as a substrate for them. Various compounds have been tested to determine their function as prebiotics. Fructooligosaccharides (FOS), galactooligosaccharides (GOS) and transgalactooligosaccharides (TOS) are the most common prebiotics. Thus, ingesting certain prebiotics can improve immune function by increasing the population of microorganisms related to human health, and animal and human studies have shown that prebiotics can reduce the population of harmful bacteria. Thus, prebiotics are ingredients that provide health benefits when fermented by the natural microflora in the subject's gut or by probiotic bacteria ingested simultaneously with the prebiotic.

[0004] It is generally known that certain types of prebiotics and probiotics are combined to produce certain synergistic effects. In particular, the effect of combining prebiotic GOS with probiotic microorganisms such as lactobacilli and bifidobacteria has been demonstrated (see, for example, YUTAKA KANAMORI, MD et al., Digestive Diseases and Sciences, Vol. 46, No. 9 (September 2001), pp. 2010-2016 ((C) 2001)). It has been demonstrated that the synbiotic effect between prebiotics and probiotics is due to the consumption of prebiotics by probiotics in the subject's digestive (GI) tract.

[0005] The manufacturing procedures for the probiotic lactic acid producing bacteria are typically standardized and involve fermenting the bacteria in a growth medium containing a carbohydrate source such as a sugar, e.g., glucose, fructose, sucrose, lactose or dextrose. After fermentation, the probiotic bacteria are usually cryoprotected and frozen or lyophilized and packaged into the final product.

[0006] Ingestion of probiotic lactobacilli has various effects in vivo. One such important effect is the bioavailability of essential minerals, which can be significantly affected by the presence of such probiotic lactobacilli. The content of minerals in the body depends mainly on their supply from food intake, and they are mainly absorbed in the digestive tract, but the simple supply of minerals through food intake does not necessarily result in sufficient mineral bioavailability. The main factors that affect mineral bioavailability are the content of minerals in food, the synergistic and antagonistic interactions between minerals in food and in the digestive tract, the presence of complexing or chelating compounds in food, as well as the health status of the organism and its age. Intestinal microflora, probiotics and prebiotics can also significantly affect the bioavailability of minerals, thereby increasing or decreasing the absorption of such minerals.

[0007] Minerals are essential for all living species, although specific requirements vary between species. Minerals have many important functions in the human organism, including iron, calcium, and magnesium, among others. Mineral deficiency, i.e., low or suboptimal amounts of such minerals in the body, typically due to ingesting less than the recommended RDA amount of such minerals, can lead to disease, and mineral overload can result. Therefore, it is crucial to obtain the right amount of minerals in the right ratio for optimal health. Although most natural foods provide these minerals in the right balance, there are situations in which they are not enough to maintain health.

[0008] Recent studies have brought new information about the interaction between microbiota and minerals, and have mainly concerned the effect of gut bacteria on calcium and iron metabolism.Given the increasing prevalence of osteoporosis among the aging population, as well as among postmenopausal women and men, new data on calcium metabolism, and therefore bone metabolism and its improvement, are important for the development of this field.In contrast, the results of studies on the adverse interactions between iron and gut microbiota show that there is a great need to further test the bioavailability of this mineral in order to make new recommendations on iron supplementation.

[0009] Also, a common problem with oral administration of probiotic bacteria is that they reach their intended location in the digestive tract in insufficient amounts, and / or that the activity of the probiotic bacteria is insufficient in those locations of the intestinal tract where they exert their effect. Thus, the survival rate, viability and engraftment of probiotic bacteria in the digestive tract are significant challenges for those who manufacture probiotic products. As a result, the dosage of probiotic bacteria must be increased and / or more frequent administration is required to obtain the desired probiotic effect. This further leads to problems with unnecessary costs, undesirable frequency of intake, and possibly reduced health benefits if the probiotic bacteria do not provide sufficient desired effects. Therefore, it is desirable to improve the probiotic effect without increasing the dosage. Summary of the Invention

[0010] It is an object of the present invention to provide more efficient means, methods and treatments to improve the viability, persistence and / or probiotic effect of certain specific probiotic microorganisms, such as probiotic L. reuteri strains, when administering such probiotic L. reuteri strains to a subject or patient in need thereof. Furthermore, it is a particular object to prevent and / or treat mineral deficiency in a subject or patient in need thereof, in particular of a mineral deficiency selected from magnesium, iron and / or calcium.

[0011] These objectives are solved by the present invention as addressed below.

[0012] The invention is defined below and in the independent claims. Further embodiments of the invention are defined herein and in the dependent claims.

[0013] In a first aspect, the present invention provides a method for preparing a preconditioned probiotic L. reuteri strain, the method comprising the steps of: a) culturing a probiotic L. reuteri strain in the presence of GOS, thereby preconditioning the probiotic L. reuteri strain; and b) harvesting the preconditioned probiotic L. reuteri strain from the growth medium.

[0014] In step a) of the method of the invention, the probiotic L. reuteri strain is cultured in the presence of GOS, resulting in the formation of a preconditioned L. reuteri strain.

[0015] According to a second aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain, the preconditioned probiotic L. reuteri strain being prepared by culturing the L. reuteri strain in the presence of GOS.

[0016] In a third aspect, the present invention provides a non-therapeutic use of an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention, or of a composition according to the present invention, for increasing or enhancing the survival, persistence and / or probiotic effect of a probiotic L. reuteri strain in the gastrointestinal tract of a healthy subject.

[0017] According to a fourth aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention for use as a medicament.

[0018] According to a fifth aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention for (therapeutic) use in the prevention and / or treatment of a disease in a subject in need thereof, the subject suffering in this aspect from a disruption of the intestinal barrier or a disruption of the mucosal lining.

[0019] According to a sixth aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention for use in the prevention and / or treatment of mineral deficiency in a subject in need thereof.

[0020] According to a seventh aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention for use in the prevention and / or treatment of bone loss.

[0021] According to an eighth aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention for use in promoting bone or tooth growth and development in children. Further preferred embodiments are described hereinafter and in the dependent claims. [Brief description of the drawings]

[0022] [Figure 1] Diagram of the short colonic SHIME® model, a dynamic in vitro simulator model of the human digestive system and an experimental approach simulating small intestinal and colonic incubation. The SHIME® model allows the cultivation of complex gut microbial ecosystems under conditions representative of different gut regions. [Diagram 2] Figure 2 shows the solubility of magnesium and calcium in the colon at 24 hours and 48 hours using the preconditioned probiotic L. reuteri strain of the present invention in the short colonic SHIME® model. The results show that the preconditioned L. reuteri strain enhances the solubility of magnesium and calcium in the colon. This suggests that the preconditioned L. reuteri strain can be used to promote the absorption of magnesium and calcium in subjects who need it. [Diagram 3]Figure 1 shows the colonic iron bioaccessibility at 48 hours using the preconditioned probiotic L. reuteri strain of the present invention in the short colonic SHIME® model.The results show that the preconditioned L. reuteri strain enhances the iron bioaccessibility in the colon.This suggests that the preconditioned L. reuteri strain can be used to promote iron absorption in subjects who need it. [Figure 4] Figure 2 shows the bacterial acid stress resistance. The results show that the preconditioned L. reuteri strains are more resistant to acid stress (normalized values). The results suggest that the preconditioned L. reuteri strains have a higher survival rate and persistence in the gastrointestinal tract. [Diagram 5] Figure 2 shows bile stress resistance for preconditioned L. reuteri strains. The graph shows that preconditioned L. reuteri strains have increased resistance to bile (normalized values). The results suggest that preconditioned L. reuteri has higher survival and persistence in the gastrointestinal tract. [Figure 6] We show that preconditioned L. reuteri strains result in improved protection of epithelial integrity from the deleterious effects of enterotoxigenic Escherichia coli (ETEC). The results suggest that preconditioned L. reuteri has a protective effect on the epithelial barrier in the gastrointestinal tract. Furthermore, the results also suggest that preconditioned L. reuteri improves mineral absorption, given the reduction in cellular damage essential for this process. [Figure 7]Figure 1 shows early differentiation of osteoblasts on day 7, measuring alkaline phosphatase (ALP) activity. The results show that preconditioning of L. reuteri strains led to increased ALP activity in osteoblasts, suggesting that preconditioned L. reuteri strains can stimulate osteoblast differentiation. [Figure 8] Figure 2 shows osteocalcin (oc) expression (at the mRNA level), a marker of mineralization expressed by differentiated osteoblasts at day 21. Results show that oc expression is enhanced by preconditioned L. reuteri strains, but not by (non-preconditioned) L. reuteri strains, suggesting that preconditioned L. reuteri strains can stimulate bone mineralization. [Figure 9] MC3T3 migration velocity, a measure of cell mobility, is shown at day 4. The results show that cell migration is significantly reduced by preconditioned L. reuteri but not by non-preconditioned L. reuteri strains, suggesting that preconditioned L. reuteri strains can stimulate osteoblast differentiation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention provides a method for enhancing the viability, persistence and / or probiotic effect of Lactobacillus reuteri (L. reuteri) strains in mammals or birds using specific substrate components during fermentation in producing the probiotic L. reuteri strains. The inventors of the present invention have developed a method comprising the use of galactooligosaccharides (GOS) during the cultivation of the probiotic L. reuteri strains, so-called preconditioning with GOS, as described herein, which surprisingly results in an increase in the probiotic effect of the L. reuteri strains, in particular an increase in the bioavailability of minerals, for example by increasing mineral solubility, accessibility and / or absorption of minerals, by improving the protective effect of the epithelial barrier integrity in the gastrointestinal tract, and / or by improving bone formation and / or bone mineralization in a subject or patient upon administration of the preconditioned probiotic L. reuteri strains. These effects are surprisingly obtained without the co-administration of GOS as a prebiotic and a preconditioned probiotic L. reuteri strain to a subject or patient in need thereof.The object of the present invention therefore relates to a method for preparing such a preconditioned probiotic L. reuteri strain with GOS, in particular to the non-therapeutic and therapeutic use of the preconditioned probiotic L. reuteri strain to increase mineral solubility, accessibility and / or absorption in the mammalian or avian digestive tract.

[0024] Surprisingly, it has been found that the preconditioned probiotic L. reuteri strain also has improved viability and / or persistence, in particular improved mucus adhesion ability, improved stress resistance against bile and gastric acid. These improved properties may also further improve the probiotic effect of the L. reuteri strain without the need for the presence of prebiotic compounds. Such improvement is particularly due to the use of GOS as a specific fermentation component for the L. reuteri strain, herein referred to as the preconditioning step. The improvement in viability, persistence and / or probiotic effect, e.g. induction of mineral (iron, magnesium and calcium) solubility in L. reuteri obtained by this additional step, as well as improved viability, improved mucus adhesion ability, improved stress resistance against bile and gastric acid, improved protective effect on epithelial barrier integrity, improved bone formation and / or bone mineralization, make it possible to reduce the dosage and / or frequency of administration or improve the effect achieved by the same dose of probiotic required to obtain the desired health effect.

[0025] Therefore, the present invention also aims to provide a solution, composition and use that increases mineral solubility, bioaccessibility and therefore mineral absorption in the digestive tract.In particular, such a solution is a composition that comprises a L. reuteri strain preconditioned with galactooligosaccharides (GOS) and is administered to a subject in need thereof, for example a subject in need of increased mineral uptake.The increase in mineral solubility, accessibility and absorption occurs in the digestive tract, for example the duodenum, jejunum, ileum and / or colon.

[0026] The present invention also aims to provide a method for preconditioning probiotic L. reuteri strains, such that by adding such preconditioned L. reuteri strains to a composition, increased mineral solubility and bioavailability in the gastrointestinal tract, as well as increased mineral absorption, can be achieved.

[0027] In view of the above, the following is disclosed:

[0028] Methods for preconditioning probiotic Lactobacillus reuteri bacterial strains According to a first aspect, the present invention provides a method for preparing a preconditioned probiotic Lactobacillus reuteri strain, the method comprising the steps of: a) cultivating or culturing a probiotic L. reuteri strain in the presence of GOS, thereby preconditioning the L. reuteri strain; and b) harvesting the preconditioned probiotic L. reuteri strain from the growth medium.

[0029] In step a) of the method of the present invention, the probiotic L. reuteri strain is cultured in the presence of GOS, thereby forming a preconditioned L. reuteri strain. In the context of the present invention, the term "preconditioning" of the probiotic L. reuteri strain preferably means growing, for example cultivating, culturing or fermenting the probiotic L. reuteri strain with GOS during the process of producing the probiotic L. reuteri strain. The culture of the probiotic L. reuteri strain in the presence of GOS results in the formation of a preconditioned probiotic L. reuteri strain.

[0030] Thus, the method of the invention preferably comprises, as a first step, a) culturing bacteria of a probiotic L. reuteri strain in the presence of GOS, thereby preconditioning the bacteria of the L. reuteri strain. In the context of the present invention, the terms "cultivation", "culturing" and "fermentation" are used interchangeably.

[0031] Then, in step b), the preconditioned bacteria of the probiotic L. reuteri strain are harvested from the growth medium. The term "preconditioned probiotic L. reuteri strain" refers to the probiotic L. reuteri strain produced, obtained or obtainable by the method of the present invention, which comprises a preconditioning step with GOS. Correspondingly, the term "preconditioned bacteria of the probiotic L. reuteri strain" refers to the probiotic L. reuteri strain bacteria produced, obtained or obtainable by the method of the present invention, which comprises a preconditioning step with GOS.

[0032] The Lactobacillus reuteri strains used throughout the present invention can generally be any L. reuteri strain, and more preferably any commercially available L. reuteri strain. In this context, the terms "Lactobacillus reuteri strain" and "Lactobacillus reuteri bacteria" can be used synonymously. It should be noted that in the past decades, DNA analysis tools have become more sophisticated, allowing scientists to discover many new bacterial species, but also to recognize that species historically grouped under the genus Lactobacillus were too diverse to fit the nomenclature convention. Therefore, in order to keep the probiotic group accurate and organized, the genus Lactobacillus has been divided into 25 different genera, including 23 novel genera. As a result, many probiotics have been given new genus names in recent years. Therefore, the alternative genus name for Lactobacillus reuteri is Limosilactobacillus reuteri. Because the reclassification process is quite recent, the previous genus nomenclature, i.e., Lactobacillus, is used in this application for clarity. However, the revised genus nomenclature and alternative names should be readily accessible to anyone using available reclassification tools.

