Tea extract and bifidobacterium animalis subsp. lactis strain combination

Abstract

The present invention relates to a Bifidobacterium animalis subsp. lactis strain and tea extract combination, and its related uses in metabolic health, in the treatment, prevention and / or improvement of metabolic syndrome, type-2 diabetes and / or a cardio-vascular diseases, and / or related conditions, disorders or symptoms in a subject, as well as its non-therapeutic use for body fat reduction in a subject.

Description

[0001] Tea extract and Bifidobacterium animalis subsp. lactis strain combination

[0002] The present invention falls within the food, feed and pharmaceutical industries. It particularly relates to a combination of a Bifidobacterium animalis subsp. lactis strain and a tea extract, with enhanced metabolic health effects, having utility in the management of metabolic and cardiovascular alterations associated with metabolic syndrome. This combination is therefore useful for exploitation in the following application areas: Food and beverages, animal feed, including pet food, nutritional supplements, infant nutrition, cosmetics (including nutricosmetics), medical foods and pharmaceutical and veterinary applications, among others.

[0003] BACKGROUND ART

[0004] Overweight and obesity are metabolic and nutritional disorders whose prevalence is increasing in industrialized and developing countries in the Western world, being there important causes of morbidity and mortality. In fact, obesity is one of the main risk factors for the development of metabolic syndrome, a condition which may include abdominal obesity, impaired fasting glucose, hypertension, type-2 diabetes, cardiovascular disease, hypertriglyceridemia and low HDL, among others.

[0005] The food and feed industries have been developing new active ingredients with the aim of reducing metabolic syndrome prevalence and helping consumers maintain a correct metabolic status.

[0006] Numerous benefits have been observed in the prevention and / or treatment of metabolic syndrome with the supplementation of botanical ingredients rich in different bioactive compounds with antioxidant and anti-inflammatory effects. Among the most outstanding are the leaves of Camellia sinensis (L.) O. Kuntze), which are traditionally consumed in the form of an infusion or in the form of a supplement as a botanical extract. Depending on the processing given to the tea leaves, different active compounds are obtained. If the tea leaves are subjected to immediate drying, green tea leaves are obtained, resulting in a product rich in catechins, theaflavins or methylxanthines, which have associated functionalities such as antioxidative, anti- inflammatory and cognitive function, among others. On the other hand, if the tea leaves undergo an enzymatic oxidation process, black tea is obtained.

[0007] Several studies relate the consumption of Camellia sinensis with improvements in weight control and metabolic alterations (Yang C.S. et al., 2016). Patent US6830765B2 describes a process to obtain a green tea extract rich in catechins standardized as epigallocatechin gallate by High Precision Liquid Chromatography (HPLC) with activity against fat absorption by inhibiting digestive lipases. In the same way, patent US7989009B2 describes a combination of botanical extracts, including extracts of white, green and black teas, with effects on weight control. More specifically, the botanical blend exerts positive improvements in the body weight and appetite of its consumers. Similarly, Chinese and South Korean patents documents CN 108619321 and KR100847355 describe, respectively, the obtention of Camellia sinensis extracts to inhibit the alpha-glucosidase enzyme, and international patent application WO0176382A1 depicts the process to obtain a Camellia sinensis extract that inhibits amylase enzyme, thus all of them reduce carbohydrate hydrolysis in the digestive tract and reduce glucose absorption.

[0008] Other studies demonstrate the involvement of the intestinal microbiota in body weight and metabolic alterations. Due to this, various strategies have been developed to intentionally manipulate the intestinal microbiota with the aim of modifying the risk of suffering obesity and suffering the metabolic alterations associated with it, highlighting the modification through diet. In this sense, numerous beneficial effects have been attributed to strains of the species Bifidobacterium animalis and particularly of the subspecies lactis in relation to the prevention and / or treatment of disorders associated with overweight, obesity and metabolic syndrome.

[0009] As indicated in patent application US20120107291 A1 , the strain B420 exerts antidiabetic activity and acts against metabolic syndrome by improving glucose tolerance after positively influencing the immune system through gut-associated lymphoid tissue. The BB-12 strain indicated in patent application US2008267933A1 demonstrated activity in weight control and metabolic syndrome through induction of satiety and improvement in insulin sensitivity., Bifidobacterium animalis subsp. lactis CECT8145 (or B. animalis subsp. lactis CECT8145), referenced in international patent application WO2015007941 A1 , has been disclosed as having the ability to induce satiety and reduce appetite, increase the capacity to resist oxidative stress, and reduce the serum concentration of total cholesterol, triglycerides and glucose. However, these are not the only strains of this species used to prevent or alleviate metabolic syndrome. The EP4001399A1 , WO2014094279A1 and CN114574407A patent documents also show improvements in insulin sensitivity in different animal models with other B. animalis subsp. lactis strains.

