Combination of microbial components for reducing uremic toxins in cardiovascular metabolism or neurodegenerative disorders

Abstract

The present invention relates to compositions for reducing or avoiding uremic toxin accumulation in cardiovascular metabolism or neurodegenerative disorders, in particular compositions comprising probiotics, prebiotics (carbohydrates or fibres) and lipids.

Description

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 439,638, filed January 18, 2023, and U.S. Provisional Application No. 63 / 480,729, filed January 20, 2023, and includes a sequence list thereof, the entire disclosure of which is incorporated herein by reference. Background Technology

[0002] This disclosure generally relates to compositions and methods that allow for the reduction or avoidance of the accumulation of uremic toxins, preferably in cardiovascular metabolic or neurodegenerative diseases, and more preferably in chronic kidney disease (CKD). Such compositions comprise, as defined herein, a specific selection of prebiotics, a specific selection of probiotics, and a specific selection of lipids.

[0003] Uremic toxins are generally classified into three categories: free, water-soluble, low-molecular-weight solutes, protein-bound solutes, and medium-molecular-weight solutes, which are typically products of protein / amino acid metabolism in body tissues, particularly the liver. Uremic toxins include, in particular, urea, indophenol sulfate, TMAO, p-cresol sulfate, 3-carboxy-4-methyl-5-propyl-2-furanopropionic acid, p-tolyl glucuronic anhydride, and uric acid. The accumulation of these systemic uremic toxins, also known as uremia or the uremia state, is a condition commonly observed in individuals with cardiovascular, metabolic, and neurological disorders, including but not limited to chronic kidney disease (CKD).

[0004] For example, chronic kidney disease (CKD) is a disease characterized by progressive and gradual loss of kidney function and can lead to the accumulation of such uremic toxins that are normally cleared by the kidneys, resulting in uremia. Due to the classification of CKD-specific risk factors, CKD patients have an increased risk of developing cardiovascular disease (CVD). The accumulation of uremic toxins in the circulation and tissues is associated with the progression of CKD and its comorbidities, including CVD (see Yong Jin Lim et al., Toxins 2021, 13, 142).

[0005] Chronic kidney disease (CKD) and cardiovascular disease (CVD) are not the only conditions that show elevated uremic toxin levels; other cardiovascular, metabolic, and neurological conditions are also involved. Furthermore, early indicators of excessive uremic toxin levels are often common and not always clearly attributable to these cardiovascular, metabolic, and neurological conditions that are the primary cause of initial findings. Generally, the accumulation of uremic toxins can lead to syndromes and symptoms, including but not limited to exhaustive fatigue, anorexia, and protein-energy depletion, ultimately impacting the quality of life of individuals with high levels of circulating uremic toxins. In this context, several publications support the causal role of uremic toxins (including indophenol sulfate, p-toluene sulfate, TMAO, and urea) in inducing tissue damage and impairment (such as in the kidneys or other organs), leading to disease progression, organ failure, and death [References: Vanholder R., Schepers E, Pletinck A, Nagler EV, Glorieux G. 2014. J Am Soc Nephrol. 25(9): 1897-907. doi: 10.1681 / ASN. 2013101062; Falconi et al. 2021. Front Physiol. 12: 686249].

[0006] Therefore, poor management of systemic uremic toxins can lead to disease progression, the development of related symptoms and syndromes (such as uremic syndrome and anorexia), poor quality of life, and even death. Thus, early intervention is of paramount importance and necessity in effectively treating this disease and positively impacting its outcomes.

[0007] As generally indicated above, there are usually two main causes of disturbances in circulating uremic toxin levels. The first cause is usually due to increased accumulation caused by poor kidney filtration. The second cause is due to alterations in production resulting from several modifications in metabolism, including those in the liver, gut, and microbiome.

[0008] In the prior art, there are currently several proposals for managing uremic toxins and the causes of uremic toxin level disturbances.

[0009] Since some uremic toxins can also originate from high protein intake in the diet, one currently effective solution for managing uremic toxins is to administer a low-protein diet to the patient. However, a low-protein diet is often unsustainable because patients typically require a balanced diet and good nutrition. For example, protein is needed to manage other complications associated with kidney disease (such as protein-energy depletion). In many cases, a low-protein diet can even be harmful, such as in cases of anorexia and muscular dystrophy, and often in the elderly, all of which require meeting a minimum protein level (or even elevated protein levels) to prevent or treat excessive muscle protein degradation (myocatabolism).

[0010] Another approach to managing uremic toxins (which can be considered the gold standard in kidney disease management) is through dialysis. However, this requires continued treatment, often necessitated by specialized care facilities or hospitals, and is therefore arduous and expensive. Furthermore, in advanced diseases such as end-stage renal disease, uremic toxins cannot be removed by dialysis. Specifically, dialysis is inefficient at removing protein-bound uremic toxins such as p-toluene sulfate, indophenol sulfate, 3-carboxy-4-methyl-5-propyl-2-furanopropyl acid, and p-toluylglucuronide.

[0011] There is also the possibility of reducing gut microbiota-derived metabolites that serve as substrates for the metabolism of uremic toxins in tissues. Such methods include, for example, (1) AST-120, a carbon-based solution designed to remove microbiota-derived precursors at the gut level; (2) using probiotics / live therapeutic agents to correct dysbiosis to break down uremic toxin precursors; and / or (3) using fiber / polysaccharides / oligosaccharides to correct dysbiosis.

[0012] However, a drawback of such solutions is that they only partially address the production of uremic toxins, and because the primary cause is not eliminated, the accumulation of uremic toxins in the body and organs may still occur or cannot be prevented. There are also reports that the accumulation of uremic toxins in systemic circulation may be at least partially independent of the production of uremic toxin precursors by the gut microbiota.

[0013] Existing technologies have also led to the development of drugs, such as SGLT2 inhibitors designed to help improve glomerular hemodynamic function. These SGLT2 inhibitors are thought to improve other local and systemic mechanisms involved in the pathogenesis of CKD. However, these drugs have not yet been fully shown to improve circulating uremic toxin levels, nor have they provided a reliable basis for treating conditions caused by such uremic toxins, such as in individuals with cardiovascular, metabolic, and neurological disorders.

[0014] Therefore, there is a need in the art to provide alternative and preferably improved treatment concepts to more effectively address such problems and avoid and / or treat the accumulation of uremic toxins in patients, preferably in individuals with cardiovascular, metabolic and neurological conditions, particularly chronic kidney disease.

[0015] The present invention, as described herein, addresses such needs and provides compositions, uses, and corresponding treatments for preventing and / or treating the accumulation of uremic toxins in such patients. The invention also provides multi-component kits for such purposes. Summary of the Invention

[0016] The aforementioned problems are addressed by the subject matter of the independent claims, and are further described in detail in the following description, embodiments and aspects thereof, and dependent claims. Where necessary and unless otherwise expressly stated, the embodiments and aspects disclosed herein may be combined with each other.

[0017] This invention relates in particular to a novel composition that targets multiple direct and indirect mechanisms that help avoid the production of uremic toxins and prevent or at least reduce the accumulation levels of uremic toxins.

[0018] According to a first embodiment, there exists a composition preferably suitable for reducing and / or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases. The composition comprises selected probiotics, selected prebiotics, and selected lipids. More specifically, the composition comprises:

[0019] (a) Probiotics, selected from:

[0020] - Probiotics lacking the gene for producing at least one of urea, uric acid, p-cresol, p-toluene sulfate, indolesulfonate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, and / or glutaric acid; and / or

[0021] - Probiotics lacking at least one bacterial enzyme selected from urease, carnitine monooxygenase and reductase, tryptophanase and / or hydroxyphenylacetate; and / or

[0022] - Probiotics expressing at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or licheninase.

[0023] (b) A prebiotic selected from carbohydrates, wherein the carbohydrates can be hydrolyzed by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme.

[0024] (c) Lipids selected from triglycerides, short-chain fatty acids and / or medium-chain fatty acids, wherein the triglycerides include butyrate, short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids or mixtures thereof.

[0025] According to the first aspect, the composition as defined herein comprises probiotics. According to the first criterion, such probiotics may be selected from probiotics lacking genes for producing at least one of urea, uric acid, p-cresol, p-toluene sulfate, indolesulfonate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, and / or glutaric acid. According to the second criterion, such probiotics may also be selected from probiotics lacking at least one bacterial enzyme selected from urease, carnitine monooxygenase and reductase, tryptophanase, and / or hydroxyphenylacetate. According to the third criterion, such probiotics may be selected from probiotics expressing at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase, and / or lichen polysaccharidease. Any of these three criteria may be used alone or in combination with the other two criteria. Preferably, all three criteria are satisfied.

[0026] Preferred probiotics meeting all three criteria may be selected from any of the following species or subspecies: *Bifidobacterium animalis* subspecies *lacis*, *Bifidobacterium longum* subspecies *infantis*, *Bifidobacterium longum* subspecies *longum*, *Enterococcus faecium*, *Lactobacillus johnsonii*, *Lactococcus lactis*, *Lactaseibacillus paracasei* (formerly classified as *Lactobacillus paracasei*), *Limosilactobacillus reuteri* (formerly classified as *Lactobacillus reuteri*), and *Lactobacillus rhamnosus*. Lactobacillus rhamnosus (previously classified as Lactobacillus rhamnosus), Staphylococcus carnosus, and / or Streptococcus thermophiles, preferably Lactobacillus johnsonii, or a probiotic having a genome sequence with at least 95% average nucleotide identity (ANI), preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%, or even 99.5% or 99.9% ANI, as defined previously.

[0027] Even more preferably, the probiotics that meet all three criteria can be selected from any of the following strains:

[0028] a. Bifidobacterium animalis subsp. lactis NCC 2818, which was deposited at CNCM [National Center for Microbial Culture Collection, Pasteur Institute, Paris, France] on June 7, 2005, and assigned accession number CNCM I-3446;

[0029] b. Bifidobacterium longum infantis subsp. NCC 341 (ATCC 15697(T)), which is available from worldwideweb.atcc.org / products / 15697;

[0030] c. Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618) (NCBI refseq; GCA_000007525.1) [NCC 2075 was deposited in CNCM on January 29, 2001, and assigned accession number CNCM I-2618];

[0031] d. Enterococcus faecium NCC 2768 (NCIMB 10415 - which is available from Cerbios-Pharma SA Barbengo Switzerland (cerbios.swiss / e-faecium-sf68-a-model-for-efficacy-safety-for-pharmaceutical-probiotics / );

[0032] e. Lactobacillus johnsonii NCC 533 [originally known as La 1, deposited in CNCM on June 30, 1992, and assigned accession number CNCM I-1225] (see also NCBIrefseq; GCA_000008065.1 and GenBank AE017198.1 (inclusive as SEQ ID NO:1))];

[0033] f. Lactococcus lactis NCC 2287 (CNCM I-4154) [NCC 2287 was deposited in CNCM on April 24, 2009, and assigned accession number CNCM I-4154];

[0034] g. Lactobacillus paracasei NCC 2461 (CNCM I-2116) [NCC 2461 was deposited in CNCM on January 12, 1999, and assigned accession number CNCM I-2116];

[0035] h. Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724) [NCC 4007 was deposited in October 2004 at CGMCC [Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center (CGMCC), P.O. Box 2714, Beijing 100080, China] with identification number CGMCC 1.3724];

[0036] i. Staphylococcus aureus NCC 1052 (CNCM I-5400) [NCC 1052 was deposited in CNCM on February 1, 2019, and assigned accession number CNCM I-5400];

[0037] j. Staphylococcus aureus NCC 971 (CNCM I-5398) [NCC 971 was deposited in CNCM on February 1, 2019, and assigned accession number CNCM I-5398]; and / or

[0038] k. Streptococcus thermophilus NCC 2496 (CNCM I-3915) [NCC 2496 was deposited in CNCM on February 5, 2008, and assigned accession number CNCM I-3915];

[0039] Probiotics may have a genome with at least 95% average nucleotide identity (ANI), preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%, or even 99.5% or 99.9% ANI, corresponding to the genome sequence defined as a. to k. above. For CNCM identification, please refer to the National Collection of Microbial Cultures at Institut Pasteur, 22 rue du docteur Roux, 75724 Paris, France. CGMCC identification refers to the China General Microbial Culture Collection (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, China.

[0040] Even more preferably, the probiotics in the composition as defined herein are selected from *Lactobacillus johnsonii* NCC 533 (CNCMI-1225) or probiotics having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% or 99.9% ANI, similar to the genome sequence of *Lactobacillus johnsonii* NCC 533 (CNCM I-1225). The NCBI reference sequence of *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) is GCA_000008065.1 (SEQ ID NO:1).

[0041] According to the second aspect, the composition as defined herein further comprises a prebiotic. This prebiotic is selected from carbohydrates, wherein the carbohydrates are hydrolyzable by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase, and / or licheninase. This carbohydrate is preferably a prebiotic selected from α-galacto-oligosaccharides, raffinose, β-galacto-oligosaccharides, and cello-oligosaccharides, or combinations thereof. Even more preferably, this prebiotic is selected from pea galactooligosaccharides (α-GOS), soybean galactooligosaccharides (α-GOS), β-galactooligosaccharides (β-GOS), bovine milk oligosaccharides (BMOS) (β-GOS), Vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, human milk oligosaccharides (HMO), soluble hydrolyzed wheat, soluble hydrolyzed oats, and / or β-glucan hydrolysates containing cellobiose, cellotriose, or cellotetraose, or combinations thereof. Preferred HMOs include 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetraose (LNnT), lactose-N-tetraose (LNT), 3'-sialylated lactose (3SL), and / or 6'-sialylated lactose (6SL), or combinations thereof. Particularly preferred HMOs include LNT, 2'FL, and / or LNnT, or combinations thereof.

[0042] According to the third aspect, the composition as defined herein further comprises a lipid (c). This lipid (c) is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, or mixtures thereof. Preferred triglycerides include triglycerides comprising butyrate, triglycerides comprising butyrate and caprylate, and triglycerides comprising butyrate and oleate. More preferably, the lipid (c) is selected from a. triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA); b. triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA); c. triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as a mixture of butyrate and oleate); d. triglycerides composed of medium-chain fatty acids; e. triglycerides (TG) composed of short-chain fatty acids; f. short-chain fatty acids that can be metabolized into ketone bodies; and / or g. short-chain and / or medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)). Any combination is possible, such as a., b., c., d., a. / b., a. / c., a. / d., a. / e., a. / f., a. / g., b. / c., b. / d., b. / e., b. / f., c. / d., c. / e., c. / f., a. / b. / c., a. / d. / c.; a. / e. / c., a. / f. / c., b. / c. / d., a. / b. / c. / d., etc. In one embodiment, the composition defined herein comprises a triglyceride consisting of butyrate and caprylate. In one embodiment, the composition defined herein comprises a triglyceride consisting of butyrate and oleate.

[0043] According to another aspect, probiotics are preferably contained in the composition as defined herein in an effective amount of 10 3 cfu to 10 12 The amount between CFU is usually in increments of 10. 4 CFU / day up to 10 11 The amount between CFU / day dose is preferably in the range of 10. 5 CFU / day up to 10 10 The amount between CFU / daily dose or 10 5 CFU / day up to 10 9 The amount between CFU / day dose is also preferably 10. 6 CFU / day up to 10 9 The amount between CFU / day dose, 10 6 CFU / day up to 10 8 The amount between CFU / day dose or in increments of 10 8 CFU / day up to 1010 The amount of CFU / day, more preferably about 10 7 CFU / day up to 10 9 CFU / day dose. The preferred daily dose is approximately 10 CFU / day. 8 CFU / day dose, 10 7 CFU / day up to 10 9 CFU / day dose or 10 8 CFU / day up to 10 9 CFU / daily dose.

[0044] Furthermore, according to another aspect, the prebiotic is preferably included in the composition as defined herein in an amount between 0.1 g / day and 30 g / day, more preferably in an amount between 2 g / day and 15 g / day.

[0045] According to another aspect, the lipids are preferably included in the composition as defined herein in an amount between 0.1 g / day and 30 g / day, more preferably in an amount between 2 g / day and 15 g / day.

[0046] According to a preferred aspect, the composition as defined herein may comprise probiotics, prebiotics, and lipids as defined above, wherein:

[0047] (a) The probiotic is selected from at least one of the probiotics defined as a. to k. above, or a probiotic having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI or even 99.5% ANI or 99.9% ANI, or combinations thereof, more preferably selected from Lactobacillus johnsonii NCC 533 (CNCM I-1225) or a probiotic having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI or even 99.5% ANI or 99.9% ANI, or a combination thereof, with the genome having ...

[0048] (b) The prebiotic is selected from α-galacto-oligosaccharides, raffinose, β-galacto-oligosaccharides and cello-oligosaccharides or combinations thereof, preferably selected from pea galacto-oligosaccharides (pea GOS, α-GOS), soybean galacto-oligosaccharides (soybean GOS, α-GOS), β-galacto-oligosaccharides (β-GOS), milk oligosaccharides (e.g., Vivinal GOS, β-GOS), cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats, human milk oligosaccharides (HMO), β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose or combinations thereof;

[0049] (c) The lipid is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, or mixtures thereof, and further wherein, in one aspect, the triglycerides comprise butyrate and / or caprylate. Preferably, the lipid is selected from:

[0050] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0051] b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA);

[0052] c. Triglycerides (TG) consisting of a mixture of butyrate and long-chain fatty acids (such as a mixture of butyrate and oleate);

[0053] d. Triglycerides composed of medium-chain fatty acids;

[0054] e. Triglycerides (TG) composed of short-chain fatty acids;

[0055] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0056] g. Short-chain and / or medium-chain fatty acids capable of being metabolized into ketone bodies, preferably C4 and / or C8 fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)); and

[0057] h. Triglycerides (TG) composed of butyrate and caprylate.

[0058] According to another aspect, the composition as defined herein is selected from food products, foods for special medical purposes (FSMP), nutritional supplements, milk-based beverages, low-volume liquid supplements, meal replacement drinks, and combinations thereof.

[0059] According to a second embodiment, there is a composition as defined herein for reducing or preventing the accumulation of uremic toxins, preferably for use in the treatment of cardiovascular metabolic or neurodegenerative diseases. The composition is thus preferably used to reduce uremic toxins in cardiovascular metabolic or neurodegenerative diseases and related comorbidities, to delay the progression of such cardiovascular metabolic or neurodegenerative diseases and / or to manage symptoms and syndromes associated with the toxic effects of uremic solutes in such cardiovascular metabolic or neurodegenerative diseases and related comorbidities.

[0060] Cardiovascular metabolic or neurodegenerative disorders and related comorbidities that can be treated with the composition particularly involve the following conditions:

[0061] • Treatment or prevention of kidney disease, including chronic and acute; dialysis and predialysis status of kidney disease; rare (kidney) diseases, genetically induced (kidney) diseases, and metabolically induced (kidney) diseases.

[0062] disease;

[0063] • Treat or prevent uremic syndromes, including protein-energy wasting syndrome, bone loss, hyperanorexia, fatigue, or inflammation;

[0064] • Delayed complications of late-stage renal disease, including anemia, hyperphosphatemia, secondary hyperparathyroidism, bone disease, and neurological disorders;

[0065] • Delayed comorbidities of kidney disease, including cardiovascular disease;

[0066] • Preventing the risk of malnutrition or managing malnutrition;

[0067] • Delaying the progression of cardiovascular and metabolic diseases;

[0068] • Preventing or managing the risk of cardiovascular disease and comorbidities such as diabetes; and / or

[0069] • To prevent or manage the risks of neurodegenerative and neurological disorders.

[0070] According to a third embodiment, there is a method for treating cardiovascular metabolic or neurodegenerative diseases as defined above, which preferably includes, as a first step, (a) preparing and providing a composition as discussed above, the composition comprising a specific selection of probiotics, a specific selection of prebiotics and a specific selection of lipids as defined above; and (b) administering such composition to a patient in need, who typically suffers from an increase in uremic toxins, typically in the case of cardiovascular metabolic or neurodegenerative diseases as defined herein.

[0071] According to a fourth embodiment, there exists a kit (multipart) suitable for reducing and / or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases as defined herein, the kit comprising compositions as described herein, such as comprising a specific selection of probiotics, a specific selection of prebiotics and a specific selection of lipids as defined above; for mixing to form one or more of the compositions disclosed herein and / or for use in one or more of the methods disclosed herein, for example as two or more liquid solutions or dry powders in separate containers. Attached Figure Description

[0072] Figure 1 This diagram illustrates the biochemical pathways for the formation of several enterobacterial-derived uremic toxins and their precursors. In this example, the production of enzymes such as urea (NH3), p-toluene sulfate (PCS), indophenol sulfate (IS), and trimethylamine-N-oxide (TMAO) is shown using the corresponding enzyme codes (ECs). Uremic toxin precursors, such as p-cresol, indole, and TMA, are byproducts of microbial metabolism of amino acids such as tryptophan, tyrosine, carnitine, and choline. These byproduct metabolites are then further chemically modified in the liver, where they are converted into uremic toxins, which reach circulation and affect target organs.

[0073] Figure 2 This is a diagram showing the production and retention of uremic toxins in the body. Figure 2 (A) demonstrates how microbiome dysbiosis can lead to the accumulation of uremic toxins in the systemic circulation, resulting in symptom exacerbation, comorbidities, and disease progression. For example... Figure 2 As shown in (B), uremic toxins have a negative impact on multiple organs, leading to clinical outcomes and symptom presentation. Figure 2 B is adapted from Rosner et al., Clin JAm Soc Nephrol. 2021.

[0074] Figure 3 This section illustrates an exemplary selection of probiotic strains used for the compositions and therapeutic purposes disclosed herein. It can be seen that probiotic strains lacking the distinct genes and key bacterial enzymes involved in the metabolism of uremic toxins have been selected during a computer-based screening process. *Lactobacillus johnsonii* NCC 533 has been shown to lack the largest number of relevant enzymes for preventing the production or accumulation of uremic toxins and to provide the optimal basis for the envisioned treatment. White cells: enzymes absent; gray cells: enzyme presence uncertain; black cells: enzyme present. Tra: tyramine; Trp: tryptophan; Tyr: tyrosine; indole-3-pyruvate: IPA; IAM: indole-3-acetamide; IAN: indole-3-acetonitrile; 4-HPPA: 4-hydroxyphenylpyruvic acid.