[0033] The presently preferred bacteria provided in the composition are preferably at least one L. reuteri strain, or a plurality, i.e. at least two L. reuteri strains, as referred to herein.

[0034] In a preferred embodiment of the invention, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 32231, L. reuteri DSM 32232, L. reuteri ATCC PTA 6127 and L. reuteri ATCC PTA 4659.

[0035] Alternatively or additionally, the L. reuteri strain used herein may be provided by at least one L. reuteri strain selected from L. reuteri DSM 17938, L. reuteri ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 or L. reuteri ATCC PTA 4659.

[0036] A currently preferred L. reuteri strain is L. reuteri DSM 17938.

[0037] In a particular embodiment, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475, preferably selected from the group consisting of L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475. Thus, in a particular embodiment, the probiotic L. reuteri strain is L. reuteri DSM 27131. In another particular embodiment, the probiotic L. reuteri strain is L. reuteri ATCC PTA 6475.

[0038] Alternatively or additionally, the L. reuteri strain used herein may be provided by at least one L. reuteri strain selected from L. reuteri ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 or L. reuteri ATCC PTA 4659.

[0039] Lactobacillus reuteri DSM 17938 was deposited in accordance with the Budapest Treaty on January 30, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany.

[0040] Lactobacillus reuteri DSM 27131 was deposited in accordance with the Budapest Treaty at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 18, 2013.

[0041] Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849 were deposited in accordance with the Budapest Treaty on July 4, 2018 at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0042] Lactobacillus reuteri DSM 32465 was deposited in accordance with the Budapest Treaty at the Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on March 21, 2017.

[0043] Lactobacillus reuteri DSM 33632, DSM 33633, DSM 33634 and DSM 33635 were deposited in accordance with the Budapest Treaty on September 9, 2020 at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0044] Lactobacillus reuteri DSM 32231 and DSM 32232 were deposited in accordance with the Budapest Treaty on December 11, 2015 at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0045] Lactobacillus reuteri ATCC PTA 5289 was deposited in accordance with the Budapest Treaty on June 25, 2003 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0046] Lactobacillus reuteri ATCC PTA 6475 was deposited in accordance with the Budapest Treaty on December 21, 2004 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0047] Lactobacillus reuteri ATCC PTA 4659 was deposited in accordance with the Budapest Treaty on September 11, 2002 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0048] Lactobacillus reuteri ATCC PTA 6127 was deposited in accordance with the Budapest Treaty on July 22, 2004 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0049] The term "GOS" as used herein means "galactooligosaccharide". Galactooligosaccharide (GOS), as used herein, typically consists of β-linked galactose moieties with galactose or glucose at the reducing end. Such GOS may contain β-(1→2), β-(1→3), β-(1→4) or β-(1→6) linked galactose moieties and have a degree of polymerization (DP) of 3 to 8 galactose units. Thus, the term GOS refers to oligosaccharides that preferably contain at least 3 galactose units, more preferably at least 4 galactose units, preferably having a degree of polymerization (DP) of 3 to 8 galactose units. Thus, the GOS added to the growth medium in step a) preferably has a degree of polymerization (DP) of 3 to 8.

[0050] Oligosaccharides occur naturally in some mammalian, e.g., bovine and human milk. GOS are commercially available and can be synthesized via a biosynthetic process from lactose by the trans-galactosidase activity of β-galactosidase. In the biosynthetic procedure, GOS formation is usually promoted by high concentrations of lactose or lactulose, incomplete lactose turnover, low water activity, and the use of enzymes that favor transgalactosylation. The linkage type of GOS resulting from such a process is specific to the enzymes used in the biosynthesis.

[0051] One particular type of carbohydrate mixture that can be advantageously used herein as a GOS source for growing probiotic bacteria is a mixture of GOS and milk oligosaccharides. In particular, a mixture of GOS and 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL) is preferably used. Indeed, such a mixture contains some oligosaccharides similar to human milk, which is particularly advantageous when the composition of the present invention is used as an infant formula or as an infant nutritional supplement. Such advantageous effects are described, for example, in Simeoni et al.; "Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp.lactis CNCM I-3446"; Environ Microbiol, 2016, 18(7): 2185-2195. Such compositions can typically be obtained by concentrating whey permeate to obtain a concentrated bovine milk oligosaccharide composition and adding GOS or generating GOS in situ from the hydrolysis of lactose by the action of β-galactosidase.

[0052] The GOS source used in step a) of the method of the invention for preconditioning the probiotic L. reuteri strain may be provided in the form of essentially pure GOS (i.e. a component having at least 90% GOS) or as part of a mixture comprising GOS, e.g. a mixture of carbohydrates.

[0053] It is preferred that a sufficient amount of GOS is provided in the growth medium.According to one embodiment, therefore, the GOS source is typically added in step a) to the growth medium in an amount of at least 0.2% by weight, preferably at least 0.5% by weight, and even more preferably at least 0.75% by weight of GOS in the growth medium.In a preferred embodiment, the GOS is provided in an amount of up to 8% by weight of GOS in the growth medium, for example up to 7% by weight of GOS, up to 6% by weight of GOS, or even up to 5% by weight of GOS, more preferably up to 4% by weight of GOS, or even up to 3% by weight of GOS, up to 2% by weight of GOS, or up to 1% by weight of GOS.The weight percentage of GOS is preferably calculated based on the growth medium (% w / w).Any combination of these upper and lower ranges is encompassed herein. More preferably, the GOS is added to the growth medium in the range of 0.2% to 8% by weight, or even 0.2% to 7% by weight, or 0.2% to 6% by weight, even more preferably 0.5% to 8% by weight, or even 0.5% to 7% by weight, or 0.5% to 6% by weight, even more preferably 0.75% to 8% by weight, or even 0.75% to 7% by weight, or 0.75% to 6% by weight, most preferably 0.75% to 7% by weight, 0.75% to 6% by weight, or even 0.75% to 5% by weight, for example 0.75% to 3% by weight, or 0.75% to 4% by weight. Preferably, the GOS is added to the growth medium in a range of 0.2% to 8% by weight, preferably in the range of 0.5% to 7% by weight, more preferably in the range of 0.75% to 6% by weight, most preferably in the range of 0.75% to 5% by weight, for example in the range of 0.75% to 4% by weight, or in the range of 0.75% to 3% by weight, preferably calculated on the growth medium (% w / w). The addition of the GOS can preferably be carried out at the beginning, during or at the end of the cultivation of the probiotic L. reuteri strain in step a) and / or all at once, stepwise or continuously.

[0054] When a mixture of carbohydrates is used as a source of GOS, the amount of GOS in such a mixture is preferably at least 20% by weight, preferably at least 30% by weight, more preferably at least 40% by weight, even more preferably at least 45% by weight and most preferably at least 48% by weight, preferably calculated on the dry weight of the mixture containing the GOS.

[0055] In one embodiment, the GOS is used as a substrate, e.g., a fermentation substrate, for the probiotic L. reuteri strain in step a) Thus, in such an embodiment, the GOS is used as a carbon source during the cultivation or fermentation of the probiotic L. reuteri strain.

[0056] Although it is not necessary for the bacteria to consume only GOS as a carbon source during fermentation, a high proportion of GOS in the GOS source promotes the consumption of GOS by the bacteria during fermentation over the consumption of other carbohydrates, resulting in an improved preconditioning effect. Smaller carbohydrates such as disaccharides (e.g. lactose) may also be present and consumed by the bacteria to aid in the preconditioning effect. It is therefore also preferred that the GOS source comprises up to 55% by weight, preferably up to 50% by weight, more preferably up to 45% by weight, most preferably up to 42% by weight of monosaccharides or disaccharides, and / or up to 40% by weight, preferably up to 30% by weight of lactose, preferably calculated based on the dry weight of the mixture containing GOS. By way of example, the mixture of carbohydrates used as the GOS source may comprise the amount of GOS defined above for the mixture, as well as lactose, glucose and / or galactose, and optionally the remainder formed by 3'-SL and / or 6'-SL.

[0057] In one embodiment, the culture medium also includes an electron acceptor, such as fructose, citrate, glycerol, and / or 1,2-propanediol.

[0058] In certain embodiments, the culture medium also contains fructose, for example, acting as an electron acceptor. In an illustrative but non-limiting example, the culture medium may contain at least 0.2% by weight of fructose, preferably at least 0.5% by weight of fructose, more preferably at least 1% by weight of fructose, and even more preferably at least 1.5% by weight of fructose, calculated based on the dry weight of the culture medium. Correspondingly, in an illustrative but non-limiting example, the culture medium preferably does not contain more than 16% by weight of fructose, preferably not more than 14% by weight of fructose, more preferably not more than 12% by weight of fructose, and even more preferably not more than 10% by weight of fructose. Any combination of these upper and lower ranges is encompassed herein. In one embodiment, the culture medium contains a weight percent ratio of electron acceptor (e.g., fructose) to GOS selected within the range of 0.25:1 to 5:1, preferably within the range of 0.5:1 to 4:1, and even more preferably within the range of 0.75 to 2.5:1. In particular, a preferred weight percent ratio of electron acceptor (eg, fructose) to GOS is within the range of about 1:1 to about 2:1.

[0059] According to a further embodiment, in step a) of the method for preparing a preconditioned probiotic L. reuteri strain, the addition of GOS to the growth medium can be carried out at any time during the culturing step, for example at the beginning of the culturing of the probiotic L. reuteri strain, during the culturing step a) of the probiotic L. reuteri strain, more preferably at the beginning of the culturing of the probiotic L. reuteri strain, all at once, stepwise or continuously. Alternatively, or even additionally, in step a) of the method for preparing a preconditioned probiotic L. reuteri strain, the addition of GOS to the growth medium can be during or at the end of the culturing step a).

[0060] The culturing step a) is carried out in a manner well known to those skilled in the art. The culturing in step a) comprises inoculating a sterile standard growth medium with a defined amount of bacteria (cfu (colony forming units)), followed by incubation at a defined temperature (usually 37° C.) and pH. In this context, the defined amount of bacteria (cfu) for starting the inoculation of the sterile standard growth medium can be determined by the skilled artisan according to the general knowledge and practice in the art. A suitable yield can be obtained using the growth medium containing the GOS described above, preferably without changing the culture conditions compared to those used by the skilled artisan for culturing the same strain using a standard growth medium.

[0061] The standard growth medium for culturing / fermenting the L. reuteri strain in step a) of the present invention can be any known standard growth medium used for lactobacilli (LAB), such as MRS medium, or any further suitable medium. Such standard growth medium is preferably commercially available, but may also be produced by adding compounds according to well-known standard recipes. Standard growth media for L. reuteri strains contain carbohydrates, such as monosaccharides selected from dextrose, sucrose, maltose, fructose or lactose, various nitrogen sources, such as peptone, yeast extract, beef extract or whey protein, minerals, mainly Mn 2+ and Mg 2+, and buffers, such as sodium acetate (CH3COONa), trisodium citrate (Na3C6H5O7) or disodium glycerophosphate (C3H7Na2O6P), which are buffers commonly used in LAB media. Other components used in standard growth media with buffering activity may include disodium phosphate (Na2HPO4), ammonium citrate (NH4C6H5O7), trisodium phosphate (Na3PO4), potassium biphosphate (KH2PO4), trimagnesium phosphate Mg3(PO4)2, calcium carbonate (CaCO3) and dipotassium phosphate (K2HPO4). Such standard growth media may also contain a wide range of growth factors, surfactants, such as lecithin or Tween® (e.g., Tween® 20, 80 and 85). Cultivation / fermentation may be performed under anaerobic or aerobic conditions depending on the strain to be produced, but preferably under anaerobic conditions. Also, the pH may or may not be controlled depending on the conditions that a particular strain is known to grow best in. The temperature and duration of the cultivation process are variable from strain to strain and are well known to those skilled in the art of culturing probiotic L. reuteri strains.

[0062] According to one embodiment, the cultivation / fermentation of the probiotic L. reuteri strain in step a) of the method of the present invention is carried out for a significant period of time to allow preconditioning of the probiotic L. reuteri strain in the presence of GOS, typically for a period of at least 1 hour, preferably for a period of 5 to 18 hours, also preferably for a period of 7 to 14 hours, more preferably for a period of 10 to 13 hours and even more preferably for a period of about 12 hours.

[0063] Furthermore, according to a further embodiment, the cultivation / fermentation of the probiotic L. reuteri strain in step a) of the method of the present invention is carried out at a temperature between 25 and 45°C, preferably between 30 and 40°C, even more preferably between 35 and 39°C, for example at a temperature of about 37°C.

[0064] Furthermore, in another embodiment, the cultivation / fermentation of the probiotic L. reuteri strain in step a) of the method of the present invention is carried out at a pH of 3.0 to 7. The pH during the preconditioning of the probiotic L. reuteri strain in the cultivation step a) may be uncontrolled or set to a constant value throughout the cultivation step a), and is typically monitored and adjusted during the cultivation step a). Thus, the pH may be 3.0 to 7, especially if the pH is not controlled during the preconditioning of the probiotic L. reuteri strain. Alternatively, when the pH is controlled during the culture step a), the pH may be adjusted to a range of about 5.0 to 7.0, for example, about 5.0 to 6.0, about 5.5 to 6.5 (e.g., about 6.0±0.2) or about 6.0 to 7.0 (e.g., about 6.6±0.2), more preferably the pH may be adjusted to a range of about 5.5 to 6.5, even more preferably the pH may be adjusted to a range of about 6.0±0.4, and most preferably the pH may be adjusted to a range of about 6.0±0.2.

[0065] After the cultivation / fermentation of the probiotic L. reuteri strain in step a) of the method of the present invention, the probiotic L. reuteri strain thus preconditioned is harvested in step b). The harvesting step, which aims to separate bacterial cells from the growth medium, is also carried out in a manner well known to those skilled in the art, for example by concentrating the bacteria, centrifugation, filtration, membrane filtration, decantation, isolation, purification, etc., preferably by centrifuging or concentrating the probiotic L. reuteri strain. Typically, the harvesting of the preconditioned probiotic L. reuteri strain is carried out in step b) after a period of at least 1 hour as defined above for the cultivation / fermentation in step a). The harvesting in step b) preferably results in an isolated or at least partially purified preconditioned probiotic L. reuteri strain.

[0066] After harvesting the preconditioned probiotic L. reuteri strains in step b), the harvested cells can be washed in an optional step c) before further processing, typically to remove traces of growth medium (after cultivation / fermentation). Washing can be performed either with saline, brine or any further suitable liquid.