[0010] Although botanical extracts and Bifidobacterium strains are disclosed with the objective of obtaining beneficial metabolic effects, there is a need in the field to develop compositions with an enhanced effect in metabolic conditions and disorders.

[0011] DESCRIPTION OF THE INVENTION

[0012] The authors of the present invention have discovered that the combination of Bifidobacterium animalis subsp. lactis CECT 8145 strain, preferably, non-viable heat- treated Bifidobacterium animalis subsp. lactis CECT 8145 strain (also referred hereinafter as “HT-BPL1”, “BPL1 HT” or “BPL1® HT”), and an extract obtained from a blend (or mixture) of green tea and black tea leaves (also referred hereinafter as “Complex Tea Extract”, “Tea Complex Extract”, “Tea Complex”, “CTE”, “TCE” or “TC”), exerts an enhanced effect or improvement of conditions associated with metabolic health and metabolic syndrome, as shown in the assays carried out in Caenorhabditis elegans (“C. elegans") and murine animal models (see Examples section).

[0013] Some of the main effects demonstrated by the inventors for this combination are the followings:

[0014] - An enhanced fat reduction effect and an enhanced decreasing body weight gain effect (wherein each mentioned effect associated to the combination is higher than the effect produced by the ingredients per separate), as shown in the assays in C. elegans; this enhanced effect in C. elegans was also shown with the viable BPL1 strain in combination with Tea Complex Extract (see Example 3); and in murine model (see Example 4). Particularly, fat reduction effect has been demonstrated to be specific of the combination of the present invention, since (i) six different botanical extracts, other than Tea Complex, were evaluated in combination with HT-BPL1 in C. elegans model, but none of those combinations produced an enhancement in fat reduction activity, and besides related fat reduction effects were lower than the one obtained with the blend of Tea Complex Extract with HT-BPL1 (Figure 7); and (ii) six different heat-treated B. animalis strains other than HT-BPL1 were evaluated in combination with Tea Complex Extract in C. elegans model, but none of those combinations produced a significant fat reduction effect, being the blend of Tea Complex Extract with HT-BPL1 the only combination which provided a significant fat reduction effect in C. elegans model, higher than other combinations including alternative B. animalis strains (Figure 8);

[0015] - An enhanced effect in reducing visceral and subcutaneous adiposity and adipocyte size, better than the ones derived from both interventions separately, as it is shown in murine assays.

[0016] - An enhanced improvement of the lipid profile: Tea Complex Extract in combination with HT-BPL1 reduces c-LDL, while such a reduction is not shown when both components or ingredients were administered separately, as it is shown in murine assays);

[0017] - An enhanced reduction of leptin levels (leptin circulating levels are solely reduced to control levels when Tea Complex Extract in combination with HT-BPL1 is administered; see murine assays);

[0018] - An increase in the gene expression of the leptin receptor and the peroxisome prol iterator-activated receptor-gamma coactivator 1 -alpha (PGC-1a) in the epididymal adipose tissue. Likewise, the combined supplementation of Tea Complex Extract and HT-BPL1 reduced the gene expression of NOX-4 and the mRNA levels of the proinflammatory cytokines IL-6, IL-1 and TNF-a, and reverted the obesity-induced overexpression of MCP-1 to control levels;

[0019] - Regarding insulin resistance and the glycemic status, the co-administration of both ingredients not only decreased circulating insulin levels and the HOMA-IR index but also reduced basal glycemia, an effect that was not found when the treatments were administered alone. In fact, only the combined treatment with Tea Complex Extract and HT-BPL1 reduced blood glucose levels and HOMA-IR to control levels. Likewise, the results of the glucose tolerance test demonstrates that the combined administration improved glucose uptake and insulin sensitivity.

[0020] In summary, although the administration of Tea Complex Extract and BPL1 , preferably, Tea Complex Extract and HT-BPL1 , separately shows positive effects on metabolic and cardiovascular health, the combination of both ingredients exerts unexpected and surprisingly enhanced effects and additional benefits.