[0075] Figure 4 This image shows the growth profile of *Lactobacillus johnsonii* NCC 533 with different carbohydrate sources (α-galacto-oligosaccharides, galactomannan, and fructans, which require α-galactosidase and β-fructofuranosidase as enzymes encoded by the probiotics) during a 48-hour incubation period. It can be seen that the addition of pea GOS and soybean GOS was the most efficient, while the addition of PHGG, fenugreek, scFOS, and inulin, although appropriate, resulted in smaller growth of *Lactobacillus johnsonii* NCC 533 (from top to bottom: soybean GOS, pea GOS, inulin, sFOS, PHGG, and fenugreek). The growth test using *Lactobacillus johnsonii* NCC 533 as a strain serves only as an example and can be applied to any of the further selected probiotics. Data were normalized with negative controls.

[0076] Figure 5 The image shows the growth profile of *Lactobacillus johnsonii* NCC 533 with different carbohydrate sources (β-galacto-oligosaccharides, which require β-galactosidase as an enzyme encoded by the probiotic) during a 48-hour incubation period. It can be seen that the addition of BMO and Vivianal GOS resulted in particularly efficient growth of *Lactobacillus johnsonii* NCC 533 (from top to bottom: BMO and...). (GOS). The use of Lactobacillus johnsonii NCC 533 as a strain growth test serves only as an example and can be applied to any of the further selected probiotics. Data were normalized with negative controls.

[0077] Figure 6 This image shows the growth profile of *Lactobacillus johnsonii* NCC533 with different carbohydrate sources (cellulose oligosaccharides, which require glucan-1,4-β-glucosidase, cellulase, and licheninase as enzymes encoded by the probiotic) during a 48-hour incubation period. It can be seen that the addition of cellobiose and cellotriose resulted in particularly efficient growth of *Lactobacillus johnsonii* NCC 533, followed by cellotetraose, soluble hydrolyzed wheat, and soluble hydrolyzed oats (from top to bottom: cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, and soluble hydrolyzed oats). The growth test using *Lactobacillus johnsonii* NCC 533 as a strain serves only as an example and can be applied to any of the further selected probiotics.

[0078] Figure 7 This paper presents an overview of an in vitro / ex vivo experimental setup for assessing differences in microbiome profiles between healthy and chronic kidney disease (CKD) donors using Prodigest's short-term single-stage colonic simulation technique.

[0079] Figure 7(A) shows a schematic diagram of the conditions and donor groups. For each donor group, two conditions were run in parallel: (1) a standard condition consisting of a basic nutritional food with minimal amino acids, simulating the amino acid content of a low-protein diet; and (2) an AA-spiked condition consisting of a nutrient medium containing a mixture of L-tryptophan, L-tyrosine, L-carnitine, choline, and L-phenylalanine. The AA mixture was chosen because these are substrates that are converted into uremic toxins and their precursors by intestinal bacteria. The AA concentration simulated the daily amino acid intake required by an adult. A total of nine healthy donors and eight CKD donors were used in this example.

[0080] The timeline of experiments and sample analysis is shown in Figure 7 (B) In this study, on day 0, the fecal microbiome was inoculated into a short-term, single-stage colonic system using ProDigest. From day 1 to day 2, a basic nutrient diet system with or without an amino acid mixture was used. A standard group was fed a basic nutrient diet, and an AA-spiked group was fed a basic nutrient diet plus an amino acid mixture. Samples were taken at different time points to measure overall fermentation activity, microbial community activity, and microbiome composition.

[0081] The readings are as follows:

[0082] (1) Overall fermentation activity: acid / base consumption

[0083] (2) Microbial community activity:

[0084] ●Lactate

[0085] ●Short-chain fatty acids (SCFA): butyric acid, propionic acid, acetic acid

[0086] ● Markers of protein hydrolysis activity: ammonium and branched SCFAs (isobutyric acid, isovaleric acid, and isohexanoic acid)

[0087] ●Uremic toxins and precursors: p-cresol, p-toluene sulfate, indole, indole-3-3-acetic acid, betaine, trimethylamine, trimethylamine-N-oxide, indolephenol, indolephenol sulfate, semialdehyde glutaric acid, uric acid, urea

[0088] (3) Microbial community composition: quantitative deepshotgun sequencing

[0089] Figure 8This illustrates an example of differences in microbial metabolic capacity between microbiomes from patients with chronic kidney disease (CKD) and healthy donors. After 48 hours of incubation in Prodigest's short-term single-stage colonic mimicry system, the CKD microbiome showed higher production of uremic toxin precursors such as p-cresol compared to the healthy microbiome, particularly in the presence of excess amino acid substrates. Figure 8 (A) indicates an imbalance in amino acid metabolism in the CKD microbiome. Furthermore, compared to a healthy microbiome, the CKD microbiome showed increased production of branched-chain fatty acids (BCFAs). Figure 8 (B) indicates that the CKD microbiome exhibits higher proteolytic activity. As expected, byproducts of protein metabolism, such as BCFA, are unaffected by excess amino acid substrates. Data are presented as mean ± SEM. *p<0.05, by Fisher's least significant difference LSD (α=5%).

[0090] Figure 9 : Shows the results using the improved Prodigest An overview of an in vitro / ex vivo experimental setup for assessing the effects of novel nutrients / co-products on the CKD microbiome and fecal microbiota from patients with chronic kidney disease (CKD).

[0091] Specifically, it consists of an improved system composed of the UpperGIT vessels, which act as the stomach and small intestine, and the colonic vessels, which represent the transverse colon. A schematic diagram of the system sample is shown in Figure 9 (A) In this embodiment, three conditions consisting of two treatment groups and one control group were run in parallel for each donor. A total of eight chronic kidney disease (CKD) donors were used in this embodiment.

[0092] The sample experimental timeline and sample analysis were depicted in Figure 9(B) In this study, on day 0, fecal microbiota from CKD donors were inoculated into a ProDigest SHIME system. From day 1 to day 10, the system was fed a basic nutritional diet (containing minimal amino acids, mimicking the amino acid content of a low-protein diet) with or without nutrient / symbiotic blend intervention. In this example, two nutrient / symbiotic blend combinations (P1 and P2) were tested. The P1 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- to medium-chain triglycerides containing butyrate and caprylate. The P2 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short- to medium-chain triglycerides containing butyrate and caprylate. During the last two days (days 8 to 10), the system was further challenged with an additional amino acid (AA) mixture containing L-tryptophan, L-tyrosine, L-carnitine, choline, and L-phenylalanine. An amino acid mixture was chosen because these are substrates that are converted by gut bacteria into uremic toxins and their precursors. Furthermore, an amino acid concentration was formulated to mimic the daily amino acid intake required by an adult. Samples were taken at different time points to measure overall fermentation activity, microbial community activity, and microbiome composition.

[0093] The readings are as follows:

[0094] (1) Overall fermentation activity: acid / base consumption

[0095] (2) Microbial community activity:

[0096] ●Lactate

[0097] ●Short-chain fatty acids (SCFA): butyric acid, propionic acid, acetic acid

[0098] ● Markers of protein hydrolysis activity: ammonium and branched SCFAs (isobutyric acid, isovaleric acid, and isohexanoic acid)

[0099] ●Uremic toxins and precursors: p-cresol, p-toluene sulfate, indole, indole-3-3-acetic acid, betaine, trimethylamine, trimethylamine-N-oxide, indolephenol, indolephenol sulfate, semialdehyde glutaric acid, uric acid, urea

[0100] (3) Microbial community composition: quantitative deep-shot sequencing

[0101] Figure 10This study demonstrates an example of the effect of novel nutrients or symbiotic blends on the production of microbiome-derived uremic toxin precursors from the fecal microbiota of patients with chronic kidney disease (CKD) in vitro / in vitro. As shown, interventions labeled P1 and P2 significantly reduced the production of clinically relevant uremic toxin precursors in the CKD microbiome compared to untreated controls. Indole (A), p-cresol (B), and trimethylamine (C) are byproducts of amino acid metabolism in the gut microbiome; therefore, it is concluded that higher levels are produced under conditions where additional amino acid substrates are available. Notably, the interventions were effective under conditions with normal and minimal amino acid levels, thus indicating a broad potential benefit for CKD patients with varying dietary requirements, such as low- to high-protein diets. Overall, these data highlight the benefits of the present invention in correcting amino acid metabolism abnormalities in the CKD patient microbiome, leading to a reduction in uremic toxin accumulation and associated clinical consequences. The P1 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate. The P2 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea galactooligosaccharides, and short- and medium-chain triglycerides containing butyrate and caprylate. The control group represented the untreated CKD microbiota. Data are presented as mean ± SEM. *p < 0.05, according to Student's t-test.

[0102] Figure 11 This study illustrates an example of the effect of novel nutrients or symbiotic blends on the production of microbiota-derived uremic toxins from fecal microbiota of patients with chronic kidney disease (CKD) in vitro / in vitro. As shown, interventions labeled P1 and P2 significantly reduced the increase in urea production in the CKD microbiota compared to the untreated control. Urea is a byproduct of protein metabolism; therefore, it follows that supplementation with additional amino acids has little effect on urea levels. Overall, these data highlight the benefits of this invention in helping to improve uremic toxin accumulation and associated clinical consequences, particularly in patients with CKD. The P1 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate, while the P2 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea galactooligosaccharides, and short- and medium-chain triglycerides containing butyrate and caprylate. The control represents the untreated CKD microbiota. Data are expressed as mean ± SEM. *p<0.05, according to Student's t-test.

[0103] Figure 12This study demonstrates the effects of novel nutrients or symbiotic blends, in vitro / ex vivo, on protein metabolism abnormalities in the CKD microbiota from patients with chronic kidney disease (CKD). As shown, the CKD microbiota treated with P1 and P2 interventions exhibited significantly lower levels of branched-chain fatty acids (A) and ammonium (B), indicating an improvement in overactive proteolytic activity, which leads to higher uremic toxin accumulation. The proteolytic activity of the gut microbiota was not significantly affected by the supplementation of additional amino acids. Overall, these data highlight the benefits of this invention in correcting dysbiosis in the CKD microbiota and thus helping to improve uremic toxin accumulation. The P1 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate, while the P2 intervention consisted of treating the CKD microbiota with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea galactooligosaccharides, and short- and medium-chain triglycerides containing butyrate and caprylate. The control group represents the untreated CKD microbiome. Data are presented as mean ± SEM. *p<0.05, according to Student's t-test.

[0104] Figure 13This study demonstrates the effects of novel nutrients or symbiotic blends on glycolytic activity and the production of beneficial microbiome-derived metabolites from the fecal microbiota of patients with chronic kidney disease (CKD) in vitro / in vitro, particularly on the production of short-chain fatty acids (SCFAs). The gut microbiota can influence host metabolic health through microbial metabolites. The balance between microbial metabolite production via glycolysis and proteolytic fermentation may be a crucial determinant of metabolic health. Indeed, dysbiosis in CKD patients has been characterized as being dominated by certain bacterial groups with a high ratio of proteolytic to glycolytic activity. The most well-studied glycolytic microbial metabolites are the short-chain fatty acids (SCFAs) acetic acid (A), propionic acid (B), and butyric acid (C). SCFAs have shown a variety of host benefits, including but not limited to improved intestinal epithelial barrier function and inflammation. Increased intestinal permeability has been shown to lead to increased availability of gut-derived uremic toxins and precursors in systemic circulation. Subsequently, increased metabolite production (which can help improve the integrity of the intestinal lining) can indirectly help prevent the accumulation of excess uremic toxins in systemic circulation, especially in CKD patients. As shown in the figure, compared with the untreated control, both P1 and P2 interventions exhibited significantly higher SCFA levels, demonstrating the ability of this invention to correct dysbiosis in the CKD microbiome, leading to potential metabolic health benefits. The P1 intervention consisted of treating the CKD microbiome with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short-to-medium chain triglycerides containing butyrate and caprylate, while the P2 intervention consisted of treating the CKD microbiome with a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea galactooligosaccharides, and short-to-medium chain triglycerides containing butyrate and caprylate. The control group represents the untreated CKD microbiome. Data are presented as mean ± SEM. *p < 0.05, according to Student's t-test.

[0105] Figure 14This section provides an overview of the animal experimental setup used to assess the effects of novel nutrients or symbiotic components on uremic toxin production and the progression of chronic kidney disease (CKD). The animal model used was a 5 / 6 nephrectomy model, which is one of the gold standards and most commonly used rodent models in CKD research, including renal drug studies. Kidneys were ablated during a two-step surgical procedure to reduce renal function (reminiscent of stage 3b and above human CKD). Additional animals underwent sham surgery and served as non-CKD animal controls. From week 3 to week 10, CKD animals were fed diets with or without the nutrient or symbiotic blend intervention. In this example, two combinations of nutrients or symbiotic blends (P1 and P2) were tested. The P1 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate. The P2 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short- and medium-chain triglycerides containing butyrate and caprylate. Metabolic parameters were evaluated starting at week 8, including glucose tolerance testing. At week 9, mice were placed in metabolic cages to collect urine for uremic toxin and renal parameter analysis. At week 10, mice were sacrificed and various tissues were collected for subsequent analysis. Tissues collected included: plasma and serum, urine, liver and heart, eWAT, scWAT, gastrocnemius muscle, soleus muscle, kidney, all intestinal segments, and cecum. Physiological and behavioral changes, as well as food and water intake, were monitored throughout the study.

[0106] Endpoint Analysis

[0107] • Blood and urine concentrations of uremic toxins (e.g., urea, indophenol sulfate, p-toluene sulfate, TMAO, uric acid, CMPF, IAA, PCG)

[0108] • Renal function assessment - blood and urine renal markers (e.g., proteinuria, creatinine, albumin, cysteine ​​protease inhibitor C) and renal histopathological analysis

[0109] • Metabolic inflammatory markers (blood and tissue cytokines IL6, TNFα, IL1β); blood CRP; fecal calprotectin and albumin)

[0110] • Intestinal function (epithelial function and blood osmotic markers, such as citrulline; intestinal histology analysis; lipid / fat fecal levels)

[0111] • Improvement of comorbid CKD (glucose regulation evaluated during oral glucose tolerance testing, fasting glucose, and insulin administration)

[0112] • Improvement in other metabolic parameters (plasma triglycerides, AST, ALT, cholesterol, LDL, or HDL)

[0113] • Improvement of comorbid CKD (collecting fat, liver, heart, intestines, and muscle for protein or RNA analysis)

[0114] • Cecal microbiome analysis - composition and function (metagenomics)

[0115] • Non-targeted metabolomics analysis of feces, urine and serum

[0116] Figure 15 This study demonstrates the effect of novel nutrients or symbiotic blends on clinically relevant plasma uremic toxin levels in animal models of chronic kidney disease (CKD). As shown, animals that underwent nephrectomy (CKD group, representing animals with CKD, reminiscent of stage 3b and above in humans) exhibited higher uremic toxin production compared to non-CKD control animals (sham-operated group, associating with healthy individuals without CKD). Group P1 consisted of intervention-treated CKD animals with a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate. Group P2 consisted of intervention-treated CKD animals with a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short- and medium-chain triglycerides containing butyrate and caprylate. Compared with untreated CKD animals, CKD animals treated with P1 and P2 interventions for 7 weeks showed significantly lower levels of uremic toxins, such as p-toluene sulfate (PCS) (A), indophenol sulfate (IS) (B), p-toluylglucuronide (PCG) (C), indoleacetic acid (IAA) (D), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) (E), and uric acid (F). The data clearly demonstrate the benefit of intervention in reducing the accumulation of uremic toxins in the systemic circulation, particularly in individuals with impaired renal function. Each data point represents one animal. Lines represent the mean. Analysis of variance was used, followed by multiple comparisons using uncorrected Fisher LSD. *p<0.05, **p<0.01, ***p<0.001.

[0117] Figure 16This demonstrates the effects of novel nutrients or symbiotic blends on renal parameters / renal function markers in animal models of chronic kidney disease (CKD). The sham-operated group represents non-CKD animals, reminiscent of healthy individuals without CKD. The CKD group represents animals with CKD, reminiscent of human CKD stage 3b and above. Group P1 consisted of CKD animals treated with an intervention consisting of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate. Group P2 consisted of CKD animals treated with an intervention consisting of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short- and medium-chain triglycerides containing butyrate and caprylate. As shown, animals undergoing nephrectomy (CKD group) exhibited impaired renal function compared to non-CKD control animals (sham-operated group, reminiscent of healthy individuals without CKD disease), as evidenced by increased (A) proteinuria (demonstrated by a higher protein-to-creatinine ratio in urine) and (B) increased urea concentrations in plasma. CKD animals treated with P1 and P2 interventions for 7 weeks showed significantly better proteinuria and plasma urea levels compared to untreated CKD animals. The data support the benefits of this invention in stabilizing renal function and can therefore be used to help prevent the vicious cycle of uremic toxin accumulation and slow the progression of renal damage and related clinical outcomes. For proteinuria, each data point represents 2 animals, while for urea levels, each data point represents 1 animal. Lines represent means. Analysis of variance was used, followed by multiple comparisons using uncorrected Fisher LSD. *p<0.05, **p<0.01, ***p<0.001.

[0118] Figure 17This study demonstrates the effects of novel nutrients or symbiotic blends on renal histology in animal models of chronic kidney disease (CKD). The sham-operated group represents non-CKD animals, reminiscent of healthy individuals without CKD. The CKD group represents animals with CKD, reminiscent of human CKD stage 3b and above. Group P1 consisted of CKD animals treated with an intervention consisting of a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, and short-to-medium chain triglycerides containing butyrate and caprylate. Group P2 consisted of CKD animals treated with an intervention consisting of a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short-to-medium chain triglycerides containing butyrate and caprylate. As shown, the residual kidneys of animals that underwent nephrectomy (CKD group) showed microscopic damage compared to non-CKD control animals (sham-operated group, reminiscent of healthy individuals without CKD). This damage was characterized by increased fibrosis (A) and impaired glomerular size (B) and volume (C). CKD animals treated with P1 and P2 interventions for 7 weeks showed reduced fibrosis and decreased glomerular size and volume. The data support the benefits of this invention in reducing renal microscopic damage and can therefore be used to help prevent the vicious cycle of uremic toxin accumulation and slow the progression of kidney damage and related clinical outcomes. Each data point represents one animal. Analysis of variance was used, followed by multiple comparisons using uncorrected Fisher LSD. *p<0.05, **p<0.01, ***p<0.001. HES: Hematoxylin and eosin staining.

[0119] Figure 18The effects of a novel nutrient or symbiotic blend on (A) body weight evolution, (B) food intake, and energy reserve depletion / protein energy expenditure in an animal model of chronic kidney disease (CKD). Some detrimental consequences of the accumulation of uremic toxins in nephropathy are loss of appetite and loss of energy reserves (including muscle and fat reserves). As shown in the figure, animals undergoing nephrectomy (CKD group, representing animals with CKD, reminiscent of stage 3b and above in humans) showed slower weight gain after surgery compared to the non-CKD control group (sham-operated group, associating with healthy individuals without CKD). Furthermore, CKD animals showed significantly lower food intake from week 2 through week 10 compared to the sham-operated group. At the end of the study, (C) CKD animals further showed a significant reduction in epididymal white adipose tissue (eWAT), indicating reduced energy reserves, reminiscent of what is seen in human CKD. CKD animals treated with P1 and P2 interventions showed significantly improved body weight evolution and food intake normalized to the same level as the non-CKD sham-operated animals. Group P1 consisted of CKD animals treated with an intervention consisting of a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, and short-to-medium chain triglycerides containing butyrate and caprylate. Group P2 consisted of CKD animals treated with an intervention consisting of a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short-to-medium chain triglycerides containing butyrate and caprylate. Furthermore, compared to untreated CKD animals, the treated animals, particularly those treated with P1, showed better fat reserves, as observed through improved epididymal white adipose tissue (eWAT). The data support the potential benefit of the intervention in alleviating uremia-related symptoms, including anorexia leading to weight loss and protein-energy depletion. For the body weight plot, the line represents the mean for all animals in each group, and SEM is used as error bars. For food intake, each data point represents one animal. Analysis of variance was used, followed by multiple comparisons using uncorrected Fisher LSD. *p<0.05, **p<0.01, ***p<0.001.

[0120] Figure 19This study demonstrates the effects of neonutrients or symbiotic blends on intestinal barrier dysfunction in animal models of chronic kidney disease (CKD). As shown, animals that underwent nephrectomy (CKD group, representing animals with CKD, reminiscent of stage 3b and above in humans) exhibited impaired intestinal barrier function compared to non-CKD control animals (sham-operated group, associating with healthy individuals without CKD). This was evidenced by lower protein expression at the tight junctions that close in the ileum. Tight junctions are specialized junctions between two adjacent cell membranes and, in the case of the intestinal lining, are important structures preventing the excessive translocation of intestinal-derived molecules, such as uremic toxin precursors, into the systemic circulation. CKD animals treated with P1 and P2 interventions for 7 weeks showed significantly higher protein expression of the closure protein compared to untreated CKD animals. The P1 group consisted of interventional CKD animals treated with a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, and short- to medium-chain triglycerides containing butyrate and caprylate. Group P2 consisted of CKD animals treated with an intervention consisting of a blend of components containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short- and medium-chain triglycerides containing butyrate and caprylate. Data support the benefits of this invention in improving intestinal dysfunction, particularly in those with renal impairment. Each data point represents one animal. Lines represent the mean. Analysis of variance was used, followed by multiple comparisons using uncorrected Fisher LSD. *p<0.05, **p<0.01, ***p<0.001.