[0067] Immediately after harvesting the preconditioned probiotic L. reuteri strain from the growth medium according to step b) or after the optional washing step c), the preconditioned probiotic L. reuteri strain may be subjected to a drying step d). Such a drying step d) may be carried out by any method known to the person skilled in the art, for example spray drying, fluidized bed drying, air convection drying, atmospheric drying, roller drying or freeze drying, lyophilizing, more preferably spray drying or lyophilizing. Spray drying may be carried out in the presence of a protective agent, for example as described in WO 2017 / 001590.

[0068] Optionally, the preconditioned probiotic L. reuteri strain obtained after harvesting from the growth medium and / or washing the preconditioned probiotic L. reuteri strain may be further mixed with a protection agent, such as a cryoprotectant, a lyoprotectant and / or a carrier, prior to the drying step, depending on the drying method used and the bacteria to be dried, as known to those skilled in the art. Thus, preferably prior to the drying step d), the harvested preconditioned probiotic L. reuteri strain is treated with a protection agent, a cryoprotectant, a lyoprotectant and / or a carrier. Such protection agents and / or carriers typically include glycerol, skimmed milk, serum albumin, peptone, yeast extract, saccharose, glucose, sorbitol, malt extract, trehalose, etc. Commercially available cryoprotectants include, for example, cryoprotectant Unipectine™ RS 150, or as described in EP 3016511, U.S. 2014 / 0004083, U.S. 2016 / 0298077, and the like.

[0069] Alternatively or additionally, the preconditioned probiotic L. reuteri strain obtained after harvesting from the growth medium or after washing or even drying, i.e. after any of steps b), c) or d), can be encapsulated for further storage and / or use, for example by forming microspheres containing the preconditioned probiotic L. reuteri strain using common encapsulating agents, for example alginate, alginate / pullulan, starch, xanthan, xanthan gellan gum mixture, gelatin, cellulose acetate phthalate, chitosan, or any further suitable encapsulating material. Encapsulation is well known and can be performed by those skilled in the art.

[0070] COMPOSITIONS COMPRISING PRECONDITIONED PROBIOTIC L. REUTERI STRAINS According to a second aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain. The preconditioned probiotic L. reuteri strain is prepared or obtained by culturing the probiotic L. reuteri strain in the presence of GOS. Such preconditioning of the probiotic L. reuteri strain in the presence of GOS is typically carried out according to the inventive method described herein under the first aspect of the present invention for preparing a preconditioned L. reuteri strain. As already disclosed in detail above, the culture / fermentation of the probiotic L. reuteri strain in the presence of GOS is preferably carried out for a sufficient time to precondition the probiotic L. reuteri strain accordingly, for example for a period of at least 1 hour, preferably according to the process defined herein.

[0071] A related aspect of the present invention defines a preconditioned probiotic L. reuteri strain obtained or obtainable by culturing a probiotic L. reuteri strain in the presence of GOS.

[0072] The composition according to this second aspect is a probiotic composition comprising an effective amount of a preconditioned probiotic L. reuteri strain.

[0073] As mentioned above, the L. reuteri strain is particularly preferably L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L.reuteri DSM 33635, L.reuteri DSM 27131, L.reuteri DSM 32465, L.reuteri DSM 32231, L.reuteri DSM 32232, L.reuteri ATCC PTA 6127 and L.reuteri ATCC PTA 4659, and preferably selected from the group comprising or consisting of L.reuteri strain DSM 17938, L.reuteri strain ATCC PTA 5289, L.reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 and L. reuteri ATCC PTA 4659, and most preferably the probiotic L. reuteri strain is the L. reuteri strain deposited under DSM 17938.

[0074] In a particular embodiment, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475, preferably selected from the group consisting of L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475. Thus, in a particular embodiment, the probiotic L. reuteri strain is L. reuteri DSM 27131. In another particular embodiment, the probiotic L. reuteri strain is L. reuteri ATCC PTA 6475.

[0075] In one embodiment, the L. reuteri strain is at least one L. reuteri strain or a plurality of L. reuteri strains as defined above, particularly preferably selected from the group consisting of L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475 and L. reuteri ATCC PTA 4659.

[0076] In one embodiment, the L. reuteri strain is at least one L. reuteri strain or strains as defined above, particularly preferably selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131 and L. reuteri DSM 32465.

[0077] Typically, an "effective amount" of a preconditioned probiotic L. reuteri strain, as defined herein, is typically about 10 3 cfu~10 12 The amount of cfu, typically 10 4 cfu~10 11 The amount of cfu, preferably 10 5 cfu~10 10 cfu, or 10 5 cfu~10 9 cfu amount, also preferably 10 6 cfu~10 9 cfu, 10 6 cfu~10 8 cfu of preconditioned probiotic L. reuteri strain. Alternatively, the amount of preconditioned L. reuteri strain may be 10 8 cfu~10 10 cfu amount, or 10 7 cfu~10 9 The preferred amount is about 10 8 Total cfu, e.g., 10 7 ~10 9 cfu, or 10 8 ~10 9 It is cfu.

[0078] The amount of preconditioned probiotic L. reuteri strain is preferably the amount of preconditioned probiotic L. reuteri strain per daily dose. For example, a dosage form or dosage unit of the composition preferably contains the amount of preconditioned probiotic L. reuteri strain.

[0079] The probiotic composition may comprise the preconditioned probiotic L. reuteri strain in a dry form as described hereinabove.In such a case, the preconditioned probiotic L. reuteri strain is preferably in a spray-dried or freeze-dried form.Thus, in one embodiment, the composition comprises an effective amount of freeze-dried or spray-dried preconditioned probiotic L. reuteri strain.

[0080] The composition of the present invention comprising an effective amount of a preconditioned probiotic L. reuteri strain as defined herein may be any type of composition in which probiotic bacteria can be incorporated, such as a food, beverage, animal feed product, human or animal nutritional supplement, pharmaceutical composition or cosmetic composition.More preferably, the composition of the present invention may be provided in an administrable form, preferably selected from the group consisting of nutritional compositions, foods such as functional food products, beverages such as functional beverage products, pharmaceutical preparations, dietary supplements, nutritional supplements, nutraceuticals and combinations thereof.In one embodiment, the administrable form is selected from the group consisting of dairy products, dairy-based products and whey protein-based beverages.

[0081] The composition of the present invention can be solid or liquid.Preferably, the composition of the present invention is in the form of a powder, a sachet, a tablet, a capsule, or may be in the form of an oil, an emulsion, such as an oil-in-water emulsion (o / w emulsion), a water-in-oil emulsion (w / o emulsion), etc.When in powder form, the composition of the present invention may be intended to be used by the end consumer in a solid (such as a powder form) or semi-solid form (such as a paste form), or alternatively, to be reconstituted into a liquid before use.

[0082] Foods and beverages as defined herein may include any product intended for oral ingestion by humans to provide nutrition and / or enjoyment. The expressions "food" and the term "beverage" generally refer to compositions that provide nutrition to a subject. This "food" is generally to be ingested orally or intraperitoneally and may include a lipid or fat source and a protein source. Similarly, a "beverage" is generally to be ingested orally, may be liquid or semi-liquid, and may include a lipid or fat source and a protein source.

[0083] Foods and beverages as defined herein may include, for example, nutritional compositions, preferably for human consumption, for example, for infants and / or young children, for pregnant or lactating women or women who wish to become pregnant, for individuals who require special nutrition due to poor health, or for the elderly. More preferably, the nutritional compositions are selected from infant formulas, infant cereals, follow-up or follow-on formulas, growing-up milk, functional milks, infant foods, infant cereal compositions, and dairy products for pregnant and lactating women or women who wish to become pregnant. Other examples of foods and beverages include sweet and savory snacks, powdered drinks, cereal products and dairy products, such as dairy products, whey protein-based products, etc. According to a particular embodiment, the composition of the invention is an infant formula, a follow-on formula, a growing-up milk, or a product for pregnant or lactating women. In a further particular embodiment, the composition of the invention may be an infant formula.

[0084] The composition of the present invention can also be in the form of animal food or animal (such as mammalian or avian) nutritional supplement.Preferably, the animal is a mammal.Examples of animals include cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds (such as poultry), etc.

[0085] The expression "infant formula" as used herein refers to a foodstuff intended for a specific nutritional use by infants during the first few months of life and which alone meets the nutritional requirements of this category of person (reference: Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae; Reg 609 / 2013 art 2.1.c and Reg 2016 / 127). It also refers to nutritional compositions intended for infants and defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialities (including Food for Special Medical Purpose). The expression "infant formula" encompasses both "starter infant formula" and "follow-up formula" or "follow-on formula", including compositions provided to premature infants.

[0086] A "follow-up formula" or "follow-on formula" is an infant nutritional composition given from the sixth month onwards. It constitutes the main liquid component in the progressively diversified diet of this category of individuals.

[0087] The expression "infant food" means a foodstuff intended for a specific nutritional use by an infant or young child during the first few years of life.

[0088] In the context of the present invention, the term "infant" refers to a child under the age of 12 months. Also, the expression "young child" refers to a child aged 1 to 3 years, also called a toddler. The expression "infant cereal composition" refers to a foodstuff intended for specific nutritional use by an infant or young child during the first few years of life.

[0089] Dietary supplements or nutritional supplements are typically present in the form of liquids, such as refrigerated liquids, oils, emulsions, such as oil-in-water emulsions (o / w emulsions), water-in-oil emulsions (w / o emulsions), etc., in the form of powders or sachets or tablets or capsules, as described above.Preferably, dietary supplements or nutritional supplements are in the form of powders, sachets, tablets, capsules or oils.Powder supplements typically include supplements that are dissolved in liquids or sprinkled on food or beverages.Such supplements are intended to provide additional nutrients and / or health benefits to the subjects who consume them, as well as other beneficial ingredients, such as the preconditioned probiotic L. reuteri strains as defined and prepared herein.The supplements according to the present invention can therefore be used to provide nutrients and / or health benefits to humans and animals, as defined above. Dietary supplements or nutritional supplements include, for example, the preconditioned probiotic L. reuteri strains defined and prepared herein as a powder supplement to be added to any type of dietary or nutritional composition.

[0090] Pharmaceutical products include powder, sachet, tablet or capsule products intended to treat or prevent an adverse medical condition or promote good health in a subject in need thereof.

[0091] Cosmetic compositions are typically intended for an aesthetic effect on the body and may be for topical use or may be administered by the oral route in the form of powders, tablets or capsules.

[0092] Optionally, the composition of the present invention may also contain GOS in addition to the preconditioned probiotic L. reuteri strains as defined and prepared herein, to further provide improved properties to the final product. Such improved properties may be a synergistic effect of the combined administration of the preconditioned probiotic L. reuteri strains and GOS. When GOS is included, such GOS is preferably as defined above. Furthermore, such GOS may be included in the composition of the present invention in an amount of at least 1 or 2 g, preferably at least 3 g, also preferably 2 to 12 g, or 2 to 10 g, 2 to 8 g, or 2 to 7 g, more preferably 3 to 12 g, also more preferably 3 to 10 g, for example 3 to 8 g, 3 to 7 g, 3 to 6 g, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 g, or any range formed thereby.

[0093] The amount of GOS added to the preconditioned probiotic L.reuteri strain is preferably the amount of GOS per daily dose.Therefore, in such an embodiment, the composition is provided in a dosage form or dosage unit.In such a case, such a dosage form or dosage unit preferably contains the amount of GOS.For example, a dosage unit of the composition contains at least 1g of GOS.This means that GOS is added to the preconditioned probiotic L.reuteri strain to prepare a dosage unit of the composition, and this dosage unit contains at least 1g of GOS.

[0094] According to a particular embodiment, GOS is not added to or is not contained in the composition of the present invention. In this particular embodiment, GOS is preferably only used to precondition the L.reuteri strain in step a) of the method of the present invention, and is not further added when preparing the composition of the present invention. Thus, in a particular embodiment, the composition of the present invention is devoid of any GOS, except for trace amounts of GOS that remain after the preconditioned probiotic L.reuteri strain is harvested from the culture medium containing GOS, optionally. Trace amounts as used herein means less than 5 mg GOS per gram of composition, preferably less than 1 mg GOS per gram of composition, more preferably less than 0.5 mg GOS per gram of composition, even more preferably less than 0.1 mg GOS per gram, and most preferably less than 0.01 mg GOS per gram of composition.

[0095] Uses of the compositions of the present invention containing preconditioned probiotic L. reuteri strains The present invention aims to provide compositions and uses which improve or increase the probiotic effect and / or increase mineral solubility and bioavailability in the gastrointestinal tract, and also mineral absorption.

[0096] As shown herein, the composition of the present invention allows the probiotic effect of L. reuteri strains in mammals or birds to be enhanced by using GOS as a specific substrate component during fermentation in the production of probiotic L. reuteri strains. The method disclosed herein specifically preconditions the probiotic L. reuteri strains by using GOS during the cultivation of the probiotic L. reuteri strains, so that an increase in the probiotic effect of the L. reuteri strains occurs in vivo, particularly during non-therapeutic and therapeutic administration as disclosed herein. This can increase the bioavailability of minerals, for example by increasing mineral solubility, accessibility and / or absorption of minerals in the digestive tract of a subject or patient, particularly upon administration of the preconditioned probiotic L. reuteri strains or compositions as described herein. This surprising effect can be obtained without the co-administration of GOS as a prebiotic and a preconditioned probiotic L. reuteri strain to a subject or patient in need thereof.

[0097] Surprisingly, the preconditioned L. reuteri strains were also found to have increased viability and persistence, in particular improved mucus adhesion ability, and improved stress resistance against bile and gastric acid. These improved properties may also further improve the probiotic effect of Lactobacillus reuteri strains without the need for the presence of prebiotic compounds. The improved viability, persistence and / or probiotic effect, exemplified by the induction of mineral (iron and calcium) solubility of the Lactobacillus reuteri strain obtained by the preconditioning step, given to a subject and / or patient in need thereof by administering the composition of the present invention comprising a preconditioned probiotic L. reuteri strain, as well as the improved mucus adhesive ability, improved stress resistance against bile and gastric acid, improved protective effect of the epithelial barrier integrity, improved bone formation and / or bone mineralization, make it possible to reduce the required probiotic dosage and / or administration frequency or to achieve an even better effect with the same dose of probiotic to obtain the desired health or therapeutic effect.