[0021] In addition, the inventors have also demonstrated that Tea Complex Extract, HT-BPL1 and the combination of both modulates gut microbiota in the context of HFHS highlighting the capacity of the blend to increase bacterial richness and beneficial bacteria like Bifidobacterium species, while preventing the increase of potential opportunistic bacteria like F. butyricus (Example 5).

[0022] Thus, in an aspect, the invention relates to a composition, hereinafter, the “composition of the invention”, comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, hereinafter also referred to as “the strain of the invention”, and an extract obtained from green tea and black tea leaves, hereinafter the “extract of the invention”.

[0023] Strain of the invention

[0024] Bifidobacterium animalis subsp. lactis strain CECT 8145 was deposited on May 14th2012 under the Budapest Treaty in the Spanish Type Culture Collection as the International Depository Authority (based in Building 3 CUE, Parc Cientffic Universitat de Valencia, C / Catedratico Agustin Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 8145. The terms “strain of the invention”, “8. animalis subsp. lactis strain 8145”, “8. animalis CECT 8145”, “BPL0001”, “BPL1™” or “BPL1” are interchangeably used in the present document to refer Bifidobacterium animalis subsp. lactis strain CECT 8145. Members of the genus Bifidobacterium are among the first microbes to colonize the human gastrointestinal tract (GIT) and are believed to exert positive health benefits on their host. Bifidobacteria have been commercially exploited as probiotic agents due to their associated health benefits and to being generally recognised as safe.

[0025] Due to their purported health-promoting properties, these bacteria have been incorporated into many functional foods as active ingredients.

[0026] Bifidobacteria naturally occur in a range of ecological niches that are either directly or indirectly connected to the animal GIT, such as the human oral cavity, the insect gut and sewage. To be able to survive in these particular ecological niches, bifidobacteria must possess specific adaptations to be competitive.

[0027] Bifidobacterium are gram-positive, non-motile, and often branched anaerobic bacteria. They are one of the major genera of bacteria that make up the GIT microbiota in mammals, and comprise over 30 different species. Among them, Bifidobacterium animalis subsp. lactis is a Gram-positive lactic acid bacterium belonging to the Actinobacteria phylum. Originally isolated from fermented milk, Bifidobacterium animalis subsp. lactis inhabits the gut of healthy adults and infants.

[0028] The present invention contemplates the use of both, viable and non-viable, cells of the strain of the invention.

[0029] In a preferred embodiment of the invention, alone or in combination with other preferred embodiments, the strain of the invention is in the form of non-viable cells.

[0030] As used herein, the term “non-viable”, “inanimate”, “inactive” or “inactivated” relates to any microorganism metabolically or physiologically inactive, i.e., not retaining its metabolic activity and the ability to elongate after the administration of nutrients. Viability is independent from the capacity of the microorganism to form colonies on solid media, i.e., from its culturability.

[0031] Some techniques known in the state of the art to render microorganisms in the form of non-viable cells are, without limitation to, thermal processing / heat, freezing or radiation, such as ultraviolet radiation. Thermal processing is likely to be used in many instances to inactivate microorganisms, as there is a long history of thermal processing in the food industry. A common procedure to inactivate microorganisms is heat treatment. Heat is lethal to living microorganisms, but each species has its own particular heat tolerance. During a thermal destruction process, such as pasteurization, tyndallisation and autoclaving, the rate of destruction is logarithmic, as is their rate of growth. Thus, bacteria subjected to heat are killed at a rate that is proportional to the number of organisms present. The process is dependent both on the temperature of exposure and the time required at this temperature to accomplish to desired rate of destruction.

[0032] In a preferred embodiment, the strain of the invention is heat-treated Bifidobacterium animalis subsp. lactis strain CECT 8145. In some parts of the document, heat-treated Bifidobacterium animalis subsp. lactis strain CECT 8145 is referred to as “HT-BPL1”, “BPL1 HT” or “BPL1® HT”.

[0033] The present invention also contemplates those bacterial strains derived from B. animalis subsp. lactis strain CECT 8145 (parental strain) that may be part of the composition of the invention retaining any of the effects disclosed in the present document for the strain of the invention (e.g., fat reduction effect or any other effect shown in the Examples section). Examples of bacterial strains derived from the parental strain comprised within the composition of the invention include genetically modified organisms which show variations in their genome compared to the genome of the strain of the invention, wherein the mutations do not affect the ability of strain and / or their components to exert the effects disclosed along the description and in the Examples section.