[0121] Figure 20 The image shows the effects of neonutrients or symbiotic blends on (A) urinary albumin, (B) urinary creatine, (C) urinary protein (albumin) to creatine ratio, and (D) LOG urinary protein (albumin) to creatine ratio at week 7 in a rat model of CKD. From left to right: sham surgery, 5 / 6 mediator, 5 / 6 Nx P1-rat intervention (P1-rat intervention consists of 10 Nx P1-rat mixtures containing 10 Nx P1-rat mixtures). 8 Lactobacillus johnsonii NCC533, 1% cellobiose, 1% blend of short- and medium-chain triglycerides containing butyrate and caprylate), 5 / 6Nx P3 intervention (intervention consisting of 10 8The formula consisted of *Lactobacillus johnsonii* NCC533, 0.3% cellobiose, 0.3% a blend of short- and medium-chain triglycerides containing butyrate and caprylate, and 20 mg / kg 5 / 6Nx lisinopril. (A) Values ​​are expressed as the mean of n = 12–18 + SEM. Dunnett test for one-way linear model. **: P < 0.01 compared to the 5 / 6Nx medium, and (B) Values ​​are expressed as the mean of n = 12–18 + SEM. Dunnett test for one-way linear model. **: P < 0.01, ***: P < 0.001 compared to the 5 / 6Nx medium. When compared to the 5 / 6Nx medium, sham surgery had a reduced urinary albumin level at week 7. When compared to the 5 / 6Nx medium, sham surgery had a reduced urinary albumin level at week 7. (C) Values ​​are expressed as the mean of n = 12–18 + SEM. Dunnett test for one-way linear model. *: P < 0.05, **: P < 0.01, compared to 5 / 6Nx mediator. (D) values ​​are expressed as the mean of n = 12–18 + SEM. Dunnett test for one-way linear model. *: P < 0.05, ***: P < 0.001, compared to 5 / 6Nx mediator. When compared to 5 / 6Nx mediator, sham surgery and 5 / 6Nx P1 had a reduced urinary ACR at week 7. When compared to 5 / 6Nx mediator, sham surgery and 20 mg / kg 5 / 6Nx lisinopril had a reduced LOG urinary ACR at week 7.

[0122] Figure 21 The image shows 5 / 6Nx rats in (from left to right) the sham-operated group, the 5 / 6 vector group, and the 5 / 6Nx P3 intervention group (intervention consisting of 10 Nx P3). 8 The study consisted of *Lactobacillus johnsonii* NCC533, 0.3% cellobiose, and a blend of 0.3% short- and medium-chain triglycerides containing butyrate and caprylate. The 5 / 6Nx P1 rat intervention group (in...) Figure 21 (A) to Figure 21 (C) indicates “5 / 6Nx P1” (P1- rat intervention consists of 10 8 The study included plasma levels of (A) indophenol sulfate (IS), (B) p-toluene sulfate (PCS), and (C) p-cresol glucuronide (PCG) in Lactobacillus johnsonii NCC533, a blend of 1% cellobiose, 1% short- and medium-chain triglycerides containing butyrate and caprylate, and 20 mg / kg 5 / 6Nx lisinopril. 5 / 6Nx P1- rat intervention reduced plasma levels of IS and pCS (IS was reduced by approximately 36% and pCS by approximately 77% compared to the 5 / 6Nx mediator group). Detailed Implementation

[0123] definition

[0124] Some definitions provided below apply to the described specification and invention. However, definitions may be found in the "Background Art," "Summary of the Invention," "Embodiments," and "Definitions" sections below.

[0125] All percentages expressed herein are by weight of the total weight of the composition, unless otherwise stated.

[0126] As used herein, “about,” “approximately,” and “substantially” should be understood to refer to a number within a certain numerical range, such as -10% to +10% of the mentioned number, preferably -5% to +5% of the mentioned number, more preferably -1% to +1% of the mentioned number, and most preferably -0.1% to +0.1% of the mentioned number.

[0127] All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be understood to support claims that involve any number or subset of numbers within that range. For example, disclosures of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0128] As used in this disclosure and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, references to “a component” or “the component” include two or more components.

[0129] The term "including / comprises" will be interpreted as inclusive rather than exclusive. Similarly, the terms "including / comprises" and "or" should be considered inclusive unless the context explicitly prohibits this interpretation. However, the compositions disclosed herein may not contain any elements not specifically disclosed herein. Therefore, the disclosure of embodiments using the term "including / comprises" includes both embodiments "consisting substantially of the specified components" and embodiments "consisting of the specified components." A composition "consisting substantially of..." contains at least 50% by weight of the reference component, preferably at least 75% by weight, more preferably at least 85% by weight, and most preferably at least 95% by weight of the reference component.

[0130] The term “and / or” used in the context of “X and / or Y” should be interpreted as “X” or “Y” or “X and Y”. Similarly, “at least one of X or Y” should be interpreted as “X” or “Y” or “X and Y”.

[0131] As used herein, the terms “example” and “such as” (especially when followed by a list of terms) are exemplary and illustrative only and should not be considered exclusive or comprehensive. As used herein, “associated” or “related” to one condition means that the conditions occur simultaneously, preferably means that the conditions are caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by another identified condition.

[0132] The terms “food,” “food product,” and “food composition” mean a product or composition intended for ingestion by an individual (such as a human) and to provide that individual with at least one nutrient. Food products typically contain at least one of a protein, lipid, and carbohydrate, and optionally contain one or more vitamins and minerals. Compositions of this disclosure (including the various embodiments described herein) may comprise, consist of, or substantially consist of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or intended for use in a diet.

[0133] "Prevention" includes reducing the risk and / or severity of a condition or disorder. The terms "treatment," "treat," and "relief" include both prophylactic or preventive treatment (preventing and / or slowing the development of a target pathological condition or disorder) and curative, therapeutic, or disease-modifying treatment, including therapeutic measures that cure, slow, or alleviate the symptoms of a diagnosed pathological condition or disorder and / or halt its progression; and the treatment of patients at risk of or suspected of having the disease, as well as the treatment of patients who are ill or have been diagnosed with a disease or medical condition. This term does not necessarily mean that an individual is treated until fully recovered. The term "treatment" also refers to the maintenance and / or promotion of health in individuals who do not have the disease but may be prone to developing unhealthy conditions. The terms "treatment" and "relief" are also intended to include intensifying or otherwise enhancing one or more major preventive or therapeutic measures. The terms "treatment" and "relief" are also intended to include dietary management of a disease or condition or dietary management for the prevention or control of a disease or condition. Treatment may be patient-related or physician-related.

[0134] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable for use as a unit dose in human and animal subjects, each unit containing a predetermined amount of the composition disclosed herein, associated with a pharmaceutically acceptable diluent, carrier, or mediator, sufficient to produce the desired effect. The specifications of a unit dosage form depend on the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0135] "Subject" or "individual" is a mammal, preferably a human. In the case of humans, the term "old age" means being at least 60 years old from birth, preferably 63 years or older, more preferably 65 years or older, and most preferably 70 years or older. In the context of humans, the term "middle-aged or older person" means being at least 45 years old from birth, preferably 50 years or older, more preferably 55 years or older, and includes elderly individuals.

[0136] As used herein, "effective amount" is the amount in an individual that prevents defects, treats diseases or medical conditions, or more generally, the amount that reduces symptoms, manages disease progression, or provides nutritional, physiological, or medical benefits to an individual. The relative terms "improving," "enhancing," "strengthening," etc., refer to the effects of the compositions disclosed herein, i.e., compositions comprising at least one probiotic, at least one prebiotic, and at least one lipid, all of which are as defined herein. As used herein, "enhancing" means an enhancement or induction relative to levels prior to application of the compositions disclosed herein.

[0137] The term "probiotics" refers to viable bacteria (live bacteria) that, when consumed, generally provide health benefits by improving or restoring the gut microbiota. "Probiotics" are preferably present in an effective amount in the composition. The manufacturing process for probiotics is generally standardized and involves the step of fermenting bacteria in a growth medium containing a carbohydrate source, such as sugars, e.g., glucose, fructose, sucrose, lactose, or dextrose. After fermentation, the probiotics are typically cryoprotected and freeze-dried or lyophilized and packaged into the final product for use in the composition. The "probiotics" used according to the invention are specifically selected in view of the requirement to reduce uremic toxins and preferably allow for the avoidance or reduction of the accumulation of such uremic toxins in the circulation, tissues, and organs, as described above.

[0138] The term "prebiotic" should generally be understood to refer to a non-digestible fibrous compound that passes undigested through the upper gastrointestinal tract and stimulates the growth or activity of beneficial bacteria in the colon by acting as its substrate. The prebiotics of this invention are specifically selected to allow stimulation of the growth or activity of "probiotics" as used according to the invention. The "prebiotic" is preferably present in an effective amount in the composition. The manufacturing process for probiotics is generally standardized and well-known to those skilled in the art.

[0139] The term “average nucleotide identity (ANI)” is a measure of nucleotide-level genomic similarity between coding regions of two genomes. ANI can be readily determined by those skilled in the art using common knowledge and available tools described in detail in the literature. For example, ANI can be assessed as described here: Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie VanLeeuwenhoek. Oct 2017; 110(10):1281-1286.

[0140] As used in this article, the term “cardiometabolic disorder” refers to any condition that involves a range of conditions that are related to or share risk factors such as overweight and obesity, dyslipidemia, and hypertension.

[0141] As used herein, the term "neurodegenerative disease" refers to any condition involving the progressive loss of functional neurons in the central nervous system. In one embodiment, the neurodegenerative disease is associated with age-related cell death. Non-limiting examples of such neurodegenerative diseases particularly include cardiovascular-metabolic or neurodegenerative diseases, preferably involving the treatment or prevention of kidney disease, including chronic and acute; dialysis and predialysis; rare (kidney) diseases, genetically induced (kidney) diseases, and metabolically induced (kidney) diseases; treatment or prevention of uremic syndromes, including protein-energy depletion, bone loss, anorexia, fatigue, or inflammation; delaying complications of advanced kidney disease, including anemia, hyperphosphatemia, secondary hyperparathyroidism, bone disease, and neurological disorders; delaying comorbidities of kidney disease, including cardiovascular disease; preventing or managing the risk of malnutrition; delaying the progression of cardiovascular-metabolic diseases; preventing or managing the risk of cardiovascular disease and comorbidities (diabetes); and / or preventing or managing the risk of neurodegenerative and neurological disorders.

[0142] In addition, neurodegenerative diseases may include or be related to the following conditions: Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS and Lou Gehrig's disease), AIDS dementia syndrome, adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia-telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobe degeneration, Kennedy's disease, Krabbe disease, and Lyme disease. Diseases including Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacute mixed degeneration of the spinal cord, tabes dorsalis, familial Tay-Sachs disease, toxic encephalopathy, infectious spongiform encephalopathy, and wobbly hedgehog syndrome.

[0143] “Sarcopenia” is defined as an age-related loss of muscle mass and function (including muscle strength and walking speed). As used in this article, “frailty” is defined as a clinically identifiable state of increased vulnerability due to age-related decline in reserves and function across multiple physiological systems, impairing the ability to cope with daily or acute stress. In the absence of a defined quantitative standard, Fried et al. operationally defined frailty as meeting three of five phenotypic criteria that indicate impaired energy: (1) weakness (grip strength of the lowest 20% of the baseline population, adjusted for sex and body mass index), (2) poor endurance and energy (self-reported exertion and energy expenditure). (3) slow (lowest 20% of the baseline group, based on time to walk 15 feet, adjusted for sex and standing height), (4) low physical activity (weighted fraction of kcal per week at baseline, lowest quintile of physical activity determined for each sex; e.g., less than 383 kcal / week for men and less than 270 kcal / week for women) and / or unintentional weight loss (less than 10 lbs in the past year). Fried LP, Tangen CM, Walston J, et al., “Frailty in older adults: evidence for a phenotype.” J. Gerontol. Biol. Sci. Med. Sci. 56(3): M146–M156 (2001). The presence of one or two of these criteria in the pre-frailty stage identifies a high risk of progression to frailty.

[0144] Cachexia is a severe wasting disease characterized by significant weight loss, anorexia, weakness, and anemia. Cachexia is a common feature of many diseases such as cancer, sepsis, chronic heart failure, rheumatoid arthritis, and acquired immunodeficiency syndrome (AIDS).

[0145] "Overweight" is defined as a person's Body Mass Index (BMI) being between 25 kg / m². 2 With 30kg / m 2 Between. "Obesity" is defined as a BMI of at least 30 kg / m². 2 For example, 30kg / m 2 Up to 39.9 kg / m 2 "Weight loss" is a reduction in total body weight. For example, weight loss can refer to a reduction in total body mass achieved to improve one or more of health, tonedness, and / or physique.

[0146] "Diabetes" encompasses both type 1 and type 2 forms of the disease. Non-limiting examples of risk factors for diabetes include: a waist circumference greater than 40 inches for men or greater than 35 inches for women; blood pressure of 130 / 85 mmHg or higher; triglycerides greater than 150 mg / dL; fasting blood glucose greater than 100 mg / dL; or high-density lipoprotein (HDL) less than 40 mg / dL for men or less than 50 mg / dL for women.

[0147] As used in this article, the term "metabolic syndrome" refers to a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. It affects one in five people in the United States, and its prevalence increases with age. Some studies suggest a prevalence of up to 25% of the population in the United States. According to the International Diabetes Foundation consensus worldwide definition (2006), metabolic syndrome is central obesity plus any two of the following:

[0148] Elevated triglycerides: >150 mg / dL (1.7 mmol / L), or specific treatment for this lipid abnormality;

[0149] Lowered HDL cholesterol: <40 mg / dL (1.03 mmol / L) in men and <50 mg / dL (1.29 mmol / L) in women, or specific treatment for this lipid abnormality;

[0150] Elevated blood pressure: systolic blood pressure >130 mmHg or diastolic blood pressure >85 mmHg, or treatment for a previously diagnosed hypertension; and

[0151] Elevated fasting blood glucose (FPG) >100 mg / dL (5.6 mmol / L), or a previously diagnosed type 2 diabetes.

[0152] Implementation Plan

[0153] According to a first embodiment, this disclosure provides a composition preferably suitable for reducing and / or preventing the accumulation of uremic toxins, preferably in cardiovascular metabolic or neurodegenerative diseases, the composition comprising a prebiotic, a probiotic and lipids as specifically selected as defined herein.

[0154] The composition provides particular health benefits to patients to be treated. These patients are typically those with cardiovascular metabolic or neurodegenerative conditions and are generally experiencing or at least at risk of experiencing an increase and accumulation of uremic toxins. The health benefits that can be advantageously provided to such patients by administering the composition are preferably a delay in the progression of the disease and comorbidities, and, in addition, the possibility of managing symptoms and syndromes associated with the toxic effects of such uremic solutes, both typically by reducing the amount of uremic toxins in such patients, which would otherwise accumulate in the patient.

[0155] Uremic toxins in the context of this invention are generally selected from, but are not limited to, urea, trimethylamine (TMA), triethylamine oxide (TMAO), indophenol and indophenol sulfate, p-cresol and p-cresol sulfate, p-tolyl glucuronic anhydride, uric acid, 3-carboxy-4-methyl-5-propyl-2-furan propionate (CMPF), and all their metabolites or precursors (see, for example...). Figure 1 The primary sources of these uremic toxins are the provision of patients with excessive amounts of amino acids (such as tryptophan or tyrosine), increased levels of L-carnitine or choline, and increased levels of urea. The accumulation of these systemic uremic toxins is a condition commonly observed in individuals with cardiovascular, metabolic, and neurological disorders, including but not limited to chronic kidney disease (CKD) and the diseases and conditions described above. The accumulation of these systemic uremic toxins also puts pressure on the treatment of these diseases, particularly in cases requiring a high-protein diet, as further protein intake, such as in wasting disorders (such as cachexia or sarcopenia) and various neurological conditions, would be quite detrimental due to the addition of additional protein, and especially additional tryptophan and tyrosine.

[0156] In a first embodiment, a composition representing a novel nutrient blend and / or symbiotic blend is provided, which allows targeting of multiple direct and indirect mechanisms that can contribute to a beneficial reduction in the production and / or accumulation of such uremic toxins, particularly through the application of a composition preferably representing a specific blend consisting of:

[0157] • Probiotics selected as defined herein (e.g., Lactobacillus johnsonii NCC 533).

[0158] • Prebiotics selected as defined herein, preferably fiber and oligosaccharides (e.g., cellobiose and pea GOS) as defined herein.

[0159] • Lipids selected as defined herein, preferably triglycerides composed of short-chain and medium-chain fatty acids as defined herein;

[0160] For example, compositions in the form of nutrient blends and / or symbiotic blends actively target and improve the microbiome (function), gut function, liver metabolism, and provide mechanisms for stabilizing kidney function and helping to reduce the systemic presence and amount of uremic toxins.

[0161] Preferably, the microbiome (function) can be improved as follows: the microbiome typically leads to the production of certain metabolites that form precursors of uremic toxins. These metabolites can be converted into uremic toxins when they are available in body tissues, particularly the liver and circulation. Importantly, patients with kidney disease have been shown to have dysbiosis characterized by impaired microbiome function that converts certain amino acids from dietary proteins into uremic toxin precursor metabolites. Therefore, the application of the composition improves the excessive availability of uremic toxin precursors in circulation by improving the dysbiosis, particularly the microbiome function. In other words, the microbiome imbalance is improved, thereby also allowing for improved metabolic processing of such uremic toxin precursor metabolites.

[0162] Furthermore, the composition advantageously allows for the enhancement or improvement of intestinal function. In this context, it is important to recognize that the microbiota from both healthy and diseased individuals continuously and normally produce uremic toxin precursors without leading to significant disease outcomes. However, patients with cardiovascular metabolic diseases, particularly kidney disease, may have impaired intestinal function, including impaired intestinal motility, intestinal epithelial barrier function, and increased inflammation, which can lead to dysregulation of intestinal accumulation and increased permeability of uremic toxin precursors across the intestinal barrier. The composition thereby improves the availability of uremic toxin precursors in systemic circulation by enhancing intestinal motility and epithelial barrier function, thus significantly contributing to the prevention of the accumulation of such uremic toxins in tissues and organs, particularly the kidneys.

[0163] The composition also provides significant benefits to liver metabolism. The liver is the central organ responsible for the production of uremic toxins. Liver enzymes utilize substrates from endogenous or other tissues, diet, and the gut microbiota to produce these uremic toxins. In this particular case, the composition allows for improved metabolism via specific preselected probiotics, along with prebiotics and lipids to be administered, leading to the conversion of uremic toxins, especially when substrate availability is excessive. This, in turn, improves the accumulation of circulating uremic toxins by enhancing liver metabolism, thereby effectively allowing the avoidance of the undesirable accumulation of these uremic toxins in the circulation and liver.

[0164] The composition also provides significant benefits for stabilizing kidney function. Under normal healthy conditions, the kidneys are able to filter out uremic toxins, thereby reducing or preventing their accumulation in the bloodstream. However, in cases where proper kidney filtration is impaired, uremic toxins can accumulate and trigger or contribute to a vicious cycle of disease progression, including further inducing dysbiosis or liver or intestinal damage. The composition thus stabilizes kidney function and prevents further decline. Therefore, the composition directly improves uremic toxin accumulation and indirectly improves uremic toxin production.

[0165] Therefore, the present invention is based on a novel combination of specifically selected probiotics, specifically selected prebiotics and specifically selected lipids, which symbiotically allow for improvement of the levels of uremic toxins (e.g., indophenol sulfate, p-toluene sulfate, PCG, CMPF, uric acid) and avoidance of their accumulation in various cardiovascular metabolic disorders (e.g., kidney disease) and neurodegenerative diseases.

[0166] In the context of this invention, this avoidance of uremic toxin accumulation specifically relates to compounds such as urea, uric acid, p-cresol, p-toluene sulfate, indole sulfate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, PCG, CMPF, glutaric acid, and / or other protein-bound uremic toxins, preferably indole sulfate, p-toluene sulfate, MCPF, PCG, urea, and / or uric acid. The measurement of such compounds in tissues, organs, and body fluids is well known to those skilled in the art.

[0167] In this context, the inventors have surprisingly discovered that compositions comprising all the ingredients required herein (i.e., specifically selected prebiotics, specifically selected prebiotics, and specifically selected lipids) not only allow for addressing isolated aspects, such as dysbiosis or improvement of microbiome function, but also represent a holistic approach to mitigating multiple targets known to lead to the production and accumulation of uremic toxins, particularly improving the microbiome (function), gut function, and liver metabolism, and providing mechanisms for stabilizing renal function and helping to reduce the systemic presence and amount of uremic toxins, preferably in cardiovascular metabolic or neurodegenerative diseases as defined herein.

[0168] The composition contains probiotics as defined above. Probiotics are considered to be live bacteria that beneficially contribute to gut and microbiome function, as defined herein. The probiotics in the composition have a significant effect on this treatment and are selected according to the following three criteria, which may be applied alone or in combination, preferably in combination.

[0169] According to the first standard, such probiotics are selected from at least one of those probiotics that lack at least one bacterial enzyme that produces urea, uric acid, p-cresol, p-toluene sulfate, indolesulfonate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, and / or glutaric acid.

[0170] The deficiency of at least one bacterial enzyme, as described above, is typically achieved by the absence or complete absence, or at least inactivation, of the corresponding portion of the genetic coding sequence of the enzyme in the probiotic genome, resulting in a lack of expression of the enzyme in vivo. If multiple copies of the bacterial enzyme are encoded by the genomic sequence, then only part or all, preferably all, of the coding sequence of the bacterial enzyme is absent or at least inactive.

[0171] Avoiding the expression of such enzymes that produce uremic toxins allows for the reduction of potentially excessive increases and loads of uremic toxins in the body, tissues, and organs, which could lead to irreversible accumulation of such toxins. Although the selected probiotics are bacteria generally considered beneficial in the gut health system, the inventors have surprisingly recognized that uremic toxins can be efficiently reduced or at least limited by selecting specific types of probiotics, prebiotics, and lipids in a symbiotic manner. Probiotics specifically selected for the composition for certain properties, such as probiotics lacking specific bacterial enzymes that may lead to an excessive increase in uremic toxins (Alternative First and Alternative Second below) and / or contributing to improved probiotic growth by promoting the expression of specific enzymes beneficial for processing specific prebiotics (fiber and carbohydrates).