[0098] Furthermore, the composition of the present invention is a composition comprising a probiotic L.reuteri strain preconditioned by GOS, which is administered to a subject or patient in need thereof, for example, a subject or patient in need of increased mineral intake. The increase in mineral solubility, accessibility and absorption occurs in the digestive tract, for example, the duodenum (first part of the small intestine), the jejunum (middle part of the small intestine), the ileum (last part of the small intestine) and / or the colon (large intestine). The present invention also aims to provide a method for preconditioning a probiotic L.reuteri strain, so that the increase in mineral solubility and bioavailability in the digestive tract and the increase in mineral absorption can also be achieved by such a probiotic L.reuteri strain preconditioned to the composition.

[0099] Minerals are essential for all living species, although specific requirements vary between species. Mineral deficiency can lead to disease, as can mineral excess. Therefore, the correct amounts of minerals in the correct ratios are necessary for optimal health. While most natural foods provide the proper balance of minerals, there are situations in which a balanced diet is not sufficient to maintain health in terms of minerals. As stated above, minerals are essential in living organisms and have many important functions.

[0100] For example, iron is responsible for the transport of oxygen and myoglobin. In addition, iron is part of many enzymes, such as catalase, peroxidase, and cytochromes. Iron is transported to the duodenum and upper small intestine by cells lining the digestive tract, which absorb iron from ingested food. 2+ It is generally absorbed in the form

[0101] Calcium is another essential mineral that is, among other things, an important bone component and enzyme activator. Calcium also participates in the conduction of bioelectrical impulses, blood clotting, muscle contraction, inflammation, and hormone secretion. Calcium is generally absorbed in the small intestine in the presence of the active form of vitamin D (calcitriol).

[0102] Magnesium is a bone component and is involved in nerve conduction and ribosomal processes, among many other functions. Magnesium is also generally absorbed in the small intestine.

[0103] Mineral deficiency of any of such minerals can lead to severe complications, pathologies and diseases. The following non-therapeutic and therapeutic uses are intended to address such mineral deficiency, e.g., by providing the composition of the present invention as a nutritional supplement or nutritional drink, non-therapeutically, by preventing or treating mineral deficiency in a healthy subject, or by administering or providing the composition defined above as, e.g., as a nutritional composition or nutritional drink, as a pharmaceutical composition, etc., to prevent or treat mineral deficiency in a subject in need thereof.

[0104] The present invention also provides methods, compositions and uses for increasing mineral solubility and bioavailability in the digestive tract, as well as mineral absorption, using probiotic L. reuteri strains preconditioned with GOS. The present invention further includes methods, compositions and uses for improving the acid and bile tolerance of probiotic L. reuteri strains in the digestive tract, particularly by administering or using probiotic L. reuteri strains preconditioned with GOS, thereby increasing the survival rate and persistence of the preconditioned probiotic L. reuteri strains in the digestive tract.

[0105] Further, alternatively or additionally, the composition of the present invention is a composition comprising a probiotic L. reuteri strain preconditioned by GOS, which is administered to a subject or patient in need thereof, such as a subject or patient in need of increased bone strength or a subject or patient at risk of developing osteoporosis.Furthermore, the composition of the present invention should be administered to a subject or patient in need thereof, who is at risk of or suffering from a disruption of the intestinal barrier or a disruption of the mucosal lining.

[0106] Listed below are exemplary, non-limiting therapeutic and non-therapeutic applications that apply to the preconditioned probiotic L. reuteri strains as well as the compositions of the present invention.

[0107] NON-THERAPEUTIC USES OF COMPOSITIONS COMPRISING PRECONDITIONED PROBIOTIC L. REUTERI STRAINS According to a third aspect, the present invention provides a non-therapeutic use of an effective amount of a preconditioned probiotic L. reuteri strain, typically in the form of an administrable composition, prepared according to the present invention or as described herein, for increasing or enhancing the survival rate, persistence and / or probiotic effect of the L. reuteri strain in the digestive tract of a healthy subject. Such an increase or enhancement of survival rate, persistence and / or probiotic effect by the preconditioned probiotic L. reuteri strain in the digestive tract of a healthy subject should typically be determined in comparison to a probiotic L. reuteri strain that has not yet been preconditioned in the digestive tract of the healthy subject.

[0108] In one embodiment, increasing the survival and / or persistence of L. reuteri results in an increased probiotic effect of the probiotic L. reuteri strain.

[0109] In one embodiment, the increased probiotic effect of the probiotic L. reuteri strain is an increased viability and / or persistence of the probiotic L. reuteri strain.

[0110] In one embodiment, the increased probiotic effect of a probiotic L. reuteri strain is an increased activity, such as the biological activity, of L. reuteri, which may result in increased effects on the surrounding environment, such as other gut microorganisms and / or host cells. More specifically, these effects may be exemplified as increased mineral absorption, increased protective effect on epithelial integrity, increased bone formation and / or bone mineralization, and / or increased osteoblast maturation and / or activity. In another embodiment, the increased probiotic effect of L. reuteri is the promotion of a healthy microflora.

[0111] Preconditioning of probiotic L. reuteri strains improves or increases the viability, persistence and / or probiotic effect of the L. reuteri strains. Such improvements or increases in viability, persistence and / or probiotic effect can be, for example: increasing the bioavailability of minerals in the intestinal tract of a subject, preferably increasing mineral solubility and / or mineral absorption; and / or increasing the stress resistance of the L. reuteri strain in the intestinal tract of the subject, preferably increasing the stress resistance to bile and gastric acid in the intestinal tract of the subject; and / or Increased adhesion of L. reuteri strains to mucus in the intestinal tract of a subject; and / or increasing the survival and / or engraftment and / or viability of L. reuteri strains in the intestinal tract of a subject; and / or increasing epithelial integrity in the intestinal tract of a subject; and / or promoting bone formation and / or bone mineralization in a subject; and / or Promoting the maturation, differentiation and / or activity of osteoblasts in a subject.

[0112] These various improved properties include, among others, those listed above and disclosed in Examples 5-11. The increased survival and persistence may be an increased stress resistance of the L. reuteri strain and / or an increased adhesion of the L. reuteri strain to mucus. The increased probiotic effect may be, for example, an increased bioavailability of the mineral by increasing mineral solubility, accessibility and / or absorption of the mineral in the digestive tract of the subject, and / or an increased epithelial integrity in the digestive tract, and / or an increased bone formation and / or bone mineralization. The increased bioavailability of the mineral may in one embodiment result in an increased enzyme function. As mentioned above, the increased stress resistance may have a positive effect on the survival and / or engraftment and / or viability of the bacteria in the digestive tract and / or the persistence of the L. reuteri strain. The increased stress resistance is preferably an increased stress resistance to bile and gastric acid in the intestinal tract of a healthy subject. Any such effects are typically compared to the effects of a probiotic L. reuteri strain that has not been preconditioned with GOS.

[0113] The increase or enhancement of the probiotic effect of a typical L. reuteri strain in the digestive tract of a healthy subject relates in particular, but not exclusively, to an increase in the bioavailability of minerals, in particular to an increase in mineral solubility, accessibility and / or absorption of minerals in the digestive tract of the subject. Such minerals are preferably essential minerals as referred to herein, such as calcium, magnesium and iron, more preferably calcium and / or iron.

[0114] Of these essential minerals, calcium is involved in the conduction of bioelectrical impulses, blood clotting, muscle contraction, prevention of inflammation, and hormone secretion. Magnesium is involved in nerve conduction and ribosomal processes, among others. Iron is a key factor in many bodily functions and processes and is necessary to maintain healthy cells, skin, hair, and nails.

[0115] Increasing the bioavailability of (such) minerals in the intestinal tract of a healthy subject, preferably by increasing mineral solubility, accessibility and / or absorption in the digestive tract of a healthy subject, thereby preferably makes it possible to increase any of such bodily functions and processes. These aspects relate in particular to non-therapeutic and cosmetic applications in which the compositions of the present invention may be applied.

[0116] In addition to such effects on mineral solubility, accessibility and / or absorption of minerals in the digestive tract of a subject, further non-therapeutic uses may include improving the survival and persistence of L. reuteri strains, for example by increasing stress resistance and / or increasing the ability to adhere to mucus in the intestinal tract of a healthy subject, and / or promoting bone formation and / or bone mineralization, and / or promoting osteoblast maturation and / or activity in a healthy subject, and / or increasing epithelial integrity in the digestive tract of a healthy subject. Again, such effects should typically be determined in comparison to a probiotic L. reuteri strain that has not yet been preconditioned in the digestive tract of a healthy subject.

[0117] In this context, the healthy subjects that are the primary targets of this non-therapeutic use may be selected from athletes, children, infants or babies, adults, elderly people, pregnant women, vegetarians, lactating women, companion animals, cats or dogs or birds, such as poultry, preferably to address or even improve such bodily functions and processes. Such healthy subjects are not normally suffering from any pathology or disease as described herein, and in particular do not suffer from any mineral deficiency.

[0118] Any of the above methods for preconditioning a probiotic L. reuteri strain and any of the features of the composition defined herein comprising a preconditioned probiotic L. reuteri strain apply mutatis mutandis to the third aspect of the invention, i.e. the non-therapeutic use of the composition of the invention as defined above.

[0119] This particularly relates to features of the method for preconditioning the probiotic L. reuteri strain, as well as optional ingredients and amounts and further features used in the composition comprising the preconditioned probiotic L. reuteri strain. The composition may be provided in any form as described above.

[0120] In this connection, it is to be noted that, for example, a composition according to the invention for non-therapeutic purposes may be solid or liquid and / or may be in the form of a powder, a sachet, a tablet, a capsule or may be in the form of an oil solution, an emulsion, an oil-in-water emulsion (o / w emulsion) or a water-in-oil emulsion (w / o emulsion).

[0121] Furthermore, the compositions of the present invention for non-therapeutic purposes may be in an administrable form preferably selected from the group consisting of nutritional compositions, pharmaceutical formulations, dietary supplements, functional foods, functional beverage products and combinations thereof, such as dairy products, dairy-based products, whey protein-based beverages.

[0122] As previously mentioned, the L. reuteri strains are selected from the group consisting of L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 33636, L. reuteri DSM 33637, L. reuteri DSM 33638, L. reuteri DSM 33639 ... 33634, L.reuteri DSM 33635, L.reuteri DSM 27131, L.reuteri DSM 32465, L.reuteri DSM 32231, L.reuteri DSM 32232, L.reuteri ATCC PTA 6127 and L.reuteri ATCC PTA 4659, and preferably selected from the group consisting of L.reuteri strain DSM 17938, L.reuteri strain ATCC PTA 5289, L.reuteri strain ATCC PTA 6475, L.reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 and L. reuteri ATCC PTA 4659, particularly preferably at least one L. reuteri strain or strains as defined above, preferably selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 and L. reuteri ATCC PTA 4659, and further preferably the probiotic L. reuteri strain is L. reuteri DSM 17938.

[0123] In a particular embodiment, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475, preferably selected from the group consisting of L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475. Thus, in a particular embodiment, the probiotic L. reuteri strain is L. reuteri DSM 27131. In another particular embodiment, the probiotic L. reuteri strain is L. reuteri ATCC PTA 6475.

[0124] Use of a composition containing a preconditioned probiotic L. reuteri strain as a pharmaceutical According to a fourth aspect, the present invention also provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention or as described herein for use as a medicament. A related aspect defines a preconditioned probiotic L. reuteri strain for use as a medicament.

[0125] As before, the efficacy of such compositions as pharmaceuticals is particularly relevant in increasing the probiotic effect in the digestive tract of a subject in need of such a pharmaceutical, typically compared to the effect of a probiotic L. reuteri strain that has not been preconditioned with GOS.

[0126] The therapeutic effects associated with the use of the compositions or preconditioned probiotic L. reuteri strains of the invention as medicaments address in particular the effects associated with any of the diseases mentioned herein, in particular application to subjects at risk of or already suffering from any of such diseases, in particular diseases explicitly mentioned herein in the context of therapeutic applications (see also below).

[0127] Any of the above methods for preconditioning a probiotic L. reuteri strain and any of the features of the composition defined herein comprising a preconditioned probiotic L. reuteri strain also apply mutatis mutandis to the fourth aspect of the invention, i.e. the use of a composition of the invention as a medicament.

[0128] This particularly relates to features of the method for preconditioning the probiotic L. reuteri strain, as well as optional ingredients and amounts and further features used in the composition comprising the preconditioned probiotic L. reuteri strain. The composition may be provided in any form as described above.

[0129] In this connection, it is particularly preferred that the composition according to the invention for use as a pharmaceutical may be solid or liquid and may be in the form of a powder, a sachet, a tablet, a capsule or may be in the form of an oil solution, an emulsion, an oil-in-water emulsion (o / w emulsion) or a water-in-oil emulsion (w / o emulsion).

[0130] Similarly, the composition of the present invention for use as a pharmaceutical may be in an administrable form preferably selected from the group consisting of nutritional compositions, pharmaceutical formulations, dietary supplements, functional foods, functional beverage products and combinations thereof, such as dairy products, dairy-based products, whey protein-based beverages.

[0131] As previously described, the L. reuteri strains were L. reuteri DSM 17938, L. reuteri ATCC PTA 5289, L. reuteri ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 33636, L. reuteri DSM 33637, L. reuteri DSM 33638, L. reuteri DSM 33639 ... 33635, L.reuteri DSM 27131, L.reuteri DSM 32465, L.reuteri DSM 32231, L.reuteri DSM 32232, L.reuteri ATCC PTA 6127 and L.reuteri ATCC PTA 4659, and is preferably selected from the group consisting of L.reuteri DSM 17938, L.reuteri ATCC PTA 5289, L.reuteri ATCC PTA 6475, L.reuteri DSM 32846, L.reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 and L. reuteri ATCC PTA 4659, and further preferably the probiotic L. reuteri strain is L. reuteri DSM 17938.

[0132] In a particular embodiment, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475, preferably selected from the group consisting of L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475. Thus, in a particular embodiment, the probiotic L. reuteri strain is L. reuteri DSM 27131. In another particular embodiment, the probiotic L. reuteri strain is L. reuteri ATCC PTA 6475.