[0034] Strains derived from B. animalis subsp. lactis strain CECT 8145 may be naturally or intentionally produced by mutagenesis methods known in the art, such as, but not limited to, growing the parent strain in the presence of mutagenic agents or stressors or genetic engineering directed to the modification, deletion and / or insertion of specific genes. In some specific embodiments, the present invention also contemplates bacterial components, metabolites and / or molecules secreted by B. animalis subsp. lactis strain CECT 8145 in combination with the extract of the invention.

[0035] Bacterial components include, without limitation to, components of the cell wall (by way of non-limiting example, peptidoglycan); nucleic acids; membrane components; and proteins, lipids, carbohydrates, and combinations thereof (such as lipoproteins, glycolipids or glycoproteins). Metabolites may include any molecule produced or modified by the bacterium as a result of its metabolic activity during growth, its use in technological processes or during its storage. Examples of these metabolites include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals or nucleic acids. Secreted molecules include any molecule secreted or released to the outside by the bacterium during growth, its use in technological processes (for example, food processing or drugs) or during its storage. Examples of these molecules include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals or nucleic acids.

[0036] Extract of the invention

[0037] The extract of the invention refers to a green tea and black tea leaves extract, particularly an extract obtained from a blend (or mixture) of green tea and black tea leaves, preferably, wherein the extract is obtained from a blend (or mixture) of green tea and black tea leaves in adequate proportion, more preferably the extract is obtained from green tea leaves in a proportion ranging from 50 to 70%, both ends included, and from black tea leaves in a proportion ranging from 30 to 50%, both ends included. In a preferred embodiment, alone or in combination with other preferred embodiments, the extract is obtained by water extraction.

[0038] In a preferred embodiment of the invention, the extract of the invention is obtained from a blend (or mixture) of green tea and black tea leaves by 60% and 40% respectively. In another preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention is obtained from a blend (or mixture) of green tea and black tea leaves, preferably in a proportion of 60% and 40%, followed by water extraction, solid-liquid separation, concentration and drying processes. More preferably, the obtention is carried out without any carrier nor preservant in the finished product, which may comply with the specifications of least 5% of methylxanthines and 10% of flavan-3-ols (% dry basis), including monomeric and oligomeric (theaflavins) components.

[0039] In a more preferred embodiment of the invention, the extract of the invention is obtained from a blend (or mixture) of green tea and black tea leaves by a process which comprises the following steps: a) subjecting the blend (or mixture) of green tea and black tea leaves to a solid liquid extraction, preferably comprising a ratio of 10-12 parts of water for 1 part of the greenblack tea leaves blend; b) solid-liquid separation; c) centrifugation; d) concentration step, preferably wherein the concentration is performed over 30 Brix previous to drying process; and preferably e) drying process;

[0040] Production steps include solid-liquid extraction, centrifugation (apart from centrifugation, the liquid extract can be also clarified depending on the finished application controlling turbidity of the finished product), concentration and drying process. Alternative organic solvents such as ethanol, ethyl acetate, etc., alone or mixed with water can achieve or improve the extraction yields of flavan-3-ols, xanthines, but water as green solvent is preferred for extraction. In addition, other technologies such as ultrasound-assisted extraction, microwave-assisted extraction, pulsed electric field, pressurized liquid extraction, counter current extraction or similar technologies can be also used for the extraction of main bioactive components of Tea Complex if the specifications are matched. For the drying process, spray drying is the preferred technology, but other drying techniques such as freeze-drying, vacuum drying, and related techniques can also be used.

[0041] The inventors of the present invention have characterized a particular extract obtained from a blend (or mixture) of green tea and black tea leaves, referred in the present document as “Complex Tea Extract”, “Tea Complex Extract”, “Tea Complex”, “CTE”, “TCE” or “TC”. Different analytical techniques were used for the characterization of the extract, mostly liquid and gas chromatography, together with spectrophotometry (as detailed in Example 1). As can be observed in Figure 1 , two main different groups of molecules, chiefly flavan-3-ols (monomeric flavanols, theaflavins) and methylxanthines were identified as TCE compounds.

[0042] In a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention comprises flavan-3-ols and methylxanthines.

[0043] In a preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of total polyphenols of, at least, 30% (dry basis), more preferably, of at least 40% (dry basis). Even more preferably, the extract of the invention is standardized to comprise a concentration of total polyphenols of, ranging from at least, 30%, 40% to 50% (dry basis).