[0172] According to the second standard, preferably in addition to the first standard, such probiotics are selected from at least one probiotic lacking at least one bacterial enzyme, wherein the at least one bacterial enzyme is selected from urease, carnitine monooxygenase and reductase, tryptophanase, and hydroxyphenylacetate. Such enzymes may preferably be selected from one of the following categories of enzymes:

[0173] EC1.14.13.239, EC: 1.14.12.17, EC: 1.14.13.25, EC: 1.14.99.-, EC:

[0174] 1.3.3.4, EC: 1.3.8.2, EC: 1.3.98.1, EC: 1.4.1.13, EC: 1.4.1.14, EC:

[0175] 1.4.3.2, EC: 2.1.1.12, EC: 2.3.1.54, EC: 2.6.1.-, EC: 2.6.1.1, EC:

[0176] 2.6.1.2、EC:2.6.1.2、EC:2.6.1.4、EC:2.6.1.44、EC:2.6.1.5、EC:

[0177] 2.6.1.57、EC 2.6.1.58、EC:2.6.1.78、EC:2.6.1.79、EC:2.6.1.9、EC:

[0178] 3.2.1.172、EC:3.5.1.111、EC:3.5.1.3、EC:3.5.1.4、EC:3.5.5.1、

[0179] EC:3.6.1.3、EC:4.1.1.105、EC:4.1.1.16、EC:4.1.1.25、EC:

[0180] 4.1.1.25、EC:4.1.1.28、EC:4.1.99.1、EC:4.3.99.4、EC:4.4.1.8、

[0181] EC:5.4.99.9、EC:6.3.5.-、EC:6.3.5.1、EC:6.3.5.7、EC:

[0182] 1.13.11.63、EC:1.13.12.3、EC:1.14.13.148、EC:1.14.13.239、EC:

[0183] 1.18.1.2、EC:1.3.3.4、EC:1.4.1.20、EC:1.4.1.4、EC:1.4.3.2、EC:

[0184] 1.5.1.34、EC:1.8.1.19、EC:1.97.1、EC:2.6.1.-、EC:2.6.1.1、EC:

[0185] 2.6.1.39、EC:2.6.1.57、EC:2.6.1.78、EC:2.6.1.79、EC:2.6.1.9、

[0186] EC:3.5.1.3、EC:3.5.2.12、EC:4.1.1.15、EC:4.1.1.83、EC:4.4.1.8

[0187] As outlined with respect to the first criterion, the absence of at least one such bacterial enzyme as described above is typically achieved by the absence or complete absence, or at least inactivation, of the corresponding portion of the genetic coding sequence of the enzyme in the probiotic genome, resulting in a lack of expression of the enzyme in vivo. If multiple copies of the bacterial enzyme are encoded by a genomic sequence, then only a portion or all, preferably all, of the coding sequence of the bacterial enzyme is absent or at least inactive.

[0188] In addition, the absence of at least one bacterial enzyme, urease, carnitine monooxygenase and reductase, tryptophanase, and hydroxyphenylacetate effectively helps to prevent further accumulation of uremic toxins, which would otherwise be excessive and accumulate in the patient's body, tissues and organs to levels higher than tolerable.

[0189] As an alternative to or supplement to either of the two criteria mentioned above, preferably additionally according to the third criterion, such probiotics are preferably selected from at least one probiotic containing at least one of the following bacterial carbohydrate enzymes: α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase, and / or lichen polysaccharide enzyme. Such enzymes may be selected from one of the following categories of enzymes:

[0190] EC3.2.1.22, EC3.2.1.23, EC3.2.1.74, EC 3.2.1.4, EC 3.2.1.26, EC 3.2.1.58, EC 3.2.1.73, EC 2.4.1.9, EC 3.2.1.84, EC 3.2.1.33, EC 3.2.1.70, EC 3.2.1.135, EC3.2.1.3, EC 3.2.1.20, EC 3.2.1.10

[0191] Expressing at least one of these bacterial carbohydrate enzymes via specifically selected probiotics is particularly helpful in processing the prebiotics applied herein, which symbiotically (given the selection of probiotics and prebiotics) support the growth of the applied probiotics, improvement of the microbiome and gut function, and stability of liver metabolism and kidney function, thereby reducing and / or preventing the accumulation of uremic toxins.

[0192] The probiotics in the composition meet at least one, preferably at least two, more preferably at least three or all of the above-described standards for the probiotics of the present invention.

[0193] As previously outlined, according to any of these criteria, the probiotic preferably meeting all three criteria may be selected from any of the following species: *Bifidobacterium animalis* subsp. *lactobacter*, *Bifidobacterium longum* subsp. *infant*, *Bifidobacterium longum* subsp. *long*, *Enterococcus faecalis*, *Lactobacillus johnsonii*, *Lactococcus lactis*, *Lactobacillus paracasei* (formerly classified as *Lactobacillus paracasei*), *Lactobacillus reuteri* (formerly classified as *Lactobacillus reuteri*), *Lactobacillus rhamnosus* (formerly classified as *Lactobacillus rhamnosus*), *Staphylococcus fleshyus*, and / or *Streptococcus thermophilus*, preferably *Lactobacillus johnsonii* or having a genome sequence with at least 95% average nucleotide identity (ANI), preferably at least 96%, with respect to any of the probiotics as previously defined. A probiotic genome with ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI or even 99.5% ANI or 99.9% ANI.

[0194] Even more preferably, the probiotics that meet all three criteria can be selected from any of the following strains:

[0195] a. Bifidobacterium animalis subsp. lactic acid NCC 2818, which was deposited in CNCM on June 7, 2005, and assigned accession number CNCM I-3446;

[0196] b. Bifidobacterium longum infantile subsp. NCC 341 (ATCC 15697(T) - available from worldwideweb.atcc.org / products / 15697);

[0197] c. Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618? (NCBI refseq; GCA_000007525.1) [NCC 2075 was deposited in CNCM on January 29, 2001, and assigned accession number CNCM I-2618];

[0198] d. Enterococcus faecium NCC 2768 (NCIMB 10415, which is available from Cerbios-Pharma SA Barbengo Switzerland (cerbios.swiss / e-faecium-sf68-a-model-for-efficacy-safety-for-pharmaceutical-probiotics / );

[0199] e. Lactobacillus johnsonii NCC 533 [originally known as La 1, deposited in CNCM on June 30, 1992, and assigned accession number CNCM I-1225] (see also NCBIrefseq; GCA_000008065.1 and GenBank AE017198.1 (inclusive as SEQ ID NO:1))];

[0200] f. Lactococcus lactis NCC 2287 (CNCM I-4154) [NCC 2287 was deposited in CNCM on April 24, 2009, and assigned accession number CNCM I-4154];

[0201] g. Lactobacillus paracasei NCC 2461 (CNCM I-2116) [NCC 2461 was deposited in CNCM on January 12, 1999, and assigned accession number CNCM I-2116];

[0202] h. Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724) [NCC 4007 was deposited in October 2004 at CGMCC [China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, China] with identification number CGMCC 1.3724];

[0203] i. Staphylococcus aureus NCC 1052 (CNCM I-5400) [NCC 1052 was deposited in CNCM on February 1, 2019, and assigned accession number CNCM I-5400];

[0204] j. Staphylococcus aureus NCC 971 (CNCM I-5398) [NCC 971 was deposited in CNCM on February 1, 2019, and assigned accession number CNCM I-5398]; and / or

[0205] k. Streptococcus thermophilus NCC 2496 (CNCM I-3915) [NCC 2496 was deposited in CNCM on February 5, 2008, and assigned accession number CNCM I-3915];

[0206] Or a probiotic having a genome with at least 95% average nucleotide identity (ANI), preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%, or even 99.5% or 99.9% ANI, of the corresponding genome sequence as defined above from a to k.

[0207] Most preferably, the probiotics in the composition as defined herein are selected from *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) or probiotics having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% or 99.9% ANI, similar to the genome sequence of *Lactobacillus johnsonii* NCC 533 (CNCM I-1225). The NCBI reference sequence of *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) is GCA_000008065.1 (SEQ ID NO:1).

[0208] Typically, a “probiotic” as defined herein as an “effective amount” may include 10 3 cfu to 10 12 The amount between CFU, usually 10 4 CFU / day up to 10 11 The amount between CFU and daily dose, preferably 10. 5 CFU / day up to 10 10 The amount between CFU / daily dose or 10 5 CFU / day up to 10 9 The amount between CFU and daily dose is also preferably 10. 6 CFU / day up to 10 9 The amount between CFU / day dose, 10 6 CFU / day up to 10 8 The amount between CFU / daily dose or 10 8 CFU / day up to 10 10 The amount of CFU / day, more preferably about 10 7 CFU / day up to 10 9 This probiotic contains CFU / day. The preferred daily dose is approximately 10 CFU / day. 8 CFU / day dose, 10 7 CFU / day up to 10 9 CFU / day dose or 10 8 CFU / day up to 10 9CFU / daily dose. The daily dose can thus be achieved by administering the composition once daily or by administering the composition multiple times, such as by administering it two, three, four, or five times, preferably no more than one to five times, more preferably no more than one to four times, and even more preferably no more than one to three times. In the case of achieving the daily dose by administering the composition once or once to five times daily, the amount of each composition is recalculated based on the required daily dose. Preferably, in the case of such multiple daily administrations, any administration or corresponding composition contains the same amount of ingredients, and therefore contains the same amount of probiotics, prebiotics, and lipids, and preferably also contains the same volume.

[0209] According to the second aspect, the composition as defined herein further comprises a prebiotic as generally defined above. This prebiotic is selected from carbohydrates, preferably fiber and oligosaccharides, wherein the carbohydrates are hydrolyzable by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or licheninase, the bacterial enzyme preferably selected from…

[0210] EC 3.2.1.22, EC 3.2.1.23, EC 3.2.1.74, EC 3.2.1.4, EC 3.2.1.26, EC3.2.1.58, EC 3.2.1.73, EC 2.4.1.9, EC 3.2.1.84, EC 3.2.1.33, EC 3.2.1.70, EC 3.2.1.135, EC 3.2.1.3, EC 3.2.1.20, EC 3.2.1.10.

[0211] Similarly, this prebiotic is selected from the family of α-galacto-oligosaccharides / raffinose (e.g., pea GOS, soybean GOS), β-galacto-oligosaccharides (e.g., β-GOS, Vivinal GOS, bovine milk oligosaccharides (BMOS)), human milk oligosaccharides (HMO), cello-oligosaccharides (COS) (e.g., cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose) or combinations thereof.

[0212] The specific selection of prebiotics in the composition works symbiotically with the specific selection of probiotics in the composition as previously defined. Such prebiotics are specific carbohydrates, preferably fiber and oligosaccharides, which preferably and beneficially support the growth of the specifically selected probiotics. More specifically, the specific carbohydrates act as substrates for the specifically selected probiotics and are capable of being hydrolyzed by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase, and / or lichen polysaccharidease, which, due to the specific selection of probiotics, is produced by the probiotics in the composition as previously defined.

[0213] According to another aspect, the prebiotic is preferably included in the composition as defined herein in an effective amount. More preferably, such effective amount of prebiotic included in the composition as defined herein is between 0.1 g / day and 30 g / day, and more preferably between 2 g / day and 15 g / day.

[0214] According to the third aspect, in addition to the selected probiotics and selected prebiotics as described above, the composition also contains selected lipids.

[0215] Such lipids include at least one lipid selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, or mixtures thereof. Preferred triglycerides include triglycerides comprising butyrate and / or caprylate, triglycerides composed of butyrate and caprylate, and triglycerides composed of butyrate and oleate. In one aspect, the triglycerides consist of butyrate and caprylate. The inventors have surprisingly discovered that these specifically selected lipids advantageously support improvements in the microbiome, gut function, liver metabolism, and kidney function in a symbiotic manner, thereby significantly contributing to the avoidance of the production and accumulation of uremic toxins.

[0216] In this context, "short-chain triglycerides" (SCTs) are preferably understood as triglycerides having two or three fatty acids, each fatty acid having one to five carbon atoms, preferably two to five carbon atoms. Such fatty acids having one to five carbon atoms, preferably two to five carbon atoms, are generally understood herein as short-chain fatty acids (SCFAs). Particularly preferred short-chain fatty acids are C4 fatty acids, most preferably butyric acid (C4:0). Short-chain triglycerides containing butyric acid are preferably SCTs containing at least one, two, or even three butyric acids.

[0217] Similarly, in this context, "medium-chain triglycerides" (MCTs) are preferably understood as triglycerides having two or three fatty acids, each with 6 to 12 carbon atoms. Such fatty acids having 6 to 12 carbon atoms are generally understood as medium-chain fatty acids (MCFAs). Preferred short-chain fatty acids are C8 fatty acids, such as caprylic acid (C8:0). Medium-chain fatty acid triglycerides containing caprylic acid can be MCTs containing at least one, at least two, or even three caprylic acids.

[0218] In the case of triglycerides comprising a mixture of butyrate and long-chain fatty acids, it is preferably understood as a triglyceride having one or two butyrate moieties and one or two long-chain fatty acids. Long-chain fatty acids are generally understood to be fatty acids having 13 to 21 carbon atoms. Exemplary triglycerides comprising butyrate and long-chain fatty acids include 1,3-dibutyryl-2-linoleoylglycerol, 1,3-dibutyryl-2-stearoylglycerol, 1-butyryl-2-oleoyl-3-palmitoylglycerol, 1-palmitoyl-2-oleoyl-3-butyrylglycerol, 1-butyryl-2-oleoyl-3-linoleoylglycerol, 1-linoleoyl-2-oleoyl-3-butyrylglycerol, 1-oleoyl-2-butyryl-3-linoleoylglycerol, 1 -Linoleoyl-2-butyroyl-3-oleoylglycerol, 1-butyroyl-2-linoleoyl-3-oleoylglycerol, 1-oleoyl-2-linoleoyl-3-butyroylglycerol, 1-butyroyl-2-stearoyl-3-oleoylglycerol, 1-oleoyl-2-stearoyl-3-butyroylglycerol, 1-butyroyl-2-oleoyl-3-stearoylglycerol and / or 1-stearoyl-2-oleoyl-3-butyroylglycerol, and mixtures of two or more thereof. In the case of triglycerides comprising a mixture of butyrate and long-chain fatty acids, the preferred long-chain fatty acid is oleic acid (C18:1).

[0219] More preferably, in the case of the composition, the lipid is selected from a. triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA); b. triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA); c. triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as a mixture of butyrate and oleate); d. triglycerides composed of medium-chain fatty acids; e. triglycerides (TG) composed of short-chain fatty acids; f. short-chain fatty acids capable of being metabolized into ketone bodies; and / or g. medium-chain fatty acids capable of being metabolized into ketone bodies, preferably C4 and / or C8 medium-chain fatty acids (such as butyrate (C4:0) and / or caprylic acid (C8:0)); in the case of the composition, exemplary triglycerides are triglycerides comprising butyrate and / or caprylic acid, triglycerides composed of butyrate and caprylic acid, and triglycerides composed of butyrate and oleate. In one aspect, the triglyceride is composed of butyrate and caprylic acid.

[0220] In the case of the composition, triglycerides comprising butyrate, short-chain fatty acids, medium-chain fatty acids, or mixtures thereof are particularly preferred as lipids. Therefore, such lipids can be...

[0221] - Triglycerides containing at least one short-chain fatty acid C1-C5, preferably C2-C5; preferably triglycerides containing at least one butyric acid (C4:0), or

[0222] - Triglycerides containing at least one medium-chain fatty acid C6-C12, such as caprylic acid (C8:0).

[0223] Preferably, triglycerides containing at least one type of caprylic acid (C8:0), or

[0224] - Contains at least one short-chain fatty acid C1-C5, preferably C2-C5 such as butyric acid (C4:0), and at least one medium-chain fatty acid C6-C12 such as caprylic acid (C8:0) triglyceride, etc.

[0225] In an even more preferred aspect, in the case of the composition, the lipid is a triglyceride containing butyric acid (C4:0) (BBB, tributylate) or caprylic acid (C8:0) (CCC, tricaprylate), preferably a mixture of triglycerides containing butyric acid (C4:0) or caprylic acid (C8:0), or optionally a triglyceride containing both butyric acid (C4:0) and caprylic acid (C8:0) in the same triglyceride. The latter triglyceride can be prepared by transesterification of a mixture of triglycerides containing butyric acid (C4:0) or caprylic acid (C8:0). Commercially available sources of BBB tributylate are (e.g., available from Sigma-Aldrich). Commercially available sources of CCC tricaprylate are, for example, Neobee 895 (e.g., available from Stepan Specialty).

[0226] Methods for preparing triglycerides composed of a mixture of butyrate and long-chain fatty acids are known in the art, for example, as described in WO2019228851, which is incorporated herein by reference in its entirety. Exemplary triglycerides comprising butyrate and long-chain fatty acids include 1,3-dibutyryl-2-linoleoylglycerol, 1,3-dibutyryl-2-stearoylglycerol, 1-butyryl-2-oleoyl-3-palmitoylglycerol, 1-palmitoyl-2-oleoyl-3-butyrylglycerol, 1-butyryl-2-oleoyl-3-linoleoylglycerol, 1-linoleoyl-2-oleoyl-3-butyrylglycerol, 1-oleoyl-2-butyryl-3-linoleoylglycerol, 1- -Linoleoyl-2-butyroyl-3-oleoylglycerol, 1-butyroyl-2-linoleoyl-3-oleoylglycerol, 1-oleoyl-2-linoleoyl-3-butyroylglycerol, 1-butyroyl-2-stearoyl-3-oleoylglycerol, 1-oleoyl-2-stearoyl-3-butyroylglycerol, 1-butyroyl-2-oleoyl-3-stearoylglycerol and / or 1-stearoyl-2-oleoyl-3-butyroylglycerol, and mixtures of two or more thereof. In the case of triglycerides comprising a mixture of butyrate and long-chain fatty acids, the preferred long-chain fatty acid is oleic acid (C18:1). Preferred triglycerides composed of a mixture of short-chain and long-chain fatty acids are triglycerides composed of a mixture of butyrate and oleate.

[0227] In the case of the composition, it is preferred to contain triglycerides containing fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain fatty acids and medium-chain fatty acids, more preferably triglycerides containing butyric acid (C4:0) and caprylic acid (C8:0) in the same triglyceride; or triglycerides containing either butyric acid (C4:0) or caprylic acid (C8:0) in the same triglyceride; or mixtures of triglycerides, one triglyceride containing butyric acid (C4:0) and the other triglyceride containing caprylic acid (C8:0).

[0228] Alternatively, but less preferably, the lipid may be selected from short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) instead of triglycerides. Thus, such lipids may be selected from at least one short-chain fatty acid C1-C5, preferably C2-C5, more preferably butyric acid (C4:0), or from at least one medium-chain fatty acid C6-C12 such as caprylic acid (C8:0), or from a mixture of at least one short-chain fatty acid C1-C5, preferably C2-C5 such as butyric acid (C4:0), and at least one medium-chain fatty acid C6-C12 such as caprylic acid (C8:0).

[0229] According to another aspect, the lipids are preferably included in the composition as defined herein in an amount between 0.1 g / day and 30 g / day, more preferably in an amount between 2 g / day and 15 g / day.

[0230] According to preferred aspects, the composition may comprise, as defined herein:

[0231] (a) Probiotics, preferably selected from such probiotics,

[0232] - Lack of a gene for producing at least one of the following: urea, uric acid, p-cresol, p-toluene sulfate, indole sulfate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, and / or glutaric acid.

[0233] - Deficiency of at least one bacterial enzyme selected from urease, carnitine monooxygenase and reductase, tryptophanase and / or hydroxyphenylacetate; and / or

[0234] - Expresses at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme;

[0235] (b) A prebiotic selected from carbohydrates, wherein the carbohydrates are hydrolyzable by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan-1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or licheninase, wherein the at least one bacterial enzyme is preferably selected from EC3.2.1.22, EC3.2.1.23, EC3.2.1.74, EC3.2.1.4, EC 3.2.1.26, EC 3.2.1.58, EC 3.2.1.73, EC 2.4.1.9, EC 3.2.1.84, EC3.2.1.33, EC 3.2.1.70, EC 3.2.1.135, EC 3.2.1.3, EC 3.2.1.20, EC 3.2.1.10, or preferably wherein the carbohydrate is selected from the family of α-galacto-oligosaccharides / raffinose (e.g., pea GOS, soybean GOS), β-galacto-oligosaccharides (e.g., b-GOS, Vivinal GOS, bovine milk oligosaccharides (BMOS)), human milk oligosaccharides (HMOs) (e.g., 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), lactose-N-tetrasaccharide (LNT), 3'-sialylated lactose (3SL) and / or 6'-sialylated lactose (6SL)), cello-oligosaccharides (COSs) (e.g., cellobiose, cellotriose, cellotetrasaccharide, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetrasaccharide) or combinations thereof;

[0236] (c) A lipid selected from triglycerides, short-chain fatty acids and / or medium-chain fatty acids, wherein the triglyceride comprises short-chain fatty acids, medium-chain fatty acids or mixtures thereof, and further wherein in one aspect, the triglyceride comprises butyrate and / or caprylate.

[0237] According to a more preferred aspect, the composition as defined herein may comprise probiotics, prebiotics, and lipids as defined above, wherein:

[0238] (a) The probiotics are selected from at least one of the probiotics defined as a. to k. below:

[0239] a. Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446);

[0240] b. Bifidobacterium longum infantile subspecies NCC 341 (ATCC 15697(T));

[0241] c. Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618) (NCBIrefseq; GCA_000007525.1);

[0242] d. Enterococcus faecalis NCC 2768 (NCIMB 10415);

[0243] e. Lactobacillus johnsonii NCC 533 (CNCM I-1225) (NCBI refseq;

[0244] GCA_000008065.1);

[0245] f. Lactococcus lactis NCC 2287 (CNCM I-4154);

[0246] g. Lactobacillus paracasei NCC 2461 (CNCM I-2116);

[0247] h. Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724);

[0248] i. Staphylococcus aureus NCC 1052 (CNCM I-5400);

[0249] j. Staphylococcus aureus NCC 971 (CNCM I-5398); and / or

[0250] k. Streptococcus thermophilus NCC 2496 (CNCM I-3915);

[0251] Or a probiotic having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% ANI or 99.9% ANI, of any one of the genome sequences of probiotics as defined above (a. to k.); or a combination of one or more of the above-mentioned probiotics;

[0252] (b) The prebiotics are selected from the family of α-galacto-oligosaccharides / raffinose (e.g., pea GOS, soybean GOS), β-galacto-oligosaccharides (e.g., β-GOS, Vivinal GOS, milk oligosaccharides), cello-oligosaccharides (COS) (e.g., cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose), human milk oligosaccharides (HMOs) (e.g., 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetraose (LNnT), lactose-N-tetraose (LNT), 3'-sialylated lactose (3SL) and / or 6'-sialylated lactose (6SL)) or combinations thereof;

[0253] (c) The lipid is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, or mixtures thereof, and further wherein, in one aspect, the triglycerides comprise butyrate and / or caprylate. Preferably, the lipid is selected from:

[0254] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0255] b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA);

[0256] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0257] d. Triglycerides composed of medium-chain fatty acids;

[0258] e. Triglycerides (TG) composed of short-chain fatty acids;

[0259] f. Short-chain fatty acids that can be metabolized into ketone bodies; and / or

[0260] g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain and medium-chain fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)).