[0133] Therapeutic Uses of Compositions Comprising Preconditioned Probiotic L. reuteri Strains According to a fifth aspect, the present invention provides a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the present invention or as described herein for use in the prevention and / or treatment of a disease in a subject in need thereof, or for reducing or inhibiting the risk of such a disease. A related aspect of the present invention defines a preconditioned probiotic L. reuteri strain for use in the prevention, reduction or inhibition of a disease in a subject in need thereof, or for reducing or inhibiting the risk of such a disease.

[0134] As used herein, the terms "treatment", "treat" and "to alleviate" include prophylactic or preventive treatments (preventing and / or delaying the onset of the targeted pathological condition or disorder), as well as therapeutic measures that cure, slow, alleviate the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and curative, therapeutic or disease-modifying treatments, including treatment of subjects at risk of or suspected of having a disease, and subjects who are ill or have been diagnosed with a disease or medical condition. The terms do not necessarily imply that a subject is treated until complete recovery. The terms "treatment" and "treat" also refer to the maintenance and / or promotion of health in individuals who are not afflicted with a disease but who may be susceptible to the development of an unhealthy condition or the like. The terms "treatment" and "treat" further include reducing the risk of developing a disease, for example in the form of prophylactic treatment. The terms "treatment", "treat" and "to alleviate" are also intended to include the strengthening or otherwise augmentation of one or more primary preventative or therapeutic measures. The terms "treatment", "treat" and "to alleviate" are further intended to include dietary management of a disease or condition, or dietary management for the prophylaxis or prevention of a disease or condition.

[0135] According to one embodiment, the present invention relates to a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the method of the present invention or as defined herein, for use in the prevention and / or treatment of a mineral deficiency in a subject in need thereof. Typically, the subject in need thereof is also typically a subject at risk of developing a mineral deficiency or a subject already suffering from a mineral deficiency.

[0136] In this particular context of the present invention, mineral deficiency preferably relates to a deficiency of calcium, magnesium and / or iron, so that as shown below it is not necessarily a deficiency of only one mineral that can lead to a particular disease, but more often it is a lack of a combination of different minerals, for example one or more minerals selected from calcium, magnesium and / or iron, which results in an overlapping combination of possible physiological consequences and different diseases / pathologies.

[0137] As mentioned above, iron deficiency is a condition that occurs when the body does not have enough mineral iron. Iron deficiency often results in abnormally low levels of red blood cells and a condition called iron deficiency anemia. As a result, iron deficiency anemia causes fatigue, lethargy and shortness of breath. Iron is also known to be very important for maintaining many bodily functions (such as immune or cognitive functions) and is necessary for maintaining healthy cells, skin, hair and nails.

[0138] Thus, in certain embodiments, the subject suffers from iron deficiency, preferably iron deficiency anemia.

[0139] The following groups of people are at highest risk for iron deficiency anemia and are therefore suitable to receive the preconditioned L. reuteri strain composition; menstruating women, especially those with heavy menstrual periods, pregnant or lactating women or women who have recently given birth, people who have undergone major surgery or physical trauma and have lost blood, people with gastrointestinal disorders such as celiac disease, inflammatory bowel diseases such as ulcerative colitis or Crohn's disease, people with peptic ulcer disease, people who have undergone bariatric procedures, especially gastric bypass surgery, vegetarians, vegans, and others whose diet does not include iron-rich foods, children who drink more than 16-24 ounces of milk per day (which not only contains very little iron, but can also reduce iron absorption and irritate the intestinal lining, causing chronic blood loss), and infants, especially those who are low birth weight or born prematurely, and / or who do not get enough iron from breast milk or formula. In general, children need extra iron during periods of accelerated growth. Any of these subject groups may be treated herein.

[0140] Thus, in one embodiment, the composition of the invention comprising an effective amount of a preconditioned probiotic L. reuteri strain used to treat iron deficiency, preferably iron deficiency, preferably iron deficiency anemia, in a subject is as prepared according to the invention or as described herein.

[0141] Furthermore, chronic iron deficiency is a known new risk factor for bone loss and osteoporosis.Calcium is another essential mineral that is an important bone component and enzyme activator.Calcium also participates in the conduction of bioelectrical impulses, blood clotting, muscle contraction, inflammation and hormone secretion.Magnesium is a bone component and participates in nerve conduction and ribosomal processes.

[0142] The compositions of the invention may also be for use in the prevention and / or treatment of iron deficiency, preferably iron deficiency anemia, in a subject in need thereof, and / or may promote cognitive function, and / or may promote oxygen transport, and / or may promote immune function, and / or may reduce fatigue or tiredness.

[0143] Thus, in one embodiment, the present invention provides a composition comprising a preconditioned L. reuteri strain for use in preventing and / or treating calcium and / or magnesium deficiency in a subject, for use in preventing and / or treating bone loss, for use in promoting bone formation and / or mineralization and / or bone strength and / or tooth development, and / or for use in promoting growth and development, particularly in children and infants, for example by promoting osteoblast maturation and / or activity, or for use in treating osteoporosis and / or osteopenia.

[0144] Furthermore, further conditions and / or diseases related to muscle problems, such as cramps, muscle spasms and dull pain, fatigue including insomnia, drowsiness and extreme tiredness, skin and nail conditions, eczema, psoriasis, redness, itchiness, skin blisters, osteoporosis and osteopenia, painful premenstrual syndrome, dental problems, depression, etc., may be prevented or treated, especially with calcium deficiency in mind. Furthermore, calcium deficiency (hypocalcemia) is also a serious problem in early infancy, making infants at this stage a particular target group. This includes neonatal hypocalcemia, which occurs in the first 2-3 days of a newborn's life, and later hypocalcemia, which begins in the first week or weeks of life. Also, here calcium levels are somewhat related to magnesium levels, so low magnesium levels will result in low calcium levels, and low magnesium levels may be an additional risk factor. Any of such subject or patient groups may be treated herein.

[0145] Furthermore, taking into account in particular magnesium deficiency, further conditions and / or diseases associated with fatigue, general weakness, muscle spasms, muscle insufficiency, abnormal heartbeat, increased irritability of the nervous system with tremors, paresthesias, palpitations, low potassium levels in the blood, hypoparathyroidism resulting in low calcium levels in the blood, chondrocalcinosis, spasticity and tetany, migraine headaches, epileptic seizures, basal ganglia calcification, and in extreme and prolonged cases coma, or intellectual disability, may be prevented or treated. Again, any of such subject or patient groups may be treated herein.

[0146] According to a further embodiment, the present invention provides a composition comprising a preconditioned probiotic L. reuteri strain for use in increasing the survival rate, persistence and / or probiotic effect of a probiotic L. reuteri strain preferably in the intestinal / gastrointestinal tract of a subject.

[0147] Such improvements or increases in viability, persistence and / or probiotic effect may manifest, for example, as: increasing the bioavailability of minerals in the intestinal tract of a subject, preferably as an increase in mineral solubility and / or mineral absorption; and / or - as an increase in the stress resistance of the L. reuteri strain in the intestinal tract of the subject, preferably as an increase in the stress resistance to bile and gastric acid in the intestinal tract of the subject; and / or as an increase in adhesion of L. reuteri strains to mucus in the intestinal tract of a subject; and / or As an increase in epithelial integrity in the intestinal tract of a subject; and / or as a stimulation of bone formation and / or bone mineralization in a subject; and / or Promoting the maturation, differentiation and / or activity of osteoblasts in a subject.

[0148] For example, such effects, including increased bioavailability of minerals in the intestinal / gastrointestinal tract of a subject, and further effects, are particularly due to the observed increase in the ability of the preconditioned L. reuteri strains described herein to positively affect and increase the bioavailability of minerals in the intestinal tract of a healthy subject, typically by increasing the mineral solubility, accessibility and / or absorption of such minerals in the gastrointestinal tract of a subject, preconditioned L. reuteri strains with GOS.In this context, particularly relevant minerals include magnesium, calcium and / or iron, preferably calcium and iron.

[0149] As mentioned above, the preconditioned probiotic L. reuteri strain used herein also positively affects and increases the stress resistance of preconditioned probiotic L. reuteri in the digestive tract of a subject in need thereof.This has been convincingly demonstrated by the present inventors through stress resistance tests using bile and acid stress test conditions.Such improvement in the stress resistance of preconditioned probiotic L. reuteri in the digestive tract of a subject in need thereof leads to an improvement in the ability of preconditioned probiotic L. reuteri to better tolerate changes in bile and acid conditions in the digestive tract, which is manifested by an increase in the survival rate and persistence of preconditioned L. reuteri strain.

[0150] The preconditioned L. reuteri strains used herein positively affect and increase adhesion (of the preconditioned probiotic L. reuteri strains) to mucus, which can positively affect the bacteria's ability to access the intestinal epithelial lining and remain in the intestine for extended periods, thus increasing its ability to persist in the gastrointestinal tract, and thus positively affect the bacteria's ability to exert its probiotic and therapeutic effects.

[0151] The preconditioned L. reuteri strain used herein also increases the epithelial integrity of the intestinal epithelium, preferably in the digestive tract. In this context, it is important to note that the main function of the intestinal barrier is to regulate the absorption of nutrients, electrolytes and water from the lumen into the circulation, while an intact barrier is essential to obtain a healthy state in order to prevent the passage of microorganisms (e.g., pathogenic microorganisms) and toxic luminal substances into the circulation. Disruption of the intestinal barrier, or other mucosal lining, is associated with a number of different diseases and conditions, such as non-alcoholic fatty liver disease (NAFLD), celiac disease, malnutrition, anxiety, stress, irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, functional gastrointestinal disorders (e.g. functional abdominal pain), diarrhea, infantile colic, infectious diseases, depression, autism spectrum disorders, diverticular disease, periodontitis, osteopenia and / or osteoporosis, preferably selected from the group consisting of IBS, Crohn's disease, ulcerative colitis, functional gastrointestinal disorders (e.g. functional abdominal pain), diarrhea, infantile colic, infectious diseases, depression, autism spectrum disorders, diverticular disease, periodontitis, osteopenia and / or osteoporosis.

[0152] In one embodiment, the disease or condition is selected from the group consisting of NAFLD, celiac disease, malnutrition, anxiety, stress, IBS, Crohn's disease, ulcerative colitis, infectious disease, depression, autism spectrum disorder, diverticular disease, diarrhea, osteopenia and osteoporosis, preferably selected from the group consisting of IBS, Crohn's disease, ulcerative colitis, infectious disease, depression, autism spectrum disorder, diverticular disease, diarrhea, osteopenia and osteoporosis.

[0153] In certain embodiments, the disease or condition is selected from the group consisting of IBS, Crohn's disease, ulcerative colitis, diverticular disease and diarrhea.

[0154] In another particular embodiment, the disease or condition is infantile colic.

[0155] Thus, according to one embodiment, the present invention also relates to a composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to the method of the present invention or as defined herein for use in the prevention and / or treatment of a disease in a subject in need thereof, the subject suffering from a disruption of the intestinal barrier or disruption of the mucosal lining. In a particular embodiment, the disruption of the intestinal barrier or disruption of the mucosal lining is associated with a disease or condition selected from irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, functional gastrointestinal disorders, such as functional abdominal pain, diarrhea, infantile colic, infectious diseases, depression, autism spectrum disorder, diverticular disease, periodontitis, osteopenia and / or osteoporosis.

[0156] A further positive effect of the preconditioned L. reuteri strain used herein is to promote bone formation and / or bone mineralization, and / or promote osteoblast maturation, differentiation and / or activity. This has been convincingly demonstrated by the inventors by the results of increased ALP enzyme activity and reduced cell migration in osteoblasts, suggesting that the preconditioned L. reuteri strain can stimulate osteoblast differentiation and also stimulate enhanced osteocalcin expression, a marker of mineralization expressed by differentiated osteoblasts.

[0157] Therefore, the present invention also relates to a composition comprising an effective amount of a preconditioned L. reuteri strain prepared according to the method of the present invention or as defined herein for use in the prevention and / or treatment of bone loss and / or for use in promoting bone or tooth growth and development in children.

[0158] It is therefore believed that increasing the survival rate, persistence and / or probiotic effect of L. reuteri strains in the intestinal tract of a treated subject may have a particularly positive impact on the treatment of any of the diseases mentioned herein.

[0159] Thus, the medical uses claimed in this specification also include increasing the stress resistance of a L. reuteri strain in the digestive tract of a subject in need thereof, increasing adhesion to mucus and / or increasing epithelial integrity of a L. reuteri strain in the digestive tract of a subject in need thereof, increasing mineral solubility and / or mineral accessibility and / or mineral absorption in the digestive tract of a subject in need thereof, and / or increasing bone formation and / or bone mineralization in a subject in need thereof, typically in association with any of the pathologies or diseases mentioned therein.

[0160] Generally, the subject of any such treatment or the subject in need of such treatment is a mammal or bird, preferably a human, more preferably selected from children, infants or toddlers, elderly people, pregnant women, vegetarians, lactating women, companion animals, but also pets, such as cats or dogs, or birds, such as poultry, and the subject is preferably at risk of developing or already developing a mineral deficiency. Populations at risk of calcium deficiency and preferred subjects for such prevention and / or treatment include pregnant women (especially in the later stages of pregnancy), lactating women, postmenopausal women, elderly people, teenagers, and / or overweight people. Populations at risk of hypomagnesemia include diabetic patients, patients at risk of diabetes, obese and / or overweight patients, etc. Any of such subjects and patient groups may be treated herein.

[0161] In this connection, any of the above-mentioned methods for preconditioning a probiotic L. reuteri strain and any of the features of the compositions defined herein comprising a preconditioned probiotic L. reuteri strain also apply mutatis mutandis to the fifth aspect of the invention, i.e. the medical or therapeutic use of a composition of the invention in any of the treatments defined herein.

[0162] This particularly relates to features of the method for preconditioning the probiotic L. reuteri strain, as well as optional ingredients and amounts and further features used in the composition comprising the preconditioned probiotic L. reuteri strain. The composition may be provided in any form as described above.

[0163] In this context, it is particularly preferred that the compositions of the invention for medical or therapeutic use described herein may be solid or liquid and may be in the form of a powder, a sachet, a tablet, a capsule or may be in the form of an oil solution, an emulsion, an oil-in-water emulsion (o / w emulsion) or a water-in-oil emulsion (w / o emulsion).

[0164] Similarly, the compositions of the present invention for medical or therapeutic uses as described herein may be in an administrable form preferably selected from the group consisting of nutritional compositions, pharmaceutical formulations, dietary supplements, functional foods, functional beverage products and combinations thereof, such as dairy products, dairy-based products, whey protein based beverages.