[0044] In a preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of total flavan-3-ols (monomeric and oligomeric flavan-3-ols- theaflavins) of, at least, 10% (dry basis). In a more preferred embodiment, the extract of the invention is standardized to comprise a concentration of total flavan-3-ols of, at least, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%, even more preferably total flavan-3-ols concentration ranges from 10 to 20% (dry basis)

[0045] Flavan-3-ols (sometimes referred to as flavanols) are a subgroup of flavonoids. They are derivatives of flavans that possess a 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton. Flavan-3-ols are structurally diverse and include a range of compounds, such as catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, proanthocyanidins, theaflavins (oligomeric flavan-3-ols), or thearubigins (polymeric flavan-3-ols). Flavan-3-ols compounds that may be present in the extract of the invention are gallocatechin, epigallocatechin, catechin, epicatechin, epigallocatechin gallate, gallocatechin-3-gallate, epicatechin-3-gallate, catechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate, and theaflavin-3,3'-gallate. In a preferred embodiment, alone or in combination with other preferred embodiments, the flavan-3-ols are selected from the list consisting of gallocatechin, epigallocatechin, catechin, epicatechin, epigallocatechin gallate, gallocatechin-3-gallate, epicatechin- 3-gallate, catechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'- monogallate, theaflavin-3,3'-gallate, and any combination thereof.

[0046] In a more preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of gallocatechin of, at least, 0.1% (dry basis), a concentration of epigallocatechin of, at least, 0.6% (dry basis), a concentration of catechin of at least, 0.3% (dry basis), a concentration of epicatechin of, at least, 0.5%, (dry basis), a concentration of epigallocatechin gallate of, at least, 4.2% (dry basis), a concentration of gallocatechin-3-gallate of, at least, 0.5% (dry basis), a concentration of epicatechin-3-gallate of, at least, 2.3% (dry basis), a concentration of catechin-3- gallate of, at least, 0.5% (dry basis), and a concentration of total theaflavins of, at least, 0.1 % (dry basis), considering that the total concentration of flavan-3-ols (monomeric and theaflavins) in the extract of the invention is at least 10% (dry basis).

[0047] In another preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention comprises a concentration of gallocatechin of 0.2% (dry basis), a concentration of epigallocatechin of 0.8% (dry basis), a concentration of catechin of 0.3% (dry basis), a concentration of epicatechin of 0.6% (dry basis), a concentration of epigallocatechin gallate of 6.0% (dry basis), a concentration of gallocatechin-3-gallate of 0.5% (dry basis), a concentration of epicatechin-3-gallate of 2.5% (dry basis), a concentration of catechin-3-gallate of 0.6% (dry basis), and a concentration of total theaflavins of 0.1% (dry basis). Other flavan-3-ols with same UV-Vis characteristics previously shown in Figure 1 , different from the previously mentioned (most common), can be also considered for quantification / standardization. In a preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of total methylxanthines of, at least, 5% (dry basis). In a more preferred embodiment, the extract of the invention is standardized to comprise a concentration of total methylxanthines of, at least, 7%, 8%, 9%; even more preferably total methylxanthines concentration ranges from 5 to 10% (dry basis). In another preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention comprises a concentration of total methylxanthines ranging from 6 to 8% (dry basis).

[0048] Methylxanthines are a group of phytochemicals derived from the purine base xanthine and obtained from plant secondary metabolism. They are compounds formed by the methylation of xanthine, such as caffeine, theobromine, and theophylline. In a preferred embodiment, alone or in combination with other preferred embodiments, the methylxanthines are selected from the list consisting of caffeine, theobromine, theophylline, and any combination thereof.

[0049] In a more preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of caffeine of, at least, 5% (dry basis). In an even more preferred embodiment, the extract of the invention is standardized to comprise a concentration of caffeine of, at least, 6% (dry basis).

[0050] In another more preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of theobromine of, at least 0.1 % (dry basis). In an even more preferred embodiment, the extract of the invention is standardized to comprise a concentration of theobromine of, at least, 0.2% (dry basis).

[0051] In another more preferred embodiment, alone or in combination with other preferred embodiments of the invention, the extract of the invention is standardized to comprise a concentration of theophylline of, at least 0.01% (dry basis). In an even more preferred embodiment, the extract of the invention is standardized to comprise a concentration of theophylline of, at least, 0.02% (dry basis). In a still more preferred embodiment, the extract of the invention is standardized to comprise a concentration of caffeine of, at least 5% (dry basis), a concentration of theobromine of, at least 0.2% (dry basis), and a concentration of theophylline of, at least, 0.01 % (dry basis).