[0261] According to even more preferred aspects, the composition as defined herein may comprise probiotics, prebiotics, and lipids as defined above, wherein:

[0262] (a) The probiotic is selected from Lactobacillus johnsonii NCC 533 (CNCMI-1225) or a probiotic having a genome sequence (SEQ ID NO:1) of Lactobacillus johnsonii NCC 533 (CNCM I-1225) with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI or even 99.5% ANI or 99.9% ANI.

[0263] (b) The prebiotics are selected from the family of α-galacto-oligosaccharides / raffinose (e.g., pea GOS, soybean GOS), β-galacto-oligosaccharides (e.g., β-GOS, Vivinal GOS, milk oligosaccharides), cello-oligosaccharides (COS) (e.g., cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose) or combinations thereof;

[0264] (c) The lipid is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, or mixtures thereof, and further wherein, in one aspect, the triglycerides comprise butyrate and / or caprylate. More preferably, the lipid is selected from:

[0265] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0266] b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA);

[0267] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0268] d. Triglycerides composed of medium-chain fatty acids;

[0269] e. Triglycerides (TG) composed of short-chain fatty acids;

[0270] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0271] g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain and medium-chain fatty acids.

[0272] According to, or even more preferred, aspects, as defined herein, the composition may comprise probiotics, prebiotics, and lipids as defined above, wherein:

[0273] (a) The probiotic is selected from Lactobacillus johnsonii NCC 533 (CNCMI-1225) or a probiotic having a genome sequence (SEQ ID NO:1) of Lactobacillus johnsonii NCC 533 (CNCM I-1225) with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI or even 99.5% ANI or 99.9% ANI.

[0274] (b) The prebiotics are selected from pea GOS, soybean GOS, β-GOS, Vivinal GOS, milk oligosaccharides, human milk oligosaccharides, cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose or combinations thereof.

[0275] (c) Lipids are selected from:

[0276] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0277] b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA);

[0278] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0279] d. Triglycerides composed of medium-chain fatty acids;

[0280] e. Triglycerides (TG) composed of short-chain fatty acids;

[0281] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0282] g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain and medium-chain fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)).

[0283] According to another aspect, the composition as defined herein is selected from food products or nutritional compositions, dietary supplements, foods for specific medical purposes (FSMP), nutritional supplements, milk-based beverages, low-volume fluid supplements, functional food products, functional beverage products, meal replacement drinks, and combinations thereof. Furthermore, and not limited thereto, the composition may be in an applicable form, preferably selected from pharmaceutical preparations.

[0284] In this case, it is particularly preferred that the composition be a solid or liquid, and may exist in the form of powder, tablets, capsules, or in the form of oil preparations, emulsions, oil-in-water emulsions (o / w emulsions) or water-in-oil emulsions (w / o emulsions).

[0285] Each of the compounds in the composition may be applied simultaneously with other compounds (e.g., as a single unit) or separately at time intervals (e.g., as separate units). Preferably, the compounds are provided as single units.

[0286] This invention specifically provides a nutrition-based solution for managing uremic toxins associated with disease conditions and related complications. General applications of such compositions may include: in the form of medical foods or FSMP (foods for specific medical purposes) supplements; as components of medical nutrition products; as adjuncts to / concurrent with standards of care; as adjuncts to / concurrent with nephropathy medications (such as SGLT inhibitors); as adjuncts to / concurrent with nutritional components targeting renal mitochondrial dysfunction; as adjuncts to / concurrent with protein diets; as nutritional supplements; as milk-based beverages; as low-volume fluid supplements; as meal replacement drinks; and combinations thereof.

[0287] According to a second embodiment, the present invention relates to the use of compositions as defined herein for reducing or preventing the accumulation of uremic toxins (preferably in the treatment of cardiovascular metabolic or neurodegenerative diseases) or for preventing the accumulation of uremic toxins (preferably in the treatment of cardiovascular metabolic or neurodegenerative diseases). The compositions are thus preferably used for reducing uremic toxins in cardiovascular metabolic or neurodegenerative diseases and related comorbidities, for delaying the progression of such cardiovascular metabolic or neurodegenerative diseases and related comorbidities, and / or for managing symptoms and syndromes associated with the toxic effects of uremic solutes in such cardiovascular metabolic or neurodegenerative diseases and related comorbidities, and / or for preventing the accumulation of uremic toxins in the treatment of cardiovascular metabolic or neurodegenerative diseases and related comorbidities.

[0288] Cardiovascular metabolic or neurodegenerative diseases and related comorbidities that can be treated with the composition preferably include the following:

[0289] • Treatment or prevention of kidney disease, including chronic and acute; dialysis and predialysis status of kidney disease; rare (kidney) diseases, genetically induced (kidney) diseases, and metabolically induced (kidney) diseases.

[0290] disease;

[0291] • Treatment or prevention of uremic syndromes, including protein-energy wasting syndrome, bone loss, anorexia, fatigue, or inflammation;

[0292] • Delayed complications of late-stage renal disease, including anemia, hyperphosphatemia, secondary hyperparathyroidism, bone disease, and neurological disorders;

[0293] • Delayed comorbidities of kidney disease, including cardiovascular disease;

[0294] • Preventing the risk of malnutrition or managing malnutrition;

[0295] • Delaying the progression of cardiovascular and metabolic diseases;

[0296] • Preventing or managing the risk of cardiovascular disease and comorbidities such as diabetes; and / or

[0297] • To prevent or manage the risks of neurodegenerative and neurological disorders.

[0298] Generally speaking, any subject of such treatment or patient requiring such treatment is a mammal, preferably a human or pet, such as a companion animal, suffering from any of the aforementioned cardiovascular, metabolic, or neurodegenerative diseases and related comorbidities. Preferably, any subject of such treatment or patient requiring such treatment may be a child, toddler or infant, elderly person, or companion animal, but may also be a companion pet such as a cat or dog, wherein the subject is preferably at risk of developing or has already developed the disease.

[0299] According to a third embodiment, a method for treating cardiovascular metabolic or neurodegenerative diseases as defined above is provided, preferably comprising, as a first step, (a) preparing and providing a composition as discussed above, the composition comprising a specific selection of probiotics, a specific selection of prebiotics, and a specific selection of lipids as defined above; and (b) administering such composition to a patient in need, who typically suffers from an increase in uremic toxins, typically in cases of cardiovascular metabolic or neurodegenerative diseases as defined herein.

[0300] According to a fourth embodiment, a multi-part kit is provided for reducing and / or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases as defined herein. The kit comprises a composition as described herein, comprising a specific selection of probiotics, a specific selection of prebiotics, and a specific selection of lipids as defined above; for mixing to form one or more of the compositions disclosed herein and / or for use in one or more of the methods disclosed herein, for example, as two or more liquid solutions or dry powders in separate containers. The kit may also include instructions for use.

[0301] It should be understood that the various aspects and embodiments of the invention disclosed herein are examples of specific ways of making and using the invention, and these contents do not limit the scope of the invention when considered in conjunction with the claims and the detailed description herein. It should also be understood that features of various aspects and embodiments of the invention may be combined with other features of the same or different aspects and embodiments of the invention.

[0302] Unless otherwise specified, the present invention will be practiced using conventional chemical, biochemical, molecular biological, microbiological, and immunological techniques, all of which are within the capabilities of a person skilled in the art. Such techniques are described in the literature. See, for example: Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM, et al. (1995 and periodically supplemented), Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM, and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ, (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are incorporated herein by reference.

[0303] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0304] The present invention includes the following embodiments:

[0305] Implementation Plan 1

[0306] A composition for reducing or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases, wherein the composition comprises:

[0307] (a) Probiotics, selected from the following probiotic strains:

[0308] - Lack of a gene for producing at least one of the following: urea, uric acid, p-cresol, p-toluene sulfate, indole sulfate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, PCG, CMPF, betaine, and / or glutaric acid.

[0309] - Deficiency of at least one bacterial enzyme selected from urease, carnitine monooxygenase and reductase, tryptophanase and / or hydroxyphenylacetate; and / or

[0310] - Expresses at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme;

[0311] (b) A prebiotic selected from carbohydrates or fiber, wherein the carbohydrates or fiber can be hydrolyzed by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme.

[0312] (c) A lipid selected from triglycerides, short-chain fatty acids and / or medium-chain fatty acids, wherein the triglyceride comprises short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids or mixtures thereof, further wherein in one aspect the triglyceride comprises butyrate and / or caprylate, and in another aspect the triglyceride is composed of butyrate and caprylate or butyrate and oleate, further wherein preferably the triglyceride is composed of butyrate and caprylate.

[0313] Implementation Plan 2

[0314] The composition according to embodiment 1 for the stated purpose, wherein the probiotic (a) is selected from at least one of the following a. to k.:

[0315] a. Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446);

[0316] b. Bifidobacterium longum infantile subspecies NCC 341 (ATCC 15697(T));

[0317] c. Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618) (NCBI refseq; GCA_000007525.1);

[0318] d. Enterococcus faecalis NCC 2768 (NCIMB 10415);

[0319] e. Lactobacillus johnsonii NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1);

[0320] f. Lactococcus lactis NCC 2287 (CNCM I-4154);

[0321] g. Lactobacillus paracasei NCC 2461 (CNCM I-2116);

[0322] h. Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724);

[0323] i. Staphylococcus aureus NCC 1052 (CNCM I-5400);

[0324] j. Staphylococcus aureus NCC 971 (CNCM I-5398); and / or

[0325] k. Streptococcus thermophilus NCC 2496 (CNCM I-3915);

[0326] Or a probiotic having a genome with at least 95% average nucleotide identity (ANI), preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%, or even 99.5% or 99.9% ANI, of any one of the genome sequences of a probiotic as defined above from a. to k.

[0327] Implementation Plan 3

[0328] The composition according to embodiment 2 for the stated use, wherein the composition comprises two or more probiotics as defined in a. to k.

[0329] Implementation Plan 4

[0330] The composition according to embodiment 2 for the stated use, wherein the composition comprises two or more probiotics having a genome having at least 99% ANI of the genome sequence of the probiotics according to a. to k.

[0331] Implementation Plan 5

[0332] The composition according to embodiment 2 for the stated use, wherein the probiotic (a) is *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1) or a probiotic having a genome with at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% ANI or 99.9% ANI.

[0333] Implementation Plan 6

[0334] The composition for the purpose according to any one of embodiments 1 to 5, wherein the prebiotic (b) is selected from α-galacto-oligosaccharide (α-GOS) / raffinose, β-galacto-oligosaccharide (β-GOS) and cellulosic oligosaccharide (COS), human milk oligosaccharide (HMO) or combinations thereof.

[0335] Implementation Plan 7

[0336] The composition for the use according to any one of embodiments 1 to 6, wherein the prebiotic (b) is selected from soybean GOS (α-GOS), pea GOS (α-GOS), bovine milk oligosaccharide (BMOS) (β-GOS), Vivinal GOS (β-GOS), human milk oligosaccharide (HMO) (2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), lactose-N-tetrasaccharide (LNT), 3'-sialylated lactose (3SL) and / or 6'-sialylated lactose (6SL)), cellobiose, cellotriose, cellotetrasaccharide and soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose or cellotriose or cellotetrasaccharide or combination thereof.

[0337] Implementation Plan 8

[0338] The composition for the stated use according to any one of embodiments 1 to 7, wherein the lipid (c) is selected from:

[0339] a. Triglycerides (TG) composed of a mixture of butyrate (C4:0) and medium-chain fatty acids (MCFA);

[0340] b. Triglycerides (TG) composed of short-chain fatty acids and medium-chain fatty acids (SMCFA), preferably a mixture of C4 and / or C8 fatty acids;

[0341] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0342] d. Triglycerides composed of medium-chain fatty acids;

[0343] e. Triglycerides (TG) composed of short-chain fatty acids;

[0344] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0345] g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)).

[0346] Implementation Plan 9

[0347] The composition for the said use according to any one of embodiments 1 to 8, wherein:

[0348] a. The probiotic is selected from at least one of the probiotics defined as a. to k. above, or a probiotic or combination thereof having a genome with at least 90% ANI, preferably at least 95% ANI, similar to any one of the probiotics defined as a. to k. above, preferably from Lactobacillus johnsonii NCC 533 (CNCM I-1225) or a probiotic having a genome with at least 90% ANI, preferably at least 95% ANI, similar to the genome sequence of Lactobacillus johnsonii NCC 533 (CNCMI-1225);

[0349] b. The prebiotic is selected from α-galacto-oligosaccharide / raffinose, β-galacto-oligosaccharide or cello-oligosaccharide or combinations thereof, preferably selected from soybean GOS (α-GOS), pea GOS (α-GOS), milk oligosaccharide (BMOS) (β-GOS), vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose or cellotriose or cellotetraose or combinations thereof;

[0350] c. The lipid is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids, or mixtures thereof, and further wherein, in one aspect, the triglycerides comprise butyrate and / or caprylate, preferably from...

[0351] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0352] b. Triglycerides (TG) composed of short-chain fatty acids and medium-chain fatty acids (SMCFA), preferably a mixture of C4 and / or C8 short-chain fatty acids and medium-chain fatty acids;

[0353] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0354] d. Triglycerides composed of medium-chain fatty acids;

[0355] e. Triglycerides (TG) composed of short-chain fatty acids;

[0356] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0357] g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain and medium-chain fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0)).

[0358] Implementation Plan 10

[0359] The composition for the use according to claim 9, wherein the probiotic (a) is selected from Lactobacillus johnsonii NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1) or a probiotic having a genome with at least 90% ANI, preferably at least 95% ANI, of the genome sequence of Lactobacillus johnsonii NCC 533 (CNCMI-1225) (NCBI refseq; GCA_000008065.1).

[0360] Implementation Plan 11

[0361] The composition used for the stated purpose according to embodiment 7 or embodiment 8, wherein the prebiotic (b) is selected from soybean GOS (α-GOS), pea GOS (α-GOS), milk oligosaccharide (BMOS) (β-GOS), vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose, or combinations thereof.

[0362] Implementation Plan 12

[0363] The composition for the stated use according to any one of embodiments 9 to 11, wherein the lipid (c) is selected from:

[0364] a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA);

[0365] b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA);

[0366] c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate);

[0367] d. Triglycerides composed of medium-chain fatty acids;

[0368] e. Triglycerides (TG) composed of short-chain fatty acids;

[0369] f. Short-chain fatty acids that can be metabolized into ketone bodies; and

[0370] g. Medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 medium-chain fatty acids (such as butyric acid (C4:0) and / or caprylic acid (C8:0));

[0371] Implementation Plan 13

[0372] The composition for the stated use according to any one of embodiments 1 to 12, wherein 10 3 CFU / day up to 10 12 The amount between CFU / day dose is usually in increments of 10. 4 CFU / day up to 10 11 The amount between CFU / day dose is preferably in the range of 10. 5 CFU / day up to 10 10 The amount between CFU / daily dose or 10 5 CFU / day up to 10 9 The amount between CFU / day dose is also preferably 10. 6 CFU / day up to 10 9 The amount between CFU / day dose, 10 6 CFU / day up to 10 8 The amount between CFU / day dose or in increments of 10 8 CFU / day up to 10 10 The amount of CFU / day, more preferably about 10 7 CFU / day up to 10 9 CFU / daily dose contains probiotics.

[0373] Implementation Plan 14

[0374] The composition for the stated purpose according to any one of embodiments 1 to 13 contains prebiotics in an amount from 0.1 g / day to 30 g / day, preferably in an amount from 2 g / day to 15 g / day.

[0375] Implementation Plan 15

[0376] The composition for the stated purpose according to any one of embodiments 1 to 14 contains lipids in an amount from 0.1 g / day to 30 g / day, preferably in an amount from 2 g / day to 15 g / day.

[0377] Implementation Plan 16

[0378] A composition for the stated use according to any one of embodiments 1 to 15, wherein the lipid is a triglyceride composed of butyrate and caprylate or a triglyceride composed of butyrate and oleate.

[0379] Implementation Plan 17

[0380] A composition for the stated use according to any one of embodiments 1 to 15, wherein the lipid is a triglyceride composed of butyrate and caprylate.

[0381] Implementation Plan 18

[0382] The composition for the stated purpose according to any one of embodiments 1 to 17 is in the form of: food products or nutritional compositions, dietary supplements, food for a specific medical purpose (FSMP), nutritional supplements, milk-based beverages, low-volume liquid supplements, functional food products, functional beverage products, meal replacement drinks, and combinations thereof.

[0383] Implementation Plan 19

[0384] The composition for the stated use according to any one of embodiments 1 to 18, wherein the composition is provided in the form of powder, tablet, capsule, or can be in the form of oil preparation, emulsion, oil-in-water emulsion (o / w emulsion) or water-in-oil emulsion (w / o emulsion).

[0385] Implementation Plan 20

[0386] A composition for the stated use according to any one of embodiments 1 to 19, wherein the reduction of uremic toxins in cardiovascular metabolic or neurodegenerative diseases is used to delay the progression of such diseases and comorbidities and / or to manage symptoms and syndromes associated with the toxic effects of uremic solutes in such diseases and comorbidities.

[0387] Implementation Plan 21

[0388] The composition for the stated use according to any one of embodiments 1 to 19, wherein the reduction of uremic toxins in cardiovascular metabolic or neurodegenerative diseases includes:

[0389] ○ Treatment or prevention of kidney disease, including chronic and acute; dialysis and predialysis; rare diseases, genetically induced and metabolically induced;

[0390] ○ Treatment or prevention of uremic syndrome, including protein and energy depletion, bone loss, anorexia, fatigue, or inflammation;

[0391] ○ Delayed complications of late-stage renal disease, including anemia, hyperphosphatemia, secondary hyperparathyroidism, bone disease, and neurological disorders;

[0392] ○ Delayed comorbidities of kidney disease, including cardiovascular disease;

[0393] ○ Preventing the risk of malnutrition or managing malnutrition;

[0394] ○ Delaying the progression of cardiovascular and metabolic diseases;

[0395] ○ Prevention of cardiovascular disease and comorbidities (diabetes) or management of cardiovascular disease and comorbidities (diabetes); and / or

[0396] ○ To prevent or manage the risks of neurodegenerative and neurological disorders.

[0397] Implementation Plan 22

[0398] A multipart kit for reducing or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases, comprising a composition as described in any one of embodiments 1 to 17, probiotics, prebiotics and lipids in two or more separate containers, and optionally including instructions.

[0399] Implementation Plan 23

[0400] A method for treating cardiovascular metabolic or neurodegenerative diseases as defined above, the method comprising, as a first step, (a) preparing and providing a composition according to any one of embodiments 1 to 17, the composition comprising probiotics, prebiotics and lipids; and (b) administering such composition to a patient in need who suffers from an increase in uremic toxins in the case of cardiovascular metabolic or neurodegenerative diseases.

[0401] Implementation Plan 24

[0402] A method for treating cardiovascular metabolic or neurodegenerative diseases according to embodiment 21, the method comprising administering to a patient the composition according to any one of embodiments 1 to 17.

[0403] Implementation Plan 25

[0404] Use of a composition according to any one of embodiments 1 to 17 in the manufacture of a medicament for treating cardiovascular metabolic or neurodegenerative diseases according to embodiment 21.

[0405] Implementation Plan 26

[0406] The composition for the stated use according to any one of embodiments 1 to 15 and 17 to 21, the multi-part kit according to embodiment 22, the method according to embodiment 23 or 24, or the use according to embodiment 25, wherein the composition comprises 10 9 Lactobacillus johnsonii NCC533, 1% cellobiose, 1% short- and medium-chain triglycerides containing butyrate and caprylate.

[0407] Implementation Plan 27

[0408] The composition for the stated use according to any one of embodiments 1 to 15 and 17 to 21, the multi-part kit according to embodiment 22, the method according to embodiment 23 or 24, or the use according to embodiment 25, wherein the composition comprises 10 9 Lactobacillus johnsonii NCC533, 1% cellobiose, 1% pea GOS, 1% short- and medium-chain triglycerides containing butyrate and caprylate.

[0409] Implementation Plan 28

[0410] Compositions for the purposes according to any one of embodiments 1 to 19, 26 and 27, multipart kits according to embodiments 20, 26 and 27, methods according to any one of embodiments 21, 22, 26 and 27, or uses according to any one of embodiments 23, 26 and 27, wherein the composition further comprises one or more HMOs, preferably one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), lactose-N-tetrasaccharide (LNT), 3'-sialylated lactose (3SL) or 6'-sialylated lactose (6SL).

[0411] Implementation Plan 29

[0412] Compositions for the purposes according to any one of embodiments 1 to 19, 26 and 27, multipart kits according to embodiments 20, 26 and 27, methods according to any one of embodiments 21, 22, 26 and 27, or uses according to any one of embodiments 23, 26 and 27, wherein the composition further comprises one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT) or lactose-N-tetrasaccharide (LNT).

[0413] Implementation Plan 30

[0414] The composition, multipart kit, method, or use according to embodiment 28 or embodiment 29 for the stated purpose further includes one or more of the following:

[0415] a. Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446);

[0416] b. *Bifidobacterium longum* subsp. infantis NCC 341 (ATCC 15697(T)); and

[0417] c. *Bifidobacterium longum* subsp. *longum* NCC 2705 (CNCM I-2618) (NCBI refseq;

[0418] GCA_000007525.1).

[0419] Implementation Plan 31

[0420] Compositions for the stated uses according to any one of embodiments 1 to 15 and 18 to 21, multipart kits according to embodiments 20, 26 and 27, methods according to any one of embodiments 21, 22, 26 and 27, or uses according to any one of embodiments 23, 26 and 27, wherein the composition comprises triglycerides consisting of butyrate and long-chain fatty acids.