[0165] As previously described, the L. reuteri strains were L. reuteri DSM 17938, L. reuteri ATCC PTA 5289, L. reuteri ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 33636, L. reuteri DSM 33637, L. reuteri DSM 33638, L. reuteri DSM 33639 ... 33635, L.reuteri DSM 27131, L.reuteri DSM 32465, L.reuteri DSM 32231, L.reuteri DSM 32232, L.reuteri ATCC PTA 6127 and L.reuteri ATCC PTA 4659, and is preferably selected from the group consisting of L.reuteri DSM 17938, L.reuteri ATCC PTA 5289, L.reuteri ATCC PTA 6475, L.reuteri DSM 32846, L.reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 32465 and L. reuteri ATCC PTA 4659, and further preferably the probiotic L. reuteri strain is L. reuteri DSM 17938.

[0166] In a particular embodiment, the probiotic L. reuteri strain is selected from the group consisting of L. reuteri DSM 17938, L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475, preferably selected from the group consisting of L. reuteri DSM 27131 and L. reuteri ATCC PTA 6475. Thus, in a particular embodiment, the probiotic L. reuteri strain is L. reuteri DSM 27131. In another particular embodiment, the probiotic L. reuteri strain is L. reuteri ATCC PTA 6475.

[0167] According to further specific embodiments, the present invention also provides a method for treating a disease in a subject in need thereof. Such diseases and subjects are preferably as described herein. The method of treatment preferably comprises administering to the subject an effective amount of a preconditioned probiotic L.reuteri strain, preferably prepared according to the method of the present invention for preconditioning a probiotic L.reuteri strain or as described herein. Thus, the preconditioned probiotic L.reuteri strain is preferably a probiotic L.reuteri strain cultured in the presence of GOS, preferably according to the method of the present invention for preconditioning a probiotic L.reuteri strain. The administration preferably allows the prevention and / or treatment of a disease in a subject in need thereof, or the reduction or inhibition of the risk of such a disease. Any of the diseases defined above are included. An effective amount of the preconditioned probiotic L. reuteri strain may be provided in the form of the inventive composition as described herein. Any of the features described above for the inventive method for preconditioning a probiotic L. reuteri strain and for the inventive composition apply equally.

[0168] According to another particular embodiment, the present invention also provides a preconditioned probiotic L. reuteri strain, preferably prepared according to the method of the present invention for preconditioning a probiotic L. reuteri strain, or the use of a preconditioned probiotic L. reuteri strain as described herein for preparing a medicament or pharmaceutical composition for preventing and / or treating a disease in a subject in need thereof. Such diseases and subjects are preferably as described herein. Thus, the preconditioned probiotic L. reuteri strain is preferably a probiotic L. reuteri strain cultured in the presence of GOS, preferably according to the method of the present invention for preconditioning a probiotic L. reuteri strain. The use preferably allows the prevention and / or treatment of a disease in a subject in need thereof, or the reduction or inhibition of the risk of such a disease. Any of the diseases defined above are included. An effective amount of the preconditioned probiotic L. reuteri strain may be provided in the form of the inventive composition as described herein. Any of the features described above for the inventive method for preconditioning a probiotic L. reuteri strain and for the inventive composition apply equally.

[0169] Further aspects and embodiments of the invention are presented below.

[0170] A method for preparing a preconditioned probiotic Lactobacillus reuteri strain, comprising the steps of: a) culturing a probiotic L. reuteri strain in a growth medium in the presence of galactooligosaccharides (GOS), thereby preconditioning the probiotic L. reuteri strain; and b) harvesting the preconditioned probiotic L. reuteri strain from the growth medium.

[0171] The GOS is preferably present in step a) in an amount of at least 0.2% by weight, preferably at least 0.5% by weight, even more preferably at least 0.75% by weight of GOS in the growth medium, preferably up to 8% by weight of GOS, such as up to 7% by weight of GOS, up to 6% by weight of GOS, or even up to 5% by weight of GOS, even more preferably up to 4% by weight of GOS, or even up to 3% by weight of GOS, for example 0.2% by weight to 8% by weight, or even up to 5% by weight of GOS, calculated based on the growth medium at the beginning, during or at the end of the cultivation of the probiotic L. reuteri strain in step a) (% w / w). In the method according to the above, the sucrose ionizing agent is added to the growth medium in a range of 0.2% by weight to 7% by weight, or 0.2% by weight to 6% by weight, more preferably in the range of 0.5% by weight to 8% by weight, or even more preferably in the range of 0.5% by weight to 7% by weight, or 0.5% by weight to 6% by weight, even more preferably in the range of 0.75% by weight to 8% by weight, or even more preferably in the range of 0.75% by weight to 7% by weight, or 0.75% by weight to 6% by weight, most preferably in the range of 0.75% by weight to 7% by weight, in the range of 0.75% by weight to 6% by weight, or even in the range of 0.75% by weight to 5% by weight, for example, in the range of 0.75% by weight to 3% by weight, or in the range of 0.75% by weight to 4% by weight.

[0172] The method according to any of the above, wherein the GOS added to the growth medium in step a) has a degree of polymerization (DP) of 3-8.

[0173] Probiotic L. reuteri strains include the L. reuteri strain deposited under DSM 17938, the L. reuteri strain deposited under ATCC PTA 5289, the L. reuteri strain deposited under ATCC PTA 6475, the L. reuteri strain deposited under DSM 32846, the L. reuteri strain deposited under DSM 32848, the L. reuteri strain deposited under DSM 32849, the L. reuteri strain deposited under DSM 27131, the L. reuteri strain deposited under DSM 32465, and the L. reuteri strain deposited under ATCC PTA The method according to any of the above, wherein the probiotic L. reuteri strain is at least one L. reuteri strain or a plurality of L. reuteri strains selected from the group comprising or consisting of the L. reuteri strain deposited under DSM 17938.

[0174] A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain, wherein the preconditioned probiotic Lactobacillus reuteri strain has been prepared by culturing the L. reuteri strain in a growth medium in the presence of galactooligosaccharides (GOS), preferably according to any of the methods described above.

[0175] Preconditioned probiotic L. reuteri strains were 3 cfu~10 12 The amount of cfu, typically 10 per daily dose 4 cfu~10 11 cfu, preferably 10 per daily dose 5 cfu~10 10 cfu or 10 per daily dose5 cfu~10 9 cfu, also preferably 10 per daily dose 6 cfu~10 9 Amount of cfu, 10 per daily dose 6 cfu~10 8 cfu, or 10 per daily dose 8 cfu~10 10 cfu per daily dose, more preferably about 10 7 cfu~10 9 cfu, most preferably about 10 per daily dose 8 Total cfu, e.g., 10 per daily dose 7 ~10 9 cfu, or 10 per daily dose 8 ~10 9 The composition according to above, comprising in an amount of cfu.

[0176] the composition may be solid or liquid and / or in the form of a powder, tablet, capsule or may be in the form of an oil, emulsion, oil-in-water emulsion (o / w emulsion) or water-in-oil emulsion (w / o emulsion); and / or The composition is in an administrable form, preferably selected from the group consisting of nutritional compositions, pharmaceutical formulations, dietary supplements, functional foods, functional beverage products and combinations thereof, such as dairy products, dairy-based products, whey protein-based beverages; A composition according to any of the above.

[0177] An effective amount of a preconditioned probiotic L. reuteri strain prepared according to any of the above methods, or a non-therapeutic use of a composition according to any of the above, for improving or increasing the survival rate, persistence and / or probiotic effect of preferably a L. reuteri strain in the digestive tract of a healthy subject.

[0178] Improving or increasing viability, persistence and / or probiotic efficacy increasing the bioavailability, preferably increasing mineral solubility and / or mineral absorption, of minerals in the intestinal tract of healthy subjects, wherein the minerals are optionally selected from magnesium, calcium and / or iron, preferably calcium and / or iron, and / or an increase in the stress resistance of L. reuteri strains in the intestinal tract of healthy subjects, preferably an increase in the stress resistance to bile and gastric acid in the intestinal tract of healthy subjects; and / or increased adhesion of L. reuteri strains to mucus in the intestinal tract of healthy subjects; and / or an increase in epithelial integrity in the intestinal tract of healthy subjects; and / or Non-therapeutic use according to the above, which is the promotion of bone formation and / or bone mineralization, and / or the promotion of osteoblast maturation and / or activity in healthy subjects.

[0179] A composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to any of the above, or a composition according to any of the above, for use as a medicament.

[0180] A composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to any of the above, or a composition according to any of the above, for use in the prevention and / or treatment of a disease in a patient in need thereof, wherein the patient is suffering from a disruption of the intestinal barrier or a disruption of the mucosal lining, preferably wherein the disruption of the intestinal barrier or the disruption of the mucosal lining is associated with a disease or condition selected from irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, functional gastrointestinal disorders, such as functional abdominal pain, diarrhea, infantile colic, infectious diseases, depression, autism spectrum disorder, diverticular disease, periodontitis, osteopenia and / or osteoporosis.

[0181] A composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to any of the above, or a composition according to any of the above, for use in the prevention and / or treatment of mineral deficiency in a patient in need thereof, wherein the minerals are optionally selected from magnesium, calcium and / or iron, preferably calcium and / or iron.

[0182] the composition is for use in the prevention and / or treatment of iron deficiency, preferably iron deficiency anemia, in a patient in need thereof, and / or promotes cognitive function, and / or promotes oxygen transport, and / or promotes immune function, and / or reduces fatigue or tiredness, and / or The composition is for use in the prevention and / or treatment of calcium and / or magnesium deficiency in a patient in need thereof. A composition for use as described above.

[0183] A composition comprising an effective amount of a preconditioned probiotic L. reuteri strain prepared according to any of the above, or a composition according to any of the above, for use in the prevention and / or treatment of bone loss, and / or for promoting bone or tooth growth and development in children, and / or for promoting muscle function, and / or for promoting the function of the nervous system and / or bone or muscle related conditions in a patient in need thereof, and / or for promoting bone formation and / or bone mineralization and / or bone strength in a patient in need thereof.

[0184] The L. reuteri strains include the L. reuteri strains deposited under DSM 17938, the L. reuteri strains deposited under ATCC PTA 5289, the L. reuteri strains deposited under ATCC PTA 6475, the L. reuteri strains deposited under DSM 32846, the L. reuteri strains deposited under DSM 32848, the L. reuteri strains deposited under DSM 32849, the L. reuteri strains deposited under DSM 27131, the L. reuteri strains deposited under DSM 32465, and the L. reuteri strains deposited under ATCC PTA 4659, preferably the probiotic L. reuteri strain is the L. reuteri strain deposited under DSM 17938.

[0185] It will be understood that the various aspects and embodiments of the detailed description disclosed herein are illustrative of specific ways to make and use the invention, and do not limit the scope of the invention when considered in light of the claims and detailed description. It will also be understood that features from aspects and embodiments of the invention can be combined with additional features from the same or different aspects and embodiments of the invention.

[0186] As used in this detailed description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0187] All ranges stated are intended to include all numbers, integers or fractions contained within the range. EXAMPLES

[0188] Example 1 - Method for preconditioning L. reuteri strains with GOS An internally produced frozen starter culture of L. reuteri strain (L. reuteri strain DSM 17938) was used to inoculate a fermentation medium containing 4% bovine milk-derived oligosaccharides (BMOS) as a carbon source and 4% fructose (dry matter equivalent) as an electron acceptor. An overnight culture of L. reuteri strain was used to inoculate a fresh fermentation medium containing 6% BMOS as a carbon source and 6% fructose (dry matter equivalent) as an electron acceptor. Preconditioned L. reuteri strain cells were harvested by centrifugation after about 10 hours of fermentation, then mixed with a protectant and spray-dried.

[0189] The BMOS carbohydrate mixture used as the GOS source consisted of 48% GOS, 30% lactose, 8% glucose, 3.9% galactose and >0.2% 3'-SL + 6'-SL (g / 100 g dry matter equivalent).

[0190] The cell numbers of L. reuteri strains were determined by pour plating. Briefly, serial decimal dilutions of spray-dried samples were made in tryptone salts (Oxoid, LP0042) solution. Then, 100 μL of the appropriate dilution was transferred to a Petri dish and mixed with MRS agar (AES Chemunex, Bruz, France). The analysis was performed in duplicate. The plates were then incubated at 37°C in aerobic conditions for 48 h. Colony forming units per gram (cfu / g) were calculated from the number of colonies counted on the plates with the appropriate dilution by determining their arithmetic mean.

[0191] Example 2 - Methods for making compositions containing preconditioned L. reuteri strains A conventional commercial Growing Up Milk composition was supplemented with 5×10 per gram of preconditioned probiotic L. reuteri strain. 6 A milk-based infant drink, also called Growing Up Milk, containing age-appropriate minerals was prepared by adding amounts of cfu.

[0192] Example 3 - Preparation of an oil-based probiotic product containing preconditioned L. reuteri The L. reuteri DSM 17938 strain is cultured with the addition of GOS according to the method described in Example 7. After cultivation, the preconditioned L. reuteri DSM 17938 is freeze-dried using industry standard methods. The product is an oil-based formulation containing a single (pure) bacterial strain that is made for good stability and shelf life. An inherent feature of the manufacturing process is the step of drying the oil by placing it under vacuum to remove most of the water in the oil and increase the stability of the formulation. The oil used herein is a pure edible vegetable oil, preferably sunflower oil and medium chain triglyceride.

[0193] Mixing the ingredients 1. Medium chain triglycerides, e.g., Akomed R (Karlshamns AB, Karlshamn Sweden) and sunflower oil, e.g., Akosun (Karlshamns AB, Karlshamn Sweden) are mixed with silicon dioxide (Cab-o-sil M5P, M5P, Cabot) in a Bolz mixer / tank (Alfred BOLZ Apparatebau GmbH, Wangen im Allgau, Germany).

[0194] 2. Homogenization A Sine pump and dispax (Sine Pump, Arvada, Colorado) is connected to a Bolz mixer and the mixture is homogenized.

[0195] 3.Vacuum drying The mixture is dried in a Bolz tank under a vacuum of 10 mBar for 12 hours.

[0196] 4. Add L. reuteri DSM 17938 bacteria. Transfer approximately 20 kg of the dry oil mixture into a 50 liter stainless steel container. Add L. reuteri bacteria powder, preferably freeze-dried (the amount of L. reuteri bacteria used will vary depending on the amount required in the oil, but one example is 10 11 Add 0.2 kg of culture with CFU / g. Mix gently until homogenous.