[0421] Implementation Plan 32

[0422] According to embodiment 31, the composition, multipart kit, method, or use for the stated purpose, wherein the triglyceride composed of butyrate and long-chain fatty acid is one or more triglycerides comprising butyrate and long-chain fatty acid, including 1,3-dibutyryl-2-linoleoylglycerol, 1,3-dibutyryl-2-stearoylglycerol, 1-butyryl-2-oleoyl-3-palmitoylglycerol, 1-palmitoyl-2-oleoyl-3-butyrylglycerol, 1-butyryl-2-oleoyl-3-linoleoylglycerol, 1-linoleoyl-2-oleoyl-3- Butyroglycerol, 1-oleoyl-2-butyro-3-linoleoylglycerol, 1-linoleoyl-2-butyro-3-oleoylglycerol, 1-butyro-2-linoleoyl-3-oleoylglycerol, 1-oleoyl-2-linoleoyl-3-butyroglycerol, 1-butyro-2-stearoyl-3-oleoylglycerol, 1-oleoyl-2-stearoyl-3-butyroglycerol, 1-butyro-2-oleoyl-3-stearoylglycerol and / or 1-stearoyl-2-oleoyl-3-butyroglycerol, and mixtures of two or more thereof.

[0423] Implementation Plan 33

[0424] According to embodiment 31, the composition, multipart kit, method, or use for the stated purpose, wherein the triglyceride composed of butyrate and a long-chain fatty acid is a mixture of butyrate and a long-chain fatty acid, wherein the long-chain fatty acid is oleic acid (C18:1).

[0425] Implementation Plan 34

[0426] Compositions, multipart kits, methods, or uses according to any one of embodiments 31 to 33 for the stated purpose, wherein the composition further comprises one or more HMOs, preferably one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), lactose-N-tetrasaccharide (LNT), 3'-sialylated lactose (3SL), or 6'-sialylated lactose (6SL).

[0427] Implementation Plan 35

[0428] Compositions, multipart kits, methods, or uses according to any one of embodiments 31 to 33 for the stated purpose, wherein the composition further comprises one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), or lactose-N-tetrasaccharide (LNT).

[0429] Example

[0430] Method 1: Ingredient Selection

[0431] Computer-based selection of probiotic strains with low ability to produce clinically relevant uremic toxins.

[0432] Using Nestle's internal bacterial genome platform WallGene (containing all genomes from the Nestle Culture Collection) supplemented with internal BlastP (Basic Local Alignment Search Tool for proteinsequences, (Altschul et al., 1990)) search, probiotic strains with low ability to produce uremic toxins were selected from the NCC.

[0433] Specifically, a BLAST database containing all predicted proteins from probiotic strains was created. Bacterial and host metabolic pathways involved in uremic toxin metabolism were compiled from public databases such as the KEGG pathway (Kyoto Encyclopedia of Genes and Genomes) and literature. Based on EC numbers, amino acid sequences of key enzymes involved in uremic toxin metabolism were further extracted from public databases such as KEGG, UniProt, and SwissProt, and compiled into FASTA files (a list of reference protein sequences). These FASTA files containing protein sequences of key enzymes were used as reference sequences for BLASTp against proteomic profiles of available probiotic strains from the NCC. When pBLAST showed low similarity to the reference protein (e.g., identity <30%), InterProScan (EMBL-EBI) was further used to predict the presence of functional domains (Quevillon et al., 2005).

[0434] Computer-aided selection of carbohydrate substrates capable of supporting the growth of selected probiotic strains with low uremic toxin production capacity.

[0435] Carbohydrate-active enzymes (CAZys) of candidate probiotics were annotated on a computer using dbCAN (Yin et al., 2012) (Lombard et al., 2014). Based on the probiotic CAZy annotations, different carbohydrates exhibiting appropriate biochemical structures were selected using the BRENDA-Comprehensive Enzyme Information System. The list of substrates for each CAZy was then matched with a carbohydrate composition consisting of polysaccharide and / or oligosaccharide structures corresponding to those substrates in the preferred substrates.

[0436] In vitro growth assay for validating the combination of computer probiotics and carbohydrate candidates

[0437] Candidate probiotics were obtained from the Nestlé Culture Collection (NCC) or from publicly available collections of strains and cultures (ATCC, NCIMB, NCBI). Carbohydrate components were sourced from commercial suppliers or Nestlé or its subsidiaries. A complete list of exemplary carbohydrate sources is available in Table 1. Table 1 shows exemplary different genes of *Lactobacillus johnsonii* NCC 533 containing carbohydrate-active enzymes (CAZyme), enzyme code (EC) numbers of the functional enzymes, their functions, suitable and associated carbohydrate families, fiber components recommended for preclinical testing in light of the present invention, and the availability of such compounds. It can be seen that carbohydrates to be fermented, metabolized, used, decomposed, degraded, or transformed by carbohydrate-active enzymes (CAZyme) present in the *Lactobacillus johnsonii* NCC 533 genome were selected based on their biochemical structural characteristics during a computational process.

[0438]

[0439] Table 1

[0440] In vitro growth assays for probiotic and carbohydrate candidates were performed using BioLector™ (m2p-labs, Baesweiler, Germany). Each run was conducted in a 48-well flowerplate (m2p-labs, Baesweiler, Germany) with a volume of 1 mL per well. Strains were cultured in sugar-free MRS medium (MRSapi) with varying carbohydrates added (final concentration 1%). Plates were anaerobically grown in a CO2 atmosphere with shaking at 300 rpm for 48 h. All cultures were inoculated using 2% fresh overnight culture. Biomass and pH changes in each well were recorded during incubation, as measured by optical density (OD) at gain 30. After each run, all data were compiled into an Excel spreadsheet for further analysis.

[0441] Preparation of ingredient combinations for preclinical and clinical trials

[0442] The probiotics and lipid components are supplied by Nestlé or its subsidiaries (e.g., Sofinol SA, Konolfingen Nestle Factory). The carbohydrate components are sourced from various food-grade suppliers, such as Olygose (France), AIDP Inc (USA), and Savanna Ingredients GmbH (Germany). The microbial composition is produced through a multi-step process:

[0443] Step 1: Encapsulation of the lipid oil component: BiPro was hydrated into water (Ystram) with stirring, and the lipid oil (Polytron) was slowly added. The resulting mixture was heated at 82°C with stirring for 10 minutes to fix the encapsulation.

[0444] Step 2: Spray drying of lipid-prebiotic components: The prebiotics were suspended in water and mixed with the lipid suspension. The mixture was then homogenized (Ystram), pasteurized at 72°C for 2 min, and then spray-dried at 140°C. The final product was a white powder.

[0445] Step 3: Adding Probiotics: Add probiotics to the spray-dried prebiotic-lipid powder mixture. Then use Turbula to ensure the probiotics are evenly redistributed into the powder.

[0446] Method 2: In vitro / ex vivo study of the correlation between microbial components and the improvement of uremic toxin production

[0447] Preservation of fecal samples

[0448] Fecal material was collected from eight CKD and nine healthy adult donors. Fecal suspensions were prepared and mixed with a ProDigest-optimized cryoprotectant, an improved version of the cryoprotectant developed by Hoefman et al. (2013). The resulting suspensions were aliquoted, rapidly frozen, and then stored at -80°C (cryostock). Just before the experiment, the aliquots were thawed and immediately added to the reactor.

[0449] Short-term colonic assay to evaluate the differences in metabolic profiles between the microbiomes of individuals with CKD and healthy individuals.

[0450] Short-term colonic experiments are conducted in a single reactor to study overall fermentation activities, such as glycolysis and proteolysis, as well as changes in microbial metabolite production from the fecal microbiome of healthy and CKD individuals. Short-term colonic experiments represent the human microbial ecosystem (…). A simplified simulation of the ProDigest continuous simulator.

[0451] At the start of the experiment, an amino acid mixture consisting of L-tryptophan, L-tyrosine, L-carnitine, choline, and L-phenylalanine was added to the reactor along with a sugar-depleted nutrient background medium (containing the basic nutrients for the colon, including peptone, yeast extract, mucin, and L-cysteine). Then, 10% (v / v) of a frozen stock solution containing 7.5% fecal inoculum from each of the study donors (which served as the microbial source) was added to bring the total volume in the reactor to 70 mL. For each donor, a reference condition (or negative control) containing only nutrient medium (without AA spikes) was included.

[0452] The reactor was incubated at 37°C for 48 h under continuous gentle shaking (90 rpm) and an anaerobic atmosphere. The incubation was carried out in a completely independent reactor with a sufficiently large volume to ensure not only robust microbial fermentation but also the ability to collect multiple samples over time (1h, 24h, 48h).

[0453] Evaluate changes in pH, gases, short-chain fatty acids, ammonium, lactate, uremic toxins, and precursors in samples at different time points.

[0454] An adjusted SHIME setting to assess the impact of microbial components on the microbiome from CKD patients.

[0455] To optimally address the ability of interventions to correct microbiome dysbiosis in CKD patients, prodigestion is modulated by combining the upper gastrointestinal tract with a single colonic region. The colonic region was set to simulate transverse colon (TC) conditions, with a pH of 6.2 to 6.6 and a retention time of 32 hours. This allows for the maintenance of diverse microbial communities within the system, enabling both glycolysis and proteolytic fermentation processes. During this specific study, the effects of the two test products on the microbiome composition and metabolic activity of eight CKD donors were evaluated compared to negative controls for each of the tested donors, resulting in 24 different test conditions.

[0456] This study The experiment consists of two phases:

[0457] (1) Inoculation period: On day 1, a suitable fecal sample (frozen as part of study phase 1) was inoculated into the colonic reactor and allowed to grow and colonize. After this overnight incubation, the colonic reactor was fed with a basic nutrient substrate for two days to support the maximum diversity of the gut microbiota initially present in the fecal inoculum. This also allowed the microbial community to differentiate in different reactors according to local environmental conditions while still maintaining its CKD characteristics.

[0458] (2) Control / Treatment Period: During this 11-day period, the SHIME reactor was operated under nominal conditions and fed 3x / day with SHIME nutrient medium. On day 1 of this period (=d0), all cohorts were operated under nominal conditions, and samples collected on this day provided baseline parameters. Starting on day 2 of this period (=d1), the diet of the treatment cohort of each donor was supplemented with the test product, while the nominal conditions were maintained in the control cohort of each donor. On day 8 of this period, all SHIME cohorts were further supplemented with an amino acid mixture. Samples were taken from the colon reactor throughout this period to investigate the specific effects of the test product on intrinsic microbial community composition and activity compared to the negative control.

[0459] The model was fed with SHIME nutrient medium three times a day during each period. The feeding schedule is shown in the table below (Table 2).

[0460] Table 2

[0461]

[0462]

[0463] Samples were collected at different time points (d0, d1, d2, d4, d7, d8, d10) and their changes in pH, gas, short-chain fatty acids, ammonium, lactate, uremic toxins and precursors were evaluated.

[0464] Analysis of microbial community composition and activity

[0465] The large volume in the colonic region allows for the collection of sufficient fluid volume daily without disturbing the microbial community or jeopardizing the rest of the experiment. Several microbial parameters were monitored in short-term colonic and SHIME experiments.

[0466] (a) Overall fermentation activity :

[0467] • pH: The degree of acidification during the experiment is a measure of the metabolic intensity of the bacteria and is used as a parameter for treatment controls. The pH of the incubation provides a rough indication of the fermentation rate of the test product.

[0468] • Gas production: Incubation is carried out in a closed system, allowing the accumulation of gas in the headspace to be measured using a pressure gauge. Gas production is a measure of microbial activity and therefore also of fermentation rate. H2 and CO2 are the first gases to be produced; they can then be used as substrates for CH4 production, thus reducing the gas volume. H2 can also be used to reduce sulfate to H2S, which is produced by proteolytic fermentation. Therefore, N2, O2, CO2, H2, and CH4 constitute 99% of the intestinal gas volume. The remaining 1% consists of NH3, H2S, volatile amino acids, and short-chain fatty acids. Each measurement is performed in a single replicate.

[0469] • Acid / Base Consumption: The production of microbial metabolites in the colonic reactor alters the pH. Without continuous pH control (by adding acid or base), the pH will exceed fixed intervals. Acid / base consumption should be continuously monitored throughout the experiment.

[0470] (b) Microbial community activity :

[0471] Short-chain fatty acids (SCFAs): The patterns of SCFA production are used to assess microbial carbohydrate metabolism (acetate, propionate, and butyrate) or protein metabolism (branched-chain CFAs) and can be compared to typical fermentation patterns in normal GI microbiota. Quantitative analysis of SCFAs is performed by capillary gas chromatography coupled with a flame ionization detector (FID). SCFA separation is achieved by liquid-liquid extraction (De Boever et al. 2000).

[0472] • Lactate: The human gut carries bacteria that produce and consume lactate. Lactate is produced by lactic acid bacteria and lowers the pH of the environment, thus acting as an antimicrobial agent. It can also be rapidly converted into propionate and butyrate by other microorganisms. Lactate concentration is determined using the Enzytec™ kit (R-Biopharm).

[0473] • Ammonium: Ammonium is a product of protein hydrolysis and degradation. Protein hydrolysis and fermentation lead to the formation of potentially toxic or carcinogenic compounds such as p-cresol and p-phenol. Indophenol blue spectrophotometry (IPB) is used.

[0474] The method involves determining the ammonium concentration in a sample through colorimetric analysis.

[0475] Branched-chain SCFAs (BCFAs; isobutyric acid, isovaleric acid, and isohexanoic acid) are biomarkers for protein hydrolysis fermentation. Quantitative analysis of BCFAs was performed by capillary gas chromatography coupled with a flame ionization detector (FID). BCFAs were separated by liquid-liquid extraction (De Boever et al. 2000).

[0476] • Uremic toxins: Concentration levels of uremic toxins (e.g., p-cresol, p-toluene sulfate, indole, indole-3-acetic acid, betaine, trimethylamine, trimethylamine-N-oxide, indolephenol, indolesulfonate, semialdehyde glutaric acid, uric acid, and urea) were determined using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry and fluorescence detection.

[0477] • Non-targeted metabolomics: Measuring the microbial metabolome, including but not limited to bile acids and amino acids, using Prodigest's Metakey platform.

[0478] Microbial community composition:

[0479] • Quantitative deep-bird gun sequencing was used to measure community composition.

[0480] Statistical description

[0481] A paired two-tailed t-test is used for statistical comparisons between different cohorts within each donor, condition, and treatment. A difference is considered statistically significant if the p-value is less than 0.05.

[0482] Method 3: Animal experiments

[0483] animal models

[0484] mouse model

[0485] Animal experiments are conducted at CarMeN Laboratory, Direction Départementaledes Services Vétérinaires du All experiments were conducted in accordance with guidelines established by the French Ministry of Agriculture and the European Union Council Directive for the Care and Use of Laboratory Animals. C57BL / 6J mice were purchased from Janvier SA (Le Genest-Saint-Isle, France) and housed in an air-conditioned controlled environment at 21°C ± 0.5°C and 60%–70% humidity, under a 12-hour light / dark cycle (light from 07:00 to 19:00), with free access to food and water. Moderate CKD was induced by a two-step surgical procedure involving 5 / 6 nephrectomy. Additional animals underwent sham surgery and served as control mice. An overview of in vivo animal experiments can be found in [reference needed]. Figure 14 .

[0486] Animal diet and processing

[0487] From week 0 to week 3, all animals were fed a standard rodent diet (SAFEA04 standard diet for rodents). Thereafter, animals were fed either the standard diet or a customized version of the standard diet containing 1% (wt / wt) of the present invention's nutrient symbiotic blends P1 and P2 for a total of 7 weeks. The P1 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, and short-to-medium chain triglycerides containing butyrate and caprylate. The P2 intervention consisted of a blend containing *Lactobacillus johnsonii* NCC533, cellobiose, pea GOS, and short-to-medium chain triglycerides containing butyrate and caprylate. Customized diets containing the nutrient or symbiotic blends were prepared by dry-blending the powdered version of SAFE A04 with the blends. Successful incorporation was verified, including measurements of probiotic activity. After verification, the customized diets were pelleted according to the standard SAFE procedure. During pellet production of the customized diets, probiotic activity decreased by 3 logs. Therefore, to ensure animals receive sufficient live probiotics, additional probiotics are provided to the treated animals by dissolving powdered probiotic stock solution in sterile distilled water to achieve a final concentration of approximately 1.8 ± 8 CFU / ml. The viability of the probiotics in sterile drinking water is confirmed by plate bonding. The probiotic solution is changed every 2 days.

[0488] Intraperitoneal glucose tolerance test (IPGTT)

[0489] After fasting overnight, an intraperitoneal glucose tolerance test (glucose, 1 g / kg body weight) was performed. The blood glucose level was determined using a drop of blood taken from the distal part of the tail using an automated glucose monitor (Accu-Check Performa, Roche, Meylan, France).

[0490] Urine parameters

[0491] Urine was collected over 24 hours in a metabolic cage to evaluate urinary output and biomarkers of renal function, such as protein, creatinine, and albumin levels, for metabolomics analysis. Biomarkers of renal function were measured using commercial assays. Reverse HPLC coupled with a fluorescence detector or Prodigest was used. The platform measures urinary metabolomics.

[0492] Biochemical and metabolomics measurements

[0493] By using reverse HPLC coupled with a fluorescence detector or using Prodigest The technology quantifies uremic toxins in plasma and urine. Commercial assays are used to determine plasma or serum concentrations of creatinine, cholesterol, triglycerides, free fatty acids, adiponectin, insulin, cysteine ​​protease inhibitor C, and urea. (Using Prodigest) The platform performs serum metabolomics.

[0494] Kidney histology

[0495] In mice undergoing sham surgery and nephrectomy, residual kidneys were removed upon sacrifice. The kidneys were harvested and unsealed. Histological lesions of the kidneys were analyzed after hematoxylin and eosin (HES) staining and Sirius red staining. Briefly, the kidneys were fixed in 4% formalin for 24 hours and embedded in paraffin after routine treatment. The levels of interstitial inflammation, interstitial fibrosis, and glomerular sclerosis on histological slides were examined independently on a blinded basis.

[0496] Ileal histology

[0497] Ileal samples were collected at the end of the experiment. The ileum was fixed in 4% formalin for 24 hours and embedded in paraffin after routine processing. The sections were then immunofluorescently stained with rabbit antibody against the closure protein, followed by goat anti-rabbit secondary antibody. Images were captured using confocal microscopy, and the relative fluorescence intensity of the closure protein was quantified using ImageJ software.

[0498] Statistical analysis

[0499] In each experiment, multiple mice were analyzed as biological replicates. Dot plots with linear scaling show the arithmetic mean. Bar plots represent the mean ± standard error of the mean (SEM). GraphPad Prism version 9 was used for statistical analysis. For comparisons between two groups, significance was determined using a two-tailed Student's t-test or a nonparametric Mann-Whitney test. For comparisons of more than two groups, an uncorrected Fisher LSD test was performed after one-way (ANOVA). Differences were considered significant at p ≤ 0.05.

[0500] rat model

[0501] Rat model experiments were conducted at the fully AAALAC-certified Grubra facility, and all animal experiments were performed in accordance with Grubra's bioethical guidelines, which fully comply with internationally recognized principles of laboratory animal care and use. All experiments were authorized by the Danish Animal Experimentation Council. In Wistar RjHan:WI rats (Janvier, France), 5 / 6 nephrectomy (Nx) was performed under isoflurane anesthesia using a two-step surgical procedure.

[0502] Animal diet and processing

[0503] Animals were treated as follows. At week 4, a two-step nephrectomy (Nx) procedure was initiated. From day -2, animals were fed a standard rodent diet (SAFE A04 standard rodent diet). On day 1, animals were fed either a standard rodent diet or a customized diet according to their group. The customized diet included cellobiose and short- and medium-chain triglycerides containing butyrate and caprylate, and Lactobacillus johnsonii NCC533 (10 8 CFU is administered via tube feeding. This regimen is given once daily for 8 weeks.

[0504] Example 1 - Identification of the optimal commensal organism for improving the accumulation of uremic toxins

[0505] Based on the lack of uremic toxin-associated enzymes encoded in the bacterial strain genome, calculations were performed on candidate probiotics. Machine screening and identification .

[0506] The European Uremic Toxin Work Group has listed 90 compounds considered uremic toxins (Yavuz et al., 2005). In this example, gut-derived and plasma-bound uremic toxins were examined, including urea, trimethylamine N-oxide (TMAO), indole-3-acetic acid (IAA), indophenol, and p-cresol. The bacterial metabolic pathways that produce these uremic toxins were compiled from public databases such as the Kyoto Encyclopedia of Genes and Genomes and from the literature.

[0507] Different biosynthetic pathways of uremic toxins were summarized, and each enzyme potentially catalyzing different steps was depicted using its EC number (Enzyme Commission Number). Figure 1 The conversion of urea to ammonia (NH3) and the conversion of carnitine or choline to TMAO require only one or two steps. However, the conversion of tryptophan to IAA and indophenol, and the conversion of tyrosine to p-cresol, require several steps. Detailed analysis of the first and last reactions of each pathway was conducted to determine the ability of each bacterium to synthesize the selected uremic toxin. In this embodiment, the enzymes involved in these pathways are referred to hereinafter as key enzymes.

[0508] In this embodiment, the suitable probiotic strains analyzed are as follows:

[0509] ● Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446);

[0510] ● Bifidobacterium longum infantile subspecies NCC 341 (ATCC 15697(T));

[0511] ● Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618) (NCBIrefseqGCA_000007525.1);

[0512] ●Enterococcus faecium NCC 2768 (NCIMB 10415);

[0513] ● Lactobacillus johnsonii NCC 533 (CNCM I-1225) (NCBIrefseqGCA_000008065.1);

[0514] ● Lactococcus lactis NCC 2287 (CNCM I-4154);

[0515] ● Lactobacillus paracasei NCC 2461 (CNCM I-2116);

[0516] ● Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724);

[0517] ● Staphylococcus aureus NCC 1052 (CNCM I-5400);

[0518] ● Staphylococcus aureus NCC 971 (CNCM I-5398);

[0519] ●Streptococcus thermophilus NCC 2496 (CNCM I-3915);

[0520] The presence of key enzymes catalyzing uremic toxin synthesis in the selected strains is described as follows: Figure 3 In the process of creating the reference protein sequence file, sequences from SwissProt and proteins from bacteria phylogenetically as closely related as possible to the aforementioned probiotic strains were reviewed. For example... Figure 3 As observed, *Lactobacillus johnsonii* NCC 533 unusually lacks any of the key enzymes encoded in its genome, and is therefore predicted to have the lowest probability of producing any of the uremic toxins of interest. This strain was also selected for the following computational prebiotic carbohydrate selection and in vitro growth assays.

[0521] Computer-based screening of prebiotic carbohydrates based on the presence of CAZy encoded in the genomes of candidate probiotics. and identification .

[0522] Based on the structure of candidate prebiotic carbohydrates and their likelihood of being degraded, metabolized, fermented, or broken down by candidate probiotics, the computer selects candidate prebiotic carbohydrates. As an example, candidate prebiotic carbohydrates capable of being degraded by the probiotic *Lactobacillus johnsonii* NCC533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1) are analyzed and shown below. Because the enzyme activities are highly similar, these findings can be transferred to other suitable probiotic strains analyzed above.

[0523] The genome sequence of the probiotic *Lactobacillus johnsonii* was examined to identify all the carbohydrate-active enzymes (CAZy) it carries. Table 1 summarizes the CAZy-related coding regions, CAZy family annotations, EC numbers of the encoded enzymes, functions, identified fiber components, and availability of carbohydrate substrates.

[0524] Lactobacillus johnsonii exhibits the presence of all the necessary carbohydrate-active enzymes (CAZY), enabling it to degrade the following fibers: α-galacto-oligosaccharides (raffinose family), galactomannan, β-galacto-oligosaccharides, cellulosic oligosaccharides, cellulose oligosaccharides, α-galacto-oligosaccharides (raffinose family), and ScFOS / inulin.

[0525] In vitro growth test of candidate probiotics and carbohydrates .

[0526] In this embodiment, the probiotic strain Lactobacillus johnsonii was used for growth testing and evaluation to confirm computer selection and define the optimal symbiotic to help improve uremic toxin retention.

[0527] A variety of carbohydrates were tested. Cellulose polymers and oligomers containing galactose and glucose as their main components were selected. For example, α-linked and β-linked galactooligosaccharides (GOS) were tested, as well as mannan containing galactose as a side chain. Additionally, β-linked glucose polymers, including cellulose oligosaccharides, purified β-glucans of three molecular weights (high, medium, and low) from barley, and the soluble fraction of enzymatically hydrolyzed wheat fiber, were tested. The constituent monomers (glucose, fructose, galactose) of each cellulose component were also analyzed in the experiments to better understand which fractions could be metabolized by probiotic strains.

[0528] like Figure 4 As indicated, *Lactobacillus johnsonii* NCC 533 can also grow on carbohydrates belonging to the α-galacto-oligosaccharide, galactomannan, or fructan families. Specifically, it can grow on commercially available fiber from peas or soybeans. Growth was observed on these substrates. For all those carbohydrates, a pH decrease was observed, indicating that *Lactobacillus johnsonii* NCC 533 grew adequately on these substrates. Similarly, *Lactobacillus johnsonii* NCC 533 grew well in the presence of commercial fiber. Equivalent growth in the case of GOS and BMO ( Figure 5 On the other hand, inulin, ScFOS, and PHGG provide minimal support for the growth of Lactobacillus johnsonii NCC533. Figure 4 ).

[0529] In addition, such as Figure 6 As shown, *Lactobacillus johnsonii* NCC 533 can grow efficiently on various carbohydrates belonging to the cello-oligosaccharide family (from disaccharides to tetrasaccharides), especially on cellobiose and cellotriose. It can also grow efficiently on cellotetraose and soluble hydrolyzed wheat and oats containing cellobiose and cellotriose, but to a lesser extent or at a later time.

[0530] result

[0531] In this embodiment, a total of 14 probiotic strains were studied, including those mentioned above and Figure 3 The strains listed are shown. *Lactobacillus johnsonii* NCC 533 does not encode any of the key enzymes required for the production of uremic toxins, such as ammonia, TMAO, IAA, indophenol, and p-cresol. Furthermore, *Lactobacillus johnsonii* NCC 533 encodes many enzymes that metabolize different carbohydrates. Based on its CAZy profile, 15 fibers were proposed for growth studies. *Lactobacillus johnsonii* NCC 533 shows promise in several commercially available fibers, such as those from soybeans or peas. It was grown on GOS and BMOS. Furthermore, the results confirmed that it did indeed metabolize all the monomers that form those fibers (e.g., glucose, fructose, galactose). Therefore, it can be concluded that this strain is capable of completely hydrolyzing those fibers and using the constituent monomers as growth substrates. β-galacto-oligosaccharides (such as...) GOS (Glycosaminoglycans) are now widely used in infant nutrition and have consistently demonstrated significant bifidobacterium-producing effects. This type of fiber is one of the few prebiotic components recognized by the International Scientific Association of Prebiotics and Probiotics (ISAPP). On the other hand, α-galacto-oligosaccharides (GOS) are an emerging component. They are byproducts of the plant protein industry and are naturally found in legume seeds. Research on GOS is limited, but it consistently shows a positive impact on the glycolysis and short-chain fatty acid abundance of producing bacteria such as Bifidobacteria and Lactobacillus.

[0532] Lactobacillus johnsonii NCC 533 is also capable of growing on cellobiose and related oligosaccharides (cellotriose and cellotetraose). While cellobiose is now commercially available at feed grade, this is not the case for cell oligosaccharides. Cell oligosaccharides are dimers and trimers composed of any cellulose-based fiber and / or mixed-linked β-glucan from oats or barley. Therefore, components enriched with cell oligosaccharides can be produced through optimized enzymatic hydrolysis of cellulose-based fiber / cereal or mixed-linked β-glucan. In combination with other Lactobacillus strains, cellobiose and / or cell oligosaccharides in the form of β-glucan hydrolysates have shown potential to exert synbiotic effects, particularly in increased intestinal glycolytic metabolism.

[0533] Other fibers tested (e.g., inulin, ScFOS, PHGG, and high / medium / low molecular weight β-glucan) minimally support *Lactobacillus johnsonii* NCC 533, indicating that this strain may not be able to completely hydrolyze polymers into their constituent monomers. However, based on the CAZy annotation of *Lactobacillus johnsonii* NCC 533, it may partially hydrolyze them into smaller fragments or oligomers. If the strain does indeed partially hydrolyze those substrates, this would suggest the possibility of cross-feeding with other microbial inhabitants of the same ecosystem in complex environments such as the gut.

[0534] In summary, these results indicate the beneficial effects of co-administration of Lactobacillus johnsonii NCC 533 and carbohydrates belonging to multiple families, including α-galacto-oligosaccharides such as pea GOS and fiber oligosaccharides such as cellobiose.

[0535] Example 2 - In Vitro (ProDigest) Study

[0536] Research Design

[0537] Several publications have demonstrated the dysbiosis profile of the microbiome in CKD patients. Specifically, altered microbial composition and function have been characterized in CKD patients compared to healthy individuals. Therefore, in this embodiment, we tested the ability of the present invention to correct microbiome dysbiosis in CKD patients.

[0538] For this purpose, fecal microbiota were collected from healthy individuals (controls) and patients with chronic kidney disease (CKD). Table 3 provides an overview of the characteristics of eight CKD patients and nine healthy adult donors who provided fecal microbiota for in vitro / ex vivo experiments. The patients were part of a clinical study registered with ClinicalTrials.gov identifier NCT04768309. Table 3 illustrates the characteristics of these donors. These fecal microbiota were then used in the experimental setup using Prodigest's in vitro / ex vivo techniques.

[0539] Table 3

[0540]

[0541]

[0542] In this embodiment, two studies are conducted sequentially.

[0543] First, the differences in fecal microbiome profiles between healthy and chronic kidney disease (CKD) adult donors were assessed using Prodigest's short-term single-stage colonic simulation technique (see [link]). Figure 7 (and its description to obtain further experimental details).

[0544] Secondly, using the improved Prodigest Techniques to assess the effects of new nutrients or synergistic components on the fecal microbiota of patients with chronic kidney disease (CKD) (see [link]). Figure 9 (and its description to obtain further experimental details).

[0545] For the second study, eight CKD donors were used, and two component blends (P1 cohort and P2 cohort, see below; the compositions of P1 and P2 are indicated below) and a control were tested. Therefore, three conditions were implemented for each CKD microbiota:

[0546] • Control (CTRL) cohort: SHIME units fed with basic nutrient medium, used to determine baseline parameters for each donor to compare with the treatment cohort.

[0547] • P1 Cohort: During one daily cycle, the SHIME unit is fed with basal nutrient medium (intake 1 hour in the colon). During the other two cycles, this SHIME unit is fed with carbohydrate-depleted nutrient medium and supplemented with nutrients or synergistic components consisting of cellobiose, butyrate, and caprylic acid, as well as the probiotic Lactobacillus johnsonii NCC 533.

[0548] • P2 Cohort: During one cycle per day, the SHIME unit is fed with basic nutrient medium. During the other two cycles, this SHIME unit is fed with carbohydrate-depleted nutrient medium and supplemented with cellobiose, pea GOS, butyrate and caprylic acid, and the probiotic Lactobacillus johnsonii NCC 533.

[0549] Additionally, an amino acid mixture was added to a colonic concentration of 2.5 g / L to provide a substrate for testing whether the invention could improve the overproduction of uremic toxins and precursors. The amino acid mixture consisted of 23% L-phenylalanine, 20% L-tryptophan, 23% L-tyrosine, 10% L-carnitine, and 23% choline. The amino acid mixture was added on days 8 (1 h in the colon), 9 (1 h in the colon), and 10 (1 h in the colon) of the experimental run during one feeding cycle per day, and this was used for both the control and treatment cohorts for each donor.

[0550] result

[0551] Fecal microbiota from CKD patients showed abnormalities in amino acid and protein metabolism.

[0552] To verify whether in vitro / ex vivo models are reliable models for testing the efficacy of the present invention in regulating the CKD microbiome, fecal microbiota from CKD patients and healthy donors were inoculated into the Prodigest system.

[0553] like Figure 8 As shown, the CKD microbiota exhibits higher production of uremic toxin precursors (such as p-cresol), especially in the presence of excess amino acid substrates. In terms of proteolytic activity, the CKD microbiota also shows higher concentrations of proteolytic markers, such as branched-chain fatty acids.

[0554] Nutritional or symbiotic blends reduce the production of uremic toxin precursors from the microbiota of CKD patient donors.

[0555] The ability of the present invention to improve the overproduction of uremic toxins and their precursors was evaluated in the presence of low amino acid (AA: days 0 to 7) and additional AA substrates (days 8 to 10). In this embodiment, three uremic toxin precursors are shown, including indole, p-cresol, and trimethylamine (TMA) (see [link to original text]). Figure 10 It is hypothesized that the addition of AA will induce a further increase in the production of uremic toxins and their precursors by the fecal microbiota.

[0556] The gut microbiota can metabolize indole using tryptophan as a precursor molecule; this process first converts tryptophan to tryptophan and indolepyruvate, and then indolepyruvate is converted to indole. Indole is known to have profound effects on gut microbiota composition, microbial metabolism, the host immune system, the host-microbiome interface, and host immune system-gut microbiota interactions. Once produced by gut bacteria, indole is absorbed into the portal circulation and enters the liver. Hepatic hydroxylation of indole produces 3-hydroxy-indole (indolephenol), most of which is then sulfonated to indolesulfonate. Indolesulfonate is considered a uremic toxin and is most commonly believed to be a contributing factor to the progression of kidney disease and cardiovascular complications. In addition, indolesulfonate also has adverse effects on the skeletal and central nervous systems. On the day before administration of the AA preparation (d7), significantly lower indole concentrations were detected for both P1 and P2 compared to the untreated control group. Figure 10 Following administration of the AA formulation, a strong increase in indole concentration was observed in all donors, consistent with the fact that tryptophan was provided as an indole precursor molecule.

[0557] p-Cresol, a phenolic compound, is a microbial metabolite produced from tyrosine, forming the intermediates 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid. In the liver, p-cresol is sulfated and converted to the toxic metabolite p-toluene sulfate. Mechanistic studies have linked this specific metabolite to oxidative stress, endothelial dysfunction, proximal tubular injury, and insulin resistance. In this regard, a positive correlation has been demonstrated between p-toluene sulfate levels and overall mortality, cardiovascular disease, and CKD progression. Figure 10 As shown, prior to AA spiking (periods d0 to d7), the P1 and P2 treatments defined a reduction effect compared to the untreated control. As expected, administration of the AA formulation to all donors in the blank control group resulted in increased p-cresol concentration levels compared to the previous period. Interestingly, both P1 and P2 were able to improve p-cresol overproduction compared to the blank control.

[0558] Trimethylamine (TMA) is a metabolite produced by the gut microbiota in the intestinal lumen, with various dietary quaternary amines serving as precursor molecules. These precursor molecules primarily include choline and carnitine, but also include betaine, γ-butylbetaine, and other choline-containing compounds. In vivo, the produced TMA is rapidly absorbed into the portal circulation via passive diffusion, and then oxidized to trimethylamine N-oxide by heparin-containing monooxygenases. Trimethylamine N-oxide (TMAO) is involved in oxidative stress, inflammation, cardiac fibrosis, endothelial injury, and platelet inactivation. Therefore, TMAO has been identified as having biological roles in several chronic non-communicable diseases, including CKD. Figure 10As shown, an increase in TMA concentration levels was observed in the blank control group during the period from d0 to d7. P1 and P2 showed lower concentration levels compared to the blank control group, with the largest change observed at P22. Following administration of the AA formulation, a significant increase in trimethylamine concentration was observed for all donors and experimental conditions, which was associated with increased availability of the substrate for TMA production. The significant effect of P2 compared to the blank control was calculated when statistical analysis was performed on all donors at time point d10.

[0559] In summary, these data highlight the efficacy of this invention in correcting amino acid metabolism abnormalities in the microbiome of CKD patients. Notably, the invention is effective under both low and normal amino acid levels, suggesting that its benefits will be applicable to diverse patients with varying dietary restrictions, such as low-protein diets.

[0560] Nutrients or symbiotic blends reduce urea production.

[0561] Urea is a metabolite produced through the urea cycle, a metabolic pathway that removes excess endogenous and exogenous nitrogen from the body by detoxifying ammonia into urea. Typical nitrogen sources in this regard include amino acids such as ornithine, arginine, aspartic acid, and glutamic acid. Although the urea cycle primarily occurs in the liver, the gut microbiota also facilitates the mitochondrial urea cycle. High urea concentrations are commonly observed in CKD patients, and it is known to significantly alter the gut microbiota, leading to a decrease in bacterial strains that produce anti-inflammatory and fuel molecules, and an increase in bacterial strains that metabolize urea but also produce uremic toxins (including indophenol sulfate and p-cresol sulfate). Furthermore, high urea concentrations may also lead to increased intestinal permeability and a toxic environment that induces the colonization of bacteria expressing urease and uricase to reduce urea to ammonia. Ammonia increases intestinal pH, promoting an increase in pathogens. Because amino acid preparations contain a variety of nitrogen sources, spiking these preparations can induce an increase in urea concentration levels during colonic incubation. However, it should be noted that amino acids, as part of the relevant formulation, have not been reported to specifically participate in the urea cycle. In this regard, the final observed urea concentration level can be defined by the general alteration of the microbial composition of the amino acid spikes, as well as specific alterations of those bacterial strains that metabolize urea.

[0562] like Figure 11 As shown, the concentration profile trend was increasing during periods d0 to d7 and d10. Note the decreasing effects of P1 and P2 on urea concentration levels, with P1 having the strongest effect. As expected, the application of AA formulations generally had a limited effect on urea concentration levels.

[0563] This data highlights the efficacy of this invention in reducing the production of uremic toxins by the gut microbiota. Furthermore, this invention can provide relief from intestinal changes and symptoms associated with excessive urea production, including intestinal permeability and inflammation.

[0564] Nutrients or symbiotic blends increase markers of reduced protein hydrolysis and fermentation.

[0565] The production of ammonium and branched-chain SCFAs (the sum of isobutyric acid, isovaleric acid, and isohexanoic acid) is both caused by protein degradation and reflects the proteolytic activity of the gut microbiota. Since the latter is associated with direct and indirect adverse health effects (such as colon cancer), a reduction in ammonium / branched-chain SCFA production is considered beneficial.

[0566] like Figure 12 As shown, branched SCFA levels increased throughout the experiment in all cohorts for each donor. The supplementation with the two-component blends (P1 and P2, see above) systematically reduced branched SCFA production in all donors compared to the control (see also above). This was also observed in the mean effects of the eight donors on days 7 and 10, where a significant reduction in branched SCFA production was observed in the treatment cohorts compared to the blank control, with the strongest effect observed in P2.

[0567] A similar effect was observed with respect to ammonium levels. Figure 12 Throughout the experiment, both P1 and P2 strongly reduced ammonium production. This was also observed on days 7 and 10, when averaged across eight selected donors, with both treatments significantly reducing ammonium levels compared to the control. The strongest effect was observed after repeated administration of P2, resulting in an even stronger and more significant reduction compared to P1 on days 7 and 10.

[0568] This data supports the efficacy of this invention in correcting ecological imbalances in the CKD microbiome, particularly abnormal protein metabolism.

[0569] Nutrients or symbiotic blends increase the production of short-chain fatty acids (SCFAs).

[0570] SCFA production is caused by carbohydrate metabolism in the colon and is associated with a variety of health effects. The most abundant SCFAs are acetate, propionate, and butyrate. SCFAs are well known to play a key role in gut health. Acetic acid can be used as an energy source for the host and as a potential substrate for lipid synthesis in the body. In addition, it is an important byproduct of butyrate synthesis and can exert antimicrobial effects against pathogens. However, the health-promoting effects are mainly attributed to propionate and butyrate, which serve as the main energy source for the intestinal epithelium and have shown protective effects against inflammation and colon cancer. The former is also known to be transported to the liver, where it has a cholesterol-lowering effect in plasma and positively influences glycemic control. In summary, the beneficial effects of the substrates studied on SCFA production therefore include increased production of acetate, propionate, and / or butyrate. For best interpretation, SCFA levels under two test conditions are presented for each of the different SCFAs.

[0571] Acetate is one of the key metabolites in the human gut and is therefore produced by a wide range of gut microbiota, including many other Bacteroides spp. (phylum Bacteroidetes) and Bifidobacteria. For example... Figure 13 As shown, compared with the control, the application of the two-component blends (P1 and P2) of all donors increased total acetate production on average compared with the blank control. P2 exerted the strongest effect. P1, P2 and the control are as defined above.

[0572] Propionate can be produced by a variety of gut microbiota, with the most abundant producers being Bacteroides (phylum Bacteroides), Veillonella (phylum Firmicutes), and Akkermansia muciniphila (phylum Verrucomicrobia). Therefore, the treatment effect on propionate production is donor- and product-dependent. Treatment with P1 and P2 resulted in increased propionate levels, but these increases were not statistically significant due to some variable responses among the eight donors.

[0573] Butyrate is produced by members of the Clostridium genus IV and XIVa (firmbacteria). In a process known as crossfeeding, these microorganisms convert acetate and / or lactate (along with other substrates) into health-relevant butyrate. Strong stimulatory effects of P1 and P2 on butyrate production in specific donors were observed. When averaged across eight selected donors, treatment with P2 alone significantly enhanced butyrate production at the end of the control / treatment period (day 7) before AA spiking, although a similar trend was observed for P1. Finally, after AA spiking during the last 3 days of the experiment, when averaged across different donors, P1 also significantly enhanced butyrate levels compared to the control, reaching levels similar to those achieved with P2 application.

[0574] In summary, the above embodiments support the effectiveness of the present invention in improving the glycolytic activity of the CKD microbiota and enhancing the production of beneficial metabolites such as SCFA.

[0575] Example 3 - In vivo animal study :

[0576] Research Design

[0577] To test the efficacy of this invention in improving uremic toxin accumulation and related symptoms and clinical outcomes, animal models with 5 / 6 nephrectomy were used in the study. Kidneys were ablated during the two-step surgical procedure to reduce renal function (reminiscent of stage 3 and above CKD in humans). Additional animals underwent sham surgery and served as non-CKD animal controls (see [link to study]). Figure 14 (and its description, used to outline the experimental design and analysis).

[0578] From week 3 to week 10, CKD animals were fed diets with or without nutritional or symbiotic blend interventions. In this example, two nutritional / symbiotic blend combinations (P1 and P2) were tested. The P1 intervention consisted of a diet containing 10... 8 The formula consists of Lactobacillus johnsonii NCC533 (this is a correction of the concentration reported in U.S. Provisional Applications Nos. 63 / 439,638 and 63 / 480,729, which reported incorrect concentrations), 1% cellobiose, and a blend of 1% short- and medium-chain triglycerides containing butyrate and caprylate. The P2 intervention consists of a blend containing 10... 8 The composition consists of Lactobacillus johnsonii NCC533 (this is a correction of the concentration reported in U.S. Provisional Applications No. 63 / 439,638 and 63 / 480,729, which reported incorrect concentrations), 1% cellobiose, 1% pea GOS, and 1% a blend of short- and medium-chain triglycerides containing butyrate and caprylate.

[0579] In week 8, a glucose tolerance test was performed. In week 9, mice were placed in metabolic cages to collect urine for uremic toxin and renal parameter analysis. In week 10, mice were sacrificed and different tissues were collected for subsequent analysis.

[0580] result

[0581] Treatment of uremic mice with nutrients or symbiotic blends improved plasma concentrations of clinically relevant uremic toxins.

[0582] To demonstrate the importance of the nutritional or symbiotic blend of the present invention in improving uremic toxins, a 5 / 6 nephrectomy or residual kidney model was used. This model, by virtue of the reduced number of nephrons, represents one of the most commonly used animal models of progressive renal failure, mimicking the condition seen in patients with CKD. Figure 15 As shown, compared with animals with intact kidney function (sham-operated group), animals that underwent nephrectomy (CKD group) exhibited significantly higher plasma levels of clinically relevant uremic toxins (such as p-toluene sulfate (PCS), indophenol sulfate (IS), p-toluene glucuronide (PCG), indoleacetic acid (IAA), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), and uric acid). This demonstrates that CKD animals are in a state of uremia and are therefore a good model for studying the effects of uremia treatment.

[0583] like Figure 15 As shown similarly, compared with CKD animals, elevated plasma uremic toxin levels were significantly reduced in CKD animals treated with the nutrient or symbiotic blends (P1 and P2 groups, see above); thus, this strongly supports the efficacy of the blends for their intended use.

[0584] Nutritional or symbiotic blends stabilize renal function in nephrectomy animals

[0585] We further investigated the benefits of the nutrients or symbiotic blends on renal function and structure. To determine the progression of residual renal functional impairment, proteinuria and blood urea levels were evaluated. Furthermore, progressive microscopic damage was assessed histologically.