[0197] 5. Mixing The premix containing the L. reuteri bacteria is returned to the Bolz mixer.

[0198] 6.Emission The suspension is discharged into a 200 liter glass container and blanketed with nitrogen. The suspension is kept in the container until it is filled into bottles to be used for oral administration to humans.

[0199] Example 4 - Production of a probiotic product in tablet form containing preconditioned L. reuteri The L. reuteri DSM 17938 strain is cultured with the addition of GOS according to the method described in Example 7. After cultivation, the preconditioned L. reuteri DSM 17938 is freeze-dried using industry standard methods.

[0200] The following steps provide an example of a manufacturing process for tablets containing preconditioned L. reuteri DSM 17938 strain, including glucose encapsulation. It is understood that excipients, fillers, flavorings, encapsulating agents, lubricants, anti-caking agents, sweeteners and other ingredients of tablet products may be used as known in the art without affecting the efficacy of the product:

[0201] 1. Melting SOFTISAN™ 154 (SASOL GMBH, Bad Homburg, Germany) is melted in a container and heated to 70° C. to ensure complete destruction of the crystalline structure. It is then cooled to 52-55° C. (just above its hardening point).

[0202] 2. Granulation Transfer the L. reuteri strain lyophilized powder to a Diosna high shear mixer / granulator or equivalent. Slowly add the melted SOFTISAN™ 154 to the L. reuteri strain powder for approximately 1 minute. Use a chopper during addition.

[0203] 3.Wet sieving Immediately after granulation, the granules are passed through a 1 mm sieve mesh using a Tornado mill. The sieved granules are packed into alu pouches made of PVC coated aluminum foil and sealed using a heat sealer to form a pouch, along with a desiccant pouch, and kept refrigerated until blending. The granulation batch is divided into two tablet batches.

[0204] 4. Add encapsulated D-glucose (G8270, >99.5% glucose, Sigma) encapsulated using standard microencapsulation methods known in the art. The amount of sugar depends on the total CFU of the added powder of dried L. reuteri strains, with standard levels being 10 8 of total CFU, but this can also vary from 0.1 grams of sugar, or from 0.01 grams to 10 grams or even 100 grams of sugar.

[0205] 5. Mixing All ingredients are mixed in a mixer until a homogenous blend is obtained.

[0206] 6. Compression The final blend is transferred to the hopper of a rotary tablet press and compressed into tablets of 765 mg total weight in a Kilian compression machine.

[0207] 7.Bulk Packaging The tablets are packaged in alu pouches along with a desiccant pouch of molecular sieves. At least one week prior to final packaging, the alu pouches are placed in plastic buckets and stored in a cool location. The use of SOFTISAN™, a hydrogenated palm oil, allows the L. reuteri cells to be encapsulated within fat and environmentally protected.

[0208] Example 5 - Preconditioned probiotic L. reuteri strains and increased calcium and magnesium solubility in the colon a) SHIME (registered trademark) model In this experiment, we used a simplified simulation of the continuous simulator of the Human Microbial Ecosystem (SHIME®). This SHIME® model has been used extensively for more than 20 years in both scientific and industrial projects and has been validated by in vivo parameters (see, for example, Van den Abbeele et al., Arabinoxylo-Oligosaccharides and Inulin Impact Inter-Individual Variation on Microbial Metabolism and Composition, Which Immunomodulates Human cells, J. Agric. Food chem. 2018, 66, 5, 1121-1130).

[0209] For this series of experiments, a simplified SHIME® system was used, a two-stage batch system that mimics upper gastrointestinal (upper GIT, stomach and small intestine) and colonic conditions (Figure 1). These tests used a milk-based infant drink containing age-appropriate minerals, also called Growing Up Milk.

[0210] To simulate the absorption process occurring in the small intestine of infants, a dialysis technique was applied by using a cellulose membrane with a cut-off of 14 kDa. By introducing the small intestinal suspension into the dialysis membrane, molecules such as digested amino acids, sugars, micronutrients and minerals were gradually removed from the upper gastrointestinal matrix. Furthermore, to simulate the gastric pH of infants, a gradual pH drop during gastric incubation was performed, going from 5.5 to 3.0 during 1 h of incubation. Also, a fixed pH of 4.5 was performed during the first 30 min of small intestinal incubation (duodenum) to allow optimal absorption of available minerals. A pH of 7 was introduced during the following 145 min of the small intestinal phase (jejunum + ileum). The milk matrix after exposure to gastric and small intestinal conditions was transferred to the colon compartment containing the infant's fecal sample.

[0211] Fresh fecal material was collected from 12-month-old infant donors. A fecal suspension was prepared and mixed with a protective agent. At the start of short-term colonic incubation, the test components (L. reuteri strains according to Example 1 and preconditioned L. reuteri strains) were added to a sugar-depleted nutrient medium containing basic nutrients present in the colon (e.g., host-derived glycans such as mucins).

[0212] L. reuteri strain and preconditioned L. reuteri strain DSM 17938 at 1 × 10 9 CFU / reactor was inoculated.

[0213] The following colon incubations were performed: Control incubation without probiotic (upper GIT digested milk only) Upper GIT digestive milk + L. reuteri strain DSM 17938 Upper GIT digested milk + preconditioned L. reuteri strain DSM 17938

[0214] Colonic incubation was performed for 48 h under shaking (90 rpm) conditions at 37° C. All experiments were performed in triplicate to account for biological variability.

[0215] b) Mineral (calcium and magnesium) analysis Calcium and magnesium analyses were performed by a specialized laboratory at Ghent University, Belgium. Samples were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). Samples for mineral analysis in the colonic compartments were taken at 0, 24 and 48 hours. Colon dialysis was performed before the 48 hour time point to obtain values ​​for bioaccessible and insoluble mineral fractions.

[0216] As seen in Figure 2, the preconditioned probiotic L. reuteri strains enhance the bioaccessibility of magnesium and calcium in the colon at both 24 and 48 hours, suggesting that the preconditioned L. reuteri strains can be used to enhance the absorption of magnesium and calcium in subjects in need thereof.

[0217] Example 6 - Preconditioned L. reuteri strains and increased iron bioaccessibility in the colon The experiments were carried out as described in Example 5. Similarly, the analysis of iron content was carried out by a specialized laboratory at Ghent University (Belgium). Samples were measured by ICP-OES. Samples for mineral analysis in the colonic compartment were taken after 0, 24 and 48 hours. Colon dialysis was performed before the 48 hour time point to obtain values ​​for bioaccessible and insoluble mineral fractions.

[0218] As seen in Figure 3, the preconditioned probiotic L. reuteri strains enhance iron bioaccessibility in the colon, suggesting that the preconditioned L. reuteri strains can be used to enhance iron absorption in subjects in need thereof.

[0219] Example 7 - Preconditioned L. reuteri strains improve stress resistance to gastrointestinal conditions For this experiment, L. reuteri strain DSM 17938 was cultured with the addition of various sugars and electron acceptors (i.e. 3% BMOS + 3% fructose, or 3% glucose + 3% fructose) before the bacteria were freeze-dried and further tested by either of the two stress assays described below: acid tolerance (c) or bile tolerance (d). The results are shown under e).

[0220] a) Cultivation in a bioreactor 10 ml of MRS medium (Oxoid) was inoculated with 100 μl from a frozen stock stored at -70°C. The tube was incubated at 37°C for 6 hours, after which 500 μl was used to inoculate 50 ml of MRS into the tube. The tube was incubated at 37°C for 16-20 hours, after which the entire volume was used as inoculum for the bioreactor. Bioreactor culture conditions: Working volume: 1L Culture medium: 3% BMOS + 3% fructose or 3% glucose + 3% fructose in industrial MRS (iMRS) Ventilation: No ventilation Temperature: 37℃ Mixing: 250 rpm pH: 6.00+ / -0.1 Culture time: 6~10 hours

[0221] iMRS components Ingredient amount (g) yeast extract 10 Yeast Peptone 10 Sodium Citrate 2 Sodium Acetate 5 K2HPO42 MgSO4 heptahydrate 0.1 Manganese sulfate heptahydrate 0.05 Tween® 80 1 L-Cysteine ​​0.1 Purified water (800ml)

[0222] Set pH to 6.5 with NaOH. Autoclave (121° C., 15 min) followed by addition of sterile sugar solution (final volume 1000 ml). After incubation, bacteria were pelleted by centrifugation and suspended in 50 ml of 10% sucrose.

[0223] b) Freeze drying The concentrated cell suspension was dispensed into lyophilization glass vials (1 ml / vial) and the vials were frozen in an ultrafreezer at -50°C for 2 hours before being transferred to a Labconco FreeZone Stoppering Tray Freeze-Dryer. The lyophilization scheme was set up as follows: Freezing step: -40°C for 4 hours; Primary drying: -35°C, 0.102 mbar for 15 minutes, -10°C, 0.102 mbar for 12.5 hours; Secondary drying: -10°C, 0.01 mbar for 30 minutes, 25°C, 0.01 mbar for 12 hours. At the end of the process, the vials were capped under vacuum and stored at -20°C until further use.

[0224] c) Stress Tolerance Assay One vial of cultured, lyophilized L. reuteri strain DSM 17938 was rehydrated with 1 ml of phosphate-buffered saline (PBS) (pH 7.4) and then diluted 20-fold with 10 ml of synthetic gastric fluid pH 2.0* or PBS (control). The tubes were incubated at 37°C and samples were removed at 0, 50 and 90 min and serially diluted (10 -1 ~10 -6), and plated onto MRS agar plates. The plates were incubated overnight in an anaerobic jar at 37°C, after which the colonies were counted.

[0225] Synthetic gastric juice pH2.0 8.3g / l Proteose peptone (Oxoid) 3.5g / l D-glucose 2.05g / l NaCl 0.6g / l KH2PO4 0.11g / l CaCl2 0.37g / l KCl 0.05g / l bile 0.1g / l lysozyme (add before use) 13.3mg / l pepsin (add before use)

[0226] The pH is adjusted to 2.0 using 1M HCl and the gastric juice is filter sterilized.

[0227] d) Stress tolerance 8% bovine bile assay One vial of cultured and lyophilized L. reuteri strain DSM 17938 cells was rehydrated with 1 ml of PBS (pH 7.4) and then diluted 20-fold with 10 ml of MRS (Oxoid) containing 8% bovine bile or PBS (control). The tubes were incubated at 37°C, after which samples were removed at 0, 30 and 90 minutes and serially diluted (10 -1 ~10 -6 ), and plated onto MRS agar plates. The plates were incubated overnight in an anaerobic jar at 37°C, after which the colonies were counted.

[0228] result Results c) and d) show that in both assays, preconditioned L. reuteri strain DSM 17938 (i.e. cultured in 3% BMOS + 3% fructose) had improved stress resistance to both acid and bile compared to L. reuteri strain DSM 17938 cultured in 3% glucose + 3% fructose (L. reuteri strain DSM 17938). See Figures 4 and 5. Bacteria with increased stress resistance to both acid and bile perform better in the subject's digestive tract, including having improved survival and persistence, as shown here in two separate studies.

[0229] Example 8 Preconditioned L. reuteri strains improve adhesion to mucus For this experiment, L. reuteri strain DSM 17938 was cultivated in a bioreactor with the addition of various sugars and electron acceptors before the bacteria were freeze-dried according to the procedure described above in Example 7. L. reuteri strain DSM 17938 was then evaluated for its adhesion properties as described below.

[0230] Materials and Methods a) Coating of microtiter wells with mucus Mucus was prepared from porcine small intestine. 5 cm of the intestine was scraped inside with a spatula to remove material, which was then collected in 5 ml of ice-cold PBS. To remove cells and particulate matter, the resulting suspension was first centrifuged at 11,000×g for 10 min and then at 26,000×g for 15 min. Mucus preparations were stored at −20° C. until use.

[0231] The mucus material was analyzed by OD in PBS pH 6. 280=0.1 and incubated in microtiter wells (Greiner or Nunc MaxiSorb) (150 μl / well) overnight at 4° C. with gentle agitation. The wells were then washed with PBS (pH 6.0) containing 0.05% Tween® 20 (PBST), followed by blocking with 0.2 ml PBS containing 1% Tween 20 (pH 6.0) for 1 hour and a final two washes with PBST.

[0232] b) Preparation of bacteria One vial of cultured and lyophilized L. reuteri strain DSM 17938 cells was rehydrated with 1 ml PBS (pH 7.4) and washed with PBS 7.4 + Tween® 20 (0.05%). The sample was diluted to OD 0.5, then washed twice with PBST 7.4 (1 ml) and finally resuspended in PBST pH 6.0. The number of cfu / ml was analyzed by diluting the suspension in MRS broth and then plating the bacteria on MRS agar plates (Merck) that were incubated at 37° C. for 48 h under anaerobic conditions.

[0233] c) combination Bacterial suspension (150 μl) was added to each well and incubated for 4 h at 37°C with gentle agitation. After 4 h of binding, wells were washed three times with PBST (0.05% Tween 20 pH 6.0) and treated with 150 μl of trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25% porcine trypsin and 1 mM EDTA·4Na in Hanks' balanced salt solution) for 30 min at 37°C, after which the bacterial suspension was serially diluted and plated on MRS plates. Plates were incubated at 37°C for 24 h under anaerobic conditions.

[0234] result Preconditioned L. reuteri strain DSM 17938 (i.e., cultured in 3% BMOS + 3% fructose) had improved mucus binding and adhesion abilities compared to L. reuteri strain DSM 17938 cultured in 3% glucose + 3% fructose (L. reuteri strain DSM 17938). See Table 1 below.

[0235] The results show that preconditioned L. reuteri strain DSM 17938 has an improved ability to adhere to the intestinal mucosa compared to L. reuteri strain DSM 17938. This can have a positive effect on the potential of the bacterium to exert its probiotic effect, as it can gain access to the epithelial lining of the intestine and persist for longer periods in the intestine. [Table 1]

[0236] Example 9 Preconditioned L. reuteri strains protect epithelial integrity For this experiment, L. reuteri strain DSM 17938 was cultivated in a bioreactor with the addition of various sugars and electron acceptors before the bacteria were freeze-dried according to the procedure described above in Example 7. L. reuteri strain DSM 17938 was then evaluated for its ability to protect the intestinal epithelium from the harmful effects of enterotoxigenic Escherichia coli (ETEC), as described below.