[0586] Proteinuria is a symptom commonly seen in conditions affecting the kidneys. Excessive protein in the urine means that the kidney's filtration mechanism—the glomeruli—is not functioning properly, causing excess protein to escape into the urine. Proteinuria is measured by calculating the ratio of protein in the urine to creatinine.

[0587] Blood urea nitrogen levels are an indicator of kidney function. Urea is produced in the liver as a byproduct when proteins are metabolized. Healthy kidneys filter urea out of the body through urine. High urea levels often indicate acute or chronic kidney disease or kidney failure. Urea is also considered one of the toxins associated with clinically relevant uremic conditions.

[0588] like Figure 16 As shown, compared with non-CKD animals (sham surgery), nephrectomized animals (CKD group) exhibited significantly higher proteinuria, confirming the progression of kidney injury. Interestingly, compared with untreated CKD animals, CKD animals treated with nutrition or symbiotic blends (P1 and P2 groups, see above) showed significantly improved proteinuria and blood urea levels.

[0589] like Figure 17 As further shown, progressive renal histological damage was evident in CKD animals compared to sham-operated animals. Animals treated with nutrients or symbiotic blends (P1 and P2 groups, see above) exhibited significantly better histology, as evidenced by lower fibrosis and improved glomerular size and volume compared to untreated CKD animals.

[0590] Overall, the data support the potential benefits of the blends in not only reducing uremic toxin levels in the blood but also helping to stabilize kidney function, as evidenced by the low incidence of markers of kidney damage progression.

[0591] Nutritional or symbiotic blends reduce loss of appetite, weight loss, and lipid profile associated with uremic toxins and chronic kidney disease. fat quality

[0592] Uremic toxicity negatively impacts multiple organ systems and metabolic pathways, leading to organ damage and symptomatic manifestations, including neurological symptoms and protein-energy depletion. Therefore, we tested the ability of nutrients or symbiotic blends to mitigate some of the harmful consequences of uremic toxin accumulation; these include food intake and energy reserves such as muscle and fat.

[0593] like Figure 18 As shown, CKD animals treated with P1 and P2 interventions (see above) exhibited significantly improved weight evolution and food intake normalized to the same level as non-CKD sham-operated animals. There was no significant difference in energy intake between the two groups. Furthermore, compared to untreated CKD animals, the treated animals, particularly those treated with P1, showed reduced adipose mass loss observed in CKD, as indicated by better fat reserves, as shown by improved epididymal white adipose tissue (eWAT).

[0594] Overall, the data support the potential benefits of interventions in alleviating uremia-related symptoms, including loss of appetite, weight loss, and protein-energy expenditure.

[0595] Nutritional or symbiotic blends improve intestinal barrier dysfunction .

[0596] One of the harmful effects of gut dysbiosis and the accumulation of uremic toxins is the induction of alterations in intestinal barrier function. These altered intestinal permeability may also induce a vicious cycle of uremic toxin accumulation in the systemic circulation.

[0597] Therefore, we further tested the potential of novel component blends to improve some of the intestinal barrier dysfunctions described in CKD. For example... Figure 19 As shown, CKD animals exhibit impaired protein expression of important tight junctions (closing proteins) compared to non-CKD animals. Intact tight junctions are crucial for preventing excessive translocation of intestinal contents and molecules into the systemic circulation.

[0598] Therefore, data support the potential benefits of interventions that not only prevent the accumulation of uremic toxins but also alleviate some disease-related intestinal barrier dysfunction.

[0599] Example 4 - In vivo animal study :

[0600] Research Design

[0601] To further confirm the renal protective effect shown in the mouse model, rats with 5 / 6 nephrectomy were treated with a blend containing Lactobacillus johnsonii NCC533, cellobiose, and short- and medium-chain triglycerides containing butyrate and caprylate.

[0602] Nine-week-old Wistar RjHan:WI rats (n=84) underwent right 2 / 3 nephrectomy at week -4 and left total nephrectomy at week -2. Twelve additional rats underwent sham surgery. Rats were randomized to the following treatment groups based on plasma urea, plasma creatinine, and body weight measured one week prior to the start of the study:

[0603] (1) Sham surgery (n=12);

[0604] (2) 5 / 6Nx medium (n=18);

[0605] (3) 5 / 6Nx P1-rat diet (P1-rat intervention consisted of a diet containing 1% cellobiose, 1% short- and medium-chain triglycerides containing butyrate and caprylate administered in the diet, and 10% of the diet administered via tube feeding. 8 Composition of Lactobacillus johnsonii NCC533 blend (n=18);

[0606] (4) 5 / 6Nx P3-diet (P3 intervention consists of 0.3% cellobiose, 0.3% short- and medium-chain triglycerides containing butyrate and caprylate administered in food, and 10% administered via tube feeding) 8 Composition of Lactobacillus johnsonii NCC533 blend (n=18); and

[0607] (5) 20mg / kg 5 / 6Nx lisinopril.

[0608] Lisinopril is an angiotensin-converting enzyme inhibitor (ACEi), which is a standard of care for kidney protection.

[0609] The diet was administered for 8 weeks. Water intake was measured on days 9, 10, 11, 25, 26, 27, 39, 40, and 41. Urine samples were taken at week 7 for albumin and creatinine analysis. At termination, plasma samples were taken for urea, creatinine, indophenol sulfate, and p-toluene sulfate analysis.

[0610] result

[0611] Contains 10 8 A symbiotic composition of *Lactobacillus johnsonii* NCC533, 1% cellobiose, and 1% short- and medium-chain triglycerides containing butyrate and caprylate can reduce plasma uremic toxin concentrations, particularly indophenol sulfate and p-toluene sulfate (see [link to symbiotic composition]). Figure 21 ).

[0612] like Figure 20 As shown in (C), nephrectomized animals (5 / 6Nx mediator group) exhibited higher proteinuria compared to non-CKD animals (sham surgery), confirming the progression of kidney injury. 5 / 6Nx animals treated with nutrients or symbiotic blends (especially P1 blends) showed significantly improved proteinuria levels compared to untreated CKD animals.

[0613] Example 5 - Clinical Trial Summary

[0614] The following is a summary of the clinical trials that form the basis for human clinical trials.

[0615]

[0616]

[0617]

[0618]

[0619] All publications mentioned in the foregoing description are incorporated herein by reference. Various modifications and variations of the compositions, uses, and methods disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the invention protected by the claims should not be unduly limited to such specific embodiments. In fact, various modifications to the modes disclosed for practicing the invention that are apparent to those skilled in the art are intended to fall within the scope of the following claims.

Claims

1. A composition for reducing or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases, wherein the composition comprises: (a) Probiotics, wherein the probiotics are selected from the following probiotic strains: - Lack of a gene for producing at least one of the following: urea, uric acid, p-cresol, p-toluene sulfate, indole sulfate, indole, indoleacetic acid, indolephenol, trimethylamine, TMAO, dimethylglycine, betaine, and / or glutaric acid. - Deficiency of at least one bacterial enzyme selected from urease, carnitine monooxygenase and reductase, tryptophanase and / or hydroxyphenylacetate; and / or - Expresses at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme; (b) A prebiotic selected from carbohydrates or fiber, wherein the carbohydrates or fiber can be hydrolyzed by at least one bacterial enzyme selected from α-galactosidase, β-galactosidase, glucan 1,4-β-glucosidase, cellulase, β-fructofuranosidase and / or lichen polysaccharide enzyme. (c) Lipids selected from triglycerides, short-chain fatty acids and / or medium-chain fatty acids, wherein the triglycerides comprise short-chain fatty acids, medium-chain fatty acids or mixtures thereof.

2. The composition according to claim 1 for the stated purpose, wherein the probiotic (a) is selected from at least one of the following a. to k.: a. Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446); b. Bifidobacterium longum infantile subspecies NCC 341 (ATCC 15697(T)); c. *Bifidobacterium longum* subsp. *longum* NCC 2705 (CNCM I-2618) (NCBI refseq; GCA_000007525.1); d. Enterococcus faecalis NCC 2768 (NCIMB 10415); e. Lactobacillus johnsonii NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1); f. Lactococcus lactis NCC 2287 (CNCM I-4154); g. Lactobacillus paracasei NCC 2461 (CNCM I-2116); h. Lactobacillus rhamnosus NCC 4007 (CGMCC 1.3724); i. Staphylococcus aureus NCC 1052 (CNCM I-5400); j. Staphylococcus aureus NCC 971 (CNCM I-5398); and / or k. Streptococcus thermophilus NCC 2496 (CNCM I-3915); Or a probiotic having a genome with at least 95% average nucleotide identity (ANI), preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% ANI or 99.9% ANI, of any one of the genome sequences of the probiotic as defined above from a. to k.

3. The composition according to claim 2 for the said use, wherein the probiotic (a) is *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1) or has the same characteristics as *Lactobacillus johnsonii* NCC 533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1). The probiotic GCA_000008065.1 (SEQ ID NO:1) has a genome sequence having at least 95% ANI, preferably at least 96% ANI, more preferably at least 97% ANI, even more preferably at least 98% ANI, most preferably at least 99% ANI, or even 99.5% ANI or 99.9% ANI.

4. The composition for the purpose according to any one of claims 1 to 3, wherein the prebiotic (b) is selected from α-galacto-oligosaccharide (α-GOS) / raffinose, β-galacto-oligosaccharide (β-GOS) and cellulosic oligosaccharide (COS) or combinations thereof.

5. The composition for the use according to any one of claims 1 to 4, wherein the prebiotic (b) is selected from soybean GOS (α-GOS), pea GOS (α-GOS), bovine milk oligosaccharide (BMOS) (β-GOS), vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, and soluble hydrolyzed wheat, human milk oligosaccharide (HMO), soluble hydrolyzed oats, and / or β-glucan hydrolysates containing cellobiose, cellotriose, or cellotetraose, or combinations thereof.

6. The composition for the said use according to any one of claims 1 to 5, wherein the lipid (c) is selected from: a. Triglycerides (TG) composed of a mixture of butyrate (C4:0) and medium-chain fatty acids (MCFA); b. Triglycerides (TG) composed of short-chain fatty acids and medium-chain fatty acids (SMCFA), preferably a mixture of C4 and / or C8 fatty acids; c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (such as triglycerides composed of a mixture of butyrate and oleate); d. Triglycerides composed of medium-chain fatty acids; e. Triglycerides (TG) composed of short-chain fatty acids; f. Short-chain fatty acids that can be metabolized into ketone bodies; and g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 fatty acids.

7. The composition for the said use according to any one of claims 1 to 6, wherein: a. The probiotic is selected from the following: at least one of the probiotics as defined in a. to k. above, or a probiotic or combination thereof having a genome with at least 90% ANI, preferably at least 95% ANI, of any one of the genome sequences of the probiotics as defined in a. to k. above, preferably from Lactobacillus johnsonii NCC533 (CNCM I-1225), or a probiotic having a genome with at least 90% ANI, preferably at least 95% ANI, of the sequence of Lactobacillus johnsonii NCC 533 (CNCM I-1225); b. The prebiotic is selected from α-galacto-oligosaccharide / raffinose, β-galacto-oligosaccharide or cellulosic oligosaccharide or combinations thereof, preferably selected from soybean GOS (α-GOS), pea GOS (α-GOS), milk oligosaccharide (BMOS) (β-GOS), vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose or cellotriose or cellotetraose or combinations thereof; c. The lipid is selected from triglycerides, short-chain fatty acids, and / or medium-chain fatty acids, wherein the triglycerides comprise butyrate, short-chain fatty acids, medium-chain fatty acids, or mixtures thereof, preferably derived from… a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA); b. Triglycerides (TG) composed of short-chain fatty acids and medium-chain fatty acids (SMCFA), preferably a mixture of C4 and / or C8 short-chain fatty acids and medium-chain fatty acids; c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids. (such as triglycerides, which are composed of a mixture of butyrate and oleate); d. Triglycerides composed of medium-chain fatty acids; e. Triglycerides (TG) composed of short-chain fatty acids; f. Short-chain fatty acids that can be metabolized into ketone bodies; and g. Short-chain and medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 short-chain and medium-chain fatty acids.

8. The composition according to claim 7 for the stated use, wherein the probiotic (a) is selected from Lactobacillus johnsonii NCC533 (CNCM I-1225) (NCBI refseq; GCA_000008065.1) or has the same characteristics as Lactobacillus johnsonii NCC 533 (CNCMI-1225). Probiotics with a genome containing at least 90% ANI, preferably at least 95% ANI, according to the sequence (NCBIrefseq; GCA_000008065.1).

9. The composition for the said use according to claim 7 or claim 8, wherein the prebiotic (b) is selected from soybean GOS (α-GOS), pea GOS (α-GOS), milk oligosaccharide (BMOS) (β-GOS), vivinal GOS (β-GOS), cellobiose, cellotriose, cellotetraose, soluble hydrolyzed wheat, soluble hydrolyzed oats and / or β-glucan hydrolysates containing cellobiose, cellotriose or cellotetraose, or combinations thereof.

10. The composition for the said use according to any one of claims 7 to 9, wherein the lipid (c) is selected from: a. Triglycerides (TG) composed of a mixture of butyrate and medium-chain fatty acids (MCFA); b. Triglycerides (TG) composed of a mixture of short-chain fatty acids and medium-chain fatty acids (SMCFA); c. Triglycerides (TG) composed of a mixture of butyrate and long-chain fatty acids (preferably triglycerides composed of a mixture of butyrate and oleate); d. Triglycerides composed of medium-chain fatty acids; e. Triglycerides (TG) composed of short-chain fatty acids; f. Short-chain fatty acids that can be metabolized into ketone bodies; and g. Medium-chain fatty acids that can be metabolized into ketone bodies, preferably C4 and / or C8 medium-chain fatty acids.

11. The composition for the said use according to any one of claims 1 to 10, wherein 10 3 CFU / day up to 10 12 The amount between CFU / day dose is usually in increments of 10. 4 CFU / day up to 10 11 The amount between CFU / day dose is preferably in the range of 10. 5 CFU / day up to 10 10 The amount between CFU / daily dose or 10 5 CFU / day up to 10 9 The amount between CFU / day dose is also preferably 10. 6 CFU / day up to 10 9 The amount between CFU / day dose, 10 6 CFU / day up to 10 8 The amount between CFU / day dose or in increments of 10 8 CFU / day up to 10 10 The amount of CFU / day, more preferably about 10 7 CFU / day up to 10 9 CFU / day dose contains the probiotics mentioned above.

12. The composition for the stated purpose according to any one of claims 1 to 11, wherein the prebiotic is contained in an amount from 0.1 g / day to 30 g / day, preferably in an amount from 2 g / day to 15 g / day.

13. The composition for the purpose according to any one of claims 1 to 12, wherein the lipid is contained in an amount from 0.1 g / day to 30 g / day, preferably in an amount from 2 g / day to 15 g / day.

14. The composition for the said use according to any one of claims 1 to 13, wherein the lipid is a triglyceride composed of butyrate and caprylate or a triglyceride composed of butyrate and oleate.

15. The composition for the use according to any one of claims 1 to 13, wherein the lipid is a triglyceride composed of butyrate and caprylate.

16. The composition for the stated use according to any one of claims 1 to 15, wherein the composition is in the form of: food product or nutritional composition, dietary supplement, food for a specific medical purpose (FSMP), nutritional supplement, milk-based beverage, low-volume liquid supplement, functional food product, functional beverage product, meal replacement beverage, and combinations thereof.

17. The composition for the purpose according to any one of claims 1 to 16, wherein the composition is provided in the form of powder, tablet, capsule, or is available in the form of an oil preparation, emulsion, oil-in-water emulsion (o / w emulsion) or water-in-oil emulsion (w / o emulsion).

18. The composition for the stated use according to any one of claims 1 to 17, wherein the reduction of uremic toxins in cardiovascular metabolic or neurodegenerative diseases is used to delay the progression of such diseases and comorbidities and / or to manage symptoms and syndromes associated with the toxic effects of uremic solutes in such diseases and comorbidities.

19. The composition for the said use according to any one of claims 1 to 18, wherein the reduction of uremic toxins in cardiovascular metabolic or neurodegenerative diseases comprises: ○ Treatment or prevention of kidney disease, including chronic and acute; dialysis and predialysis; rare diseases, genetically induced and metabolically induced; ○ Treatment or prevention of uremic syndrome, including protein and energy depletion, bone loss, anorexia, fatigue, or inflammation; ○ Delayed complications of late-stage renal disease, including anemia, hyperphosphatemia, secondary hyperparathyroidism, bone disease, and neurological disorders; ○ Delayed comorbidities of kidney disease, including cardiovascular disease; ○ Preventing the risk of malnutrition or managing malnutrition; ○ Delaying the progression of cardiovascular and metabolic diseases; ○ Prevention of cardiovascular disease and comorbidities (diabetes) or management of cardiovascular disease and comorbidities (diabetes); and / or ○ To prevent or manage the risks of neurodegenerative and neurological disorders.

20. A multipart kit for reducing or preventing the accumulation of uremic toxins in cardiovascular metabolic or neurodegenerative diseases, comprising a composition as claimed in any one of claims 1 to 17, the probiotics, the prebiotics, and the lipids in two or more separate containers, and optionally including instructions.

21. A method for treating cardiovascular metabolic or neurodegenerative diseases as defined above, the method comprising, as a first step, (a) preparing and providing a composition according to any one of claims 1 to 17, the composition comprising the probiotic, the prebiotic, and the lipid; and (b) administering the composition to a patient in need who suffers from an increase in uremic toxins in the case of cardiovascular metabolic or neurodegenerative diseases.

22. A method of treating a cardiovascular metabolic or neurodegenerative disease according to claim 18, the method comprising administering to a patient the composition according to any one of claims 1 to 17.

23. Use of the composition according to any one of claims 1 to 17 in the manufacture of a medicament for treating cardiovascular metabolic or neurodegenerative diseases according to claim 18.

24. A composition for the use of any one of claims 1 to 13 and 16 to 19, a multipart kit according to claim 20, a treatment method according to claim 21 or claim 22, or the use according to claim 23, wherein the lipid is a triglyceride comprising butyrate and / or caprylate.

25. A composition for the said use according to any one of claims 1 to 13 and 16 to 19, a multipart kit according to claim 20 or 24, a treatment method according to any one of claims 21, 22 and 24, or the use according to claim 23 or 24, wherein the lipid is a triglyceride composed of butyrate and caprylate.

26. A composition for the use of any one of claims 1 to 14 and 16 to 19, a multipart kit according to claim 20, a treatment method according to claim 21 or claim 22, or the use according to claim 23, wherein the lipid is a triglyceride comprising butyrate and oleate.

27. The composition for the said use according to any one of claims 1 to 14 and 16 to 19, the multipart kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises Lactobacillus johnsonii NCC533, cellobiose, and short- to long-chain triglycerides containing butyrate and oleate.

28. The composition for the said use according to any one of claims 1 to 14 and 16 to 19, the multipart kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises Lactobacillus johnsonii NCC533, cellobiose, pea GOS, and short- to long-chain triglycerides containing butyrate and oleate.

29. The composition for the said use according to any one of claims 1 to 13 and 16 to 19, the multipart kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises Lactobacillus johnsonii NCC533, cellobiose, and a short- to medium-chain triglyceride containing butyrate and caprylate.

30. The composition for the said use according to any one of claims 1 to 13 and 16 to 19, the multipart kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises Lactobacillus johnsonii NCC533, cellobiose, pea GOS, and short- to medium-chain triglycerides containing butyrate and caprylate.

31. The composition for the said use according to any one of claims 1 to 13 and 16 to 19, the multi-part kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises 10 9 Lactobacillus johnsonii NCC533, 1% cellobiose, 1% short- and medium-chain triglycerides containing butyrate and caprylate.

32. The composition for the said use according to any one of claims 1 to 13 and 16 to 19, the multi-part kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the composition comprises 10 9 Lactobacillus johnsonii NCC533, 1% cellobiose, 1% pea GOS, 1% short- and medium-chain triglycerides containing butyrate and caprylate.

33. The composition for the stated use according to any one of claims 1 to 14 and 16 to 19, the multipart kit according to claim 20, the method according to claim 21 or claim 22, or the use according to claim 23, wherein the lipid is a triglyceride composed of butyrate and long-chain fatty acids, and is one or more of triglycerides comprising butyrate and long-chain fatty acids, including 1,3-dibutyryl-2-linoleoylglycerol, 1,3-dibutyryl-2-stearoylglycerol, 1-butyryl-2-oleoyl-3-palmitoylglycerol, 1-palmitoyl-2-oleoyl-3-butyrylglycerol, 1-butyryl... -2-Oleoyl-3-linoleoylglycerol, 1-linoleoyl-2-linoleoyl-3-butyroylglycerol, 1-oleoyl-2-butyroyl-3-linoleoylglycerol, 1-linoleoyl-2-butyroyl-3-oleoylglycerol, 1-butyroyl-2-linoleoyl-3-oleoylglycerol, 1-oleoyl-2-linoleoyl-3-butyroylglycerol, 1-butyroyl-2-stearoyl-3-oleoylglycerol, 1-oleoyl-2-stearoyl-3-butyroylglycerol, 1-butyroyl-2-oleoyl-3-stearoylglycerol and / or 1-stearoyl-2-oleoyl-3-butyroylglycerol, and mixtures of two or more thereof.

34. A composition, multipart kit, method, or use according to any one of claims 1 to 33 for the stated purpose, wherein the composition further comprises one or more HMOs, preferably one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), lactose-N-tetrasaccharide (LNT), 3'-sialylated lactose (3SL), or 6'-sialylated lactose (6SL).

35. A composition, multipart kit, method, or use according to any one of claims 1 to 33 for the stated purpose, wherein the composition further comprises one or more of 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3'FL), lactose-N-neotetrasaccharide (LNnT), or lactose-N-tetrasaccharide (LNT).

36. The composition, multipart kit, method, or use according to any one of claims 1 to 35, further comprising one or more of the following: a. Bifidobacterium animalis subsp. lactis NCC 2818 (CNCM I-3446); b. *Bifidobacterium longum* subsp. infantis NCC 341 (ATCC 15697(T)); and c. Bifidobacterium longum subsp. NCC 2705 (CNCM I-2618) (NCBI refseq; GCA_000007525.1).

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