[0237] Materials and Methods Epithelial cell culture (Caco-2 / HT29 MTX) Caco-2 and HT29-MTX cells were grown separately in tissue culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin and streptomycin at 37°C in an atmosphere of 5% CO2 and 90% relative humidity. Caco-2 and HT29-MTX cells were grown in 25 cm 2 Cells were grown in tissue culture flasks and split at 80-90% confluence using a 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. 2 6 x 10 per flask 4 The cells were seeded at a density of 100×.

[0238] Cell co-culture Caco-2 and HT29-MTX cells were seeded into the apical chamber of Transwell inserts (Transwell-COL collagen-coated membrane filters) at a ratio of 9:1, with 1 × 10 5 cells / cm 2 The cells were grown in 12-well Transwell plates at a final density of 1000 x 1000 cells / well. The cells were maintained under the same conditions and expanded in medium (0.5 ml apical and 1.5 ml basolateral) for 21 days. The cells were allowed to differentiate by changing the medium every other day.

[0239] Cell layer integrity Two methods were used to determine the integrity of the cell layer: transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-dextran) permeability determination.

[0240] The integrity of the cell monolayer during the experiment was determined by transepithelial electrical resistance (TEER) measurements using a Millicell electrical resistance system (Millipore, Darmstadt, Germany). Three different areas were used to detect the TEER value in each well (the average was used). 250 Ω cm 2 Wells with TEER values ​​above were used for permeability studies.

[0241] Seeded Caco-2 / HT29-MTX cells were pretreated with L. reuteri strain DSM 17938 bacterial cells (cultured with various carbon sources and lyophilized according to the culture and lyophilization procedure described above) rehydrated using PBS (pH 7.4) for 6 h at a multiplicity of bacteria (MOB) of 100 and then challenged with ETEC (a enterotoxigenic E. coli known to have destructive effects on epithelial integrity) at a multiplicity of infection (MOI) of 100 for an additional 6 h. ETEC (strain 853 / 67) was inoculated from frozen stocks stored at -70 °C and grown in 10 ml LB broth (Miller's) for 16 h at 37 °C. 1 ml of ETEC overnight culture was then reinoculated into 100 ml LB broth (Miller's) for 4-6 h at 37 °C. TEER was measured before pretreatment and challenge with ETEC, and then every hour during the entire challenge. To quantify paracellular permeability of the monolayer, 1 mg / mL of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma) was added to the apical side of the insert at the beginning of challenge with ETEC. Samples from the basolateral compartment were taken after 6 h of incubation. Diffused fluorescent tracer was then analyzed in triplicate by fluorometry (excitation, 485 nm; emission, 520 nm) using a FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

[0242] result It was found that preconditioned L. reuteri strain DSM 17938 (cultured in 3% BMOS + 3% fructose) has an improved ability to protect epithelial barrier integrity from the deleterious effects of ETEC compared to L. reuteri strain DSM 17938 cultured in 3% glucose + 3% fructose (L. reuteri strain DSM 17938). See Figure 6.

[0243] These results indicate that preconditioning with L. reuteri strain DSM 17938 improves its protective effect on barrier integrity.

[0244] Example 10 - Effects on bones In vitro experiments were performed using a mouse pre-osteoblast model: MC3T3-E1 (osteoblasts are bone-forming cells). The cells were exposed to different conditions: after 48 hours of colonic incubation, upper GIT digested milk supplemented with L. reuteri samples (with and without preconditioning) was centrifuged, filtered and then added to the culture medium (DMEM) at 1%: conditions: Digested milk (control) Digestive milk + L. reuteri strain Digested milk + preconditioned L. reuteri strain

[0245] reading: ALP activity (enzyme activity). The ALP enzyme is expressed by osteoblasts as they mature. It is therefore an indicator of bone-forming cell (osteoblast) differentiation.

[0246] · Osteocalcin gene expression is an indicator of osteoblast mineralization. Scratch assay to measure closure velocity as an index of MC3T3 cell migration

[0247] As can be seen in Figure 7, a positive effect on osteoblast differentiation was observed with a significant advantage of the preconditioned L. reuteri strain compared to the L. reuteri strain without preconditioning versus the control condition. The effect on osteoblast differentiation can be associated with a positive effect on bone formation rate and therefore growth rate.

[0248] Mineralization (late osteoblast activity) is higher in the preconditioned L. reuteri strains as shown in Figure 8. Higher mineralization may increase bone strength.

[0249] As shown in Figure 9, preconditioned L. reuteri significantly reduced cell migration compared to the control, whereas the non-preconditioned L. reuteri strain did not significantly affect this parameter. This reduction in migration coincides with the increased differentiation of bone cells when exposed to preconditioned L. reuteri, as shown in Figure 7 (ALP).

[0250] Example 11 - Various preconditioned L. reuteri strains show improved stress resistance to bile In this experiment, three different L. reuteri bacterial strains were tested: L. reuteri DSM 17938, L. reuteri ATCC PTA 6475 and L. reuteri DSM 27131. Each of the L. reuteri strains was inoculated from frozen stocks stored at -70°C and grown in 10 ml of MRS broth (Oxoid) for 16 hours at 37°C. One ml of the overnight culture was re-inoculated into 100 ml of IMRS (see Example 7 above) containing either 3% BMOS + 3% fructose or 3% glucose + 3% fructose for 16 hours at 37°C. The cells were then pelleted by centrifugation and resuspended in 10% sucrose (5 ml; 20x concentrated) prior to lyophilization.

[0251] Freeze drying The concentrated cell suspension was dispensed into lyophilization glass vials (1 ml / vial) and the vials were frozen in an ultrafreezer at -50°C for 2 hours before being transferred to a Labconco FreeZone Stoppering Tray Freeze-Dryer. The lyophilization scheme was set up as follows: Freezing step: -40°C for 4 hours; Primary drying: -35°C, 0.102 mbar for 15 minutes, -10°C, 0.102 mbar for 12.5 hours; Secondary drying: -10°C, 0.01 mbar for 30 minutes, 25°C, 0.01 mbar for 12 hours. At the end of the process, the vials were capped under vacuum and stored at -20°C until further use.

[0252] Stress Tolerance 8% Bovine Bile Assay One vial each of cultured and lyophilized L. reuteri strains DSM 17938, ATCC PTA 6475 or DSM 27131 cells (control and preconditioned cells) was rehydrated in 1 ml PBS (pH 7.4) and then diluted 20-fold with 10 ml MRS (Oxoid) containing 8% calf bile or PBS (control). The tubes were incubated at 37°C and samples were taken for further processing at 0, 30 min (ATCC PTA 6475 and DSM 27131) and 90 min (ATCC PTA 6475, DSM 27131 and DSM 17938). These samples were serially diluted (10 -1 ~10 -6 ), plated onto MRS agar plates, which were incubated overnight at 37°C in anaerobic jars, and cell viability was analyzed by counting colonies.

[0253] result The results of this example show that all preconditioned L. reuteri strains (L. reuteri strains DSM 17938, L. reuteri ATCC PTA 6475 and L. reuteri DSM 27131) (i.e., cultured in 3% BMOS + 3% fructose) have improved stress resistance to bile compared to the corresponding control strains that were not preconditioned, i.e., cultured in 3% glucose + 3% fructose in all assays. See Tables 2A and 2B below. As shown herein, bacteria with increased stress resistance to bile are predicted to perform better in the subject's gastrointestinal tract, e.g., have improved survival and persistence to gastrointestinal conditions. The results shown here show that in the relevant assays, all three L. reuteri bacterial strains have improved stress resistance. [Table 2] [Table 3]

Claims

1. 1. A method for preparing a preconditioned probiotic Lactobacillus reuteri strain, comprising: The method comprises the steps of: a) culturing a probiotic L. reuteri strain in the presence of galactooligosaccharides (GOS) in a growth medium supplemented with GOS, thereby preconditioning the probiotic L. reuteri strain; and b) harvesting the preconditioned probiotic L. reuteri strain from the growth medium The above method.

2. GOS is added to the growth medium in step a) in an amount of at least 0.2% by weight, preferably at least 0.5% by weight, more preferably at least 0.75% by weight of GOS in the growth medium; and / or GOS is added to the growth medium in step a) in an amount of up to 8% GOS by weight in the growth medium, preferably up to 7% GOS by weight, more preferably up to 6% GOS by weight and even more preferably up to 5% GOS by weight, e.g. up to 4% GOS by weight or up to 3% GOS by weight, and / or 2. The method according to claim 1, wherein GOS is added to the growth medium in step a) in an amount selected in the range of 0.2% to 8% by weight of the growth medium, preferably in the range of 0.5% to 7% by weight, more preferably in the range of 0.75% to 6% by weight, and most preferably in the range of 0.75% to 5% by weight, such as 0.75% to 4% by weight, or 0.75% to 3% by weight of GOS.

3. 3. The method according to claim 1 or 2, wherein the GOS added to the growth medium in step a) has a degree of polymerization (DP) of 3 to 8.

4. Probiotic L. reuteri strains include L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. At least one L. reuteri strain selected from the group consisting of L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 27131, L. reuteri DSM 32465, L. reuteri DSM 32231, L. reuteri DSM 32232, L. reuteri ATCC PTA 6127 and L. reuteri strain ATCC PTA 4659. Preferably the probiotic L. reuteri strain is selected from the group consisting of L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. The method according to any one of claims 1 to 3, wherein the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32465 and L. reuteri strain ATCC PTA 4659, and more preferably L. reuteri strain DSM 17938.

5. Optionally, but preferably, drying the harvested preconditioned probiotic L. reuteri strain by spray drying or freeze drying the harvested preconditioned probiotic L. reuteri strain. The method of any one of claims 1 to 4, further comprising:

6. 1. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain, A preconditioned probiotic L. reuteri strain was prepared by culturing the L. reuteri strain in the presence of galactooligosaccharides (GOS) in a growth medium supplemented with GOS; composition.

7. in a form selected from the group consisting of a powder, a sachet, a tablet, a capsule, an oil, an emulsion, an oil-in-water emulsion (o / w emulsion) and a water-in-oil emulsion (w / o emulsion); and / or 7. The composition of claim 6, in an administrable form selected from the group consisting of a nutritional composition, a pharmaceutical formulation, a dietary supplement, a functional food and a functional beverage product.

8. L. reuteri strains include L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32847, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 33632, L. At least one L. reuteri strain selected from the group consisting of L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 27131, L. reuteri DSM 32465, L. reuteri DSM 32231, L. reuteri DSM 32232, L. reuteri ATCC PTA 6127 and L. reuteri strain ATCC PTA 4659. Preferably the probiotic L. reuteri strain is selected from the group consisting of L. reuteri strain DSM 17938, L. reuteri strain ATCC PTA 5289, L. reuteri strain ATCC PTA 6475, L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L.

8. The composition according to claim 6 or 7, wherein the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32465 and L. reuteri strain ATCC PTA 4659, and more preferably L. reuteri strain DSM 17938.

9. A composition according to any one of claims 6 to 8, comprising an effective amount of freeze-dried or spray-dried pre-conditioned probiotic Lactobacillus reuteri strain.

10. Non-therapeutic use of an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to the method of any one of claims 1 to 5 for improving or increasing the survival rate, persistence and / or probiotic effect of preferably L. reuteri strains in the gastrointestinal tract of healthy subjects, comprising Optionally, but preferably, improving or increasing the survival rate, persistence and / or probiotic effect is achieved by: - increasing the bioavailability, preferably mineral solubility and / or mineral absorption, of minerals in the intestinal tract of healthy subjects, where the minerals are preferably selected from the group consisting of magnesium, calcium and iron, more preferably selected from the group consisting of calcium and iron, and / or - increasing the stress resistance of L. reuteri strains in the intestinal tract of healthy subjects, preferably increasing the stress resistance to bile and gastric acid in the intestinal tract of healthy subjects; and / or - increased adhesion of L. reuteri strains to mucus in the intestinal tract of healthy subjects; and / or - an increase in epithelial integrity in the intestinal tract of healthy subjects; and / or - promoting bone formation and / or bone mineralization in healthy subjects, and / or promoting osteoblast maturation, differentiation and / or activity; Non-therapeutic use.

11. A composition according to any one of claims 6 to 9 for improving or increasing the viability, persistence and / or probiotic effect of preferably a L. reuteri strain in the gastrointestinal tract of a healthy subject, comprising: Optionally, but preferably, improving or increasing the survival rate, persistence and / or probiotic effect is achieved by: - increasing the bioavailability, preferably mineral solubility and / or mineral absorption, of minerals in the intestinal tract of healthy subjects, where the minerals are preferably selected from the group consisting of magnesium, calcium and iron, more preferably selected from the group consisting of calcium and iron, and / or - increasing the stress resistance of L. reuteri strains in the intestinal tract of healthy subjects, preferably increasing the stress resistance to bile and gastric acid in the intestinal tract of healthy subjects; and / or - increased adhesion of L. reuteri strains to mucus in the intestinal tract of healthy subjects; and / or - an increase in epithelial integrity in the intestinal tract of healthy subjects; and / or - promoting bone formation and / or bone mineralization in healthy subjects, and / or promoting osteoblast maturation, differentiation and / or activity; composition.

12. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 9, for use as a medicament.

13. 10. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to any one of claims 1 to 5 for use in the prevention and / or treatment of a disease in a subject in need thereof, the subject suffering from a disruption of the intestinal barrier or disruption of the mucosal lining, preferably wherein the disruption of the intestinal barrier or disruption of the mucosal lining is associated with a disease or condition selected from the group consisting of: non-alcoholic fatty liver disease (NAFLD), celiac disease, malnutrition, anxiety, stress, irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, functional gastrointestinal disorders, such as functional abdominal pain, diarrhea, infantile colic, infectious diseases, depression, autism spectrum disorder, diverticular disease, periodontitis, osteopenia and / or osteoporosis, or a composition according to any one of claims 6 to 9.

14. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 9, for use in the prevention and / or treatment of a mineral deficiency in a subject in need thereof, optionally but preferably wherein said subject is suffering from iron deficiency, preferably iron deficiency anemia, wherein the mineral is preferably selected from the group consisting of magnesium, calcium and iron, and more preferably selected from the group consisting of calcium and iron.

15. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 9, for use in the prevention and / or treatment of bone loss.

16. A composition comprising an effective amount of a preconditioned probiotic Lactobacillus reuteri strain prepared according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 9, for use in promoting bone or tooth growth and development in children.

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