[0052] In another still more preferred embodiment, the extract of the invention comprises a concentration of caffeine, theobromine and theophylline, which sum is ranging from 5% to 10% (dry basis).

[0053] Furthermore, in the characterisation of the Tea Complex, the inventors have identified a complete profile of volatile components. Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention further comprises volatile compounds selected from the list consisting of, hexanal, E-2- hexenal, hexanol, E-3-hexenol, linalool, linalool oxides, methyl salicylate, geraniol, benzyl alcohol, phenethyl alcohol, benzaldehyde and any combination thereof.

[0054] The CTE preferably comprises characteristic volatiles from Camellia sinensis (L.), which may be classified in two groups according to Inarejos-Garcia et al., Authentication of Commercial Powdered Tea Extracts (Camellia sinensis L.) by Gas Chromatography. ACS Food Science and Technology, 1 , 4, 596-604 (2021); Group I (1-penten-3-ol, hexanal, hexanol, E-2-hexenal, Z-3-hexenol- E-2-hexenol) and Group II (linalool, linalool oxides, methyl salicylate, geraniol, benzaldehyde, a-ionone, - ionone). Apart from these volatiles, other could be also found in CTE because the product is 100% developed from Camellia sinensis (L.), chiefly limonene, hotrienol, safranal, benzyl acetate, eugenol.

[0055] CTE preferably comprises volatiles from at least from both groups of volatiles; in a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention further comprises some or all volatiles selected from hexanal, hexanol, E-3-hexenol (Group I), linalool oxides, benzaldehyde, linalool, methyl salicylate, benzyl alcohol, and phenethyl alcohol (Group II). In another preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention further comprises some or all volatiles selected from hexanal, hexanol, E-3-hexenol (Group I), linalool oxides, benzaldehyde, linalool, methyl salicylate, benzyl alcohol, and phenethyl alcohol (Group II). In another preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention further comprises some or all of other volatiles characteristics from Camellia sinensis (L.) selected from limonene, hotrienol, safranal, benzyl acetate, eugenol and any combination thereof.

[0056] Concentrations of the components in the extract of the invention are given in the form of wt% of the component by weight relative to the total weight of the dry basis of the extract.

[0057] “Dry weight basis” (also mentioned herein as “dry basis”) can be defined as the amount of dry solid in the sample after drying. Dry weight basis is referred to the percentage of a substance after removing the moisture from the substance.

[0058] The term “standardized” as used in the field of naturally derived nutritional products refers to the process for delivering a product with a specific minimum level of one or more plant constituents. Standardization represents the level of concentration of particularly desired elements from a plant source. Methods for standardizing plant substances, including black tea and / or green tea extracts, are well known in the art. Furthermore, the measurement of particular plant constituents on which standardization is based is also well known in the art.

[0059] In a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention has fat reduction capacity, and / or lipid profile improvement capacity, and / or any other effect disclosed in the Example section.

[0060] In a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention is a powdered extract.

[0061] Formulations and administration doses In each case, the presentation of the composition of the invention, which comprises the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and the extract of the invention, will be adapted to the type of administration used. For instance, the composition may be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount in some embodiments. The composition of the invention can be formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders, granules, solutions, suppositories, gels, softgels or microspheres.

[0062] In a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention is formulated in liquid form or in solid form.

[0063] In a more preferred embodiment, the solid formulation is selected from the group consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, soft chews, gummies, capsules, sachets, powders, suppositories, gels, softgels, microspheres, granules, coated particles, coated tablets, tablets, g astro- resista nt tablets, gastro-resistant capsules , dispersible strips, films, dry kibble, baked treats, soft chews, and wet canned food.

[0064] In another more preferred embodiment, the liquid formulation is selected from the group consisting of oral solutions, suspensions, droplets, emulsions and syrups.

[0065] Likewise, various systems are known that can be used for sustained-release administration of the composition of the invention, including, for example, the encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. The suitable sustained-release forms as well as materials and methods for their preparation are well known in the state of the art. Thus, the orally administrable form of the composition of the invention is in a sustained-release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semisynthetic or synthetic polymers, water-insoluble or modified, waxes, fats, fatty alcohols, fatty acids, natural, semisynthetic or synthetic plasticizers or a combination of two or more of the same. Enteric coatings can be applied using conventional processes known to those skilled in the art.

[0066] In addition to what it described above, the present invention also encompasses the possibility that the composition of the invention may be administered to a subject together with other components or compounds, although these are not part of the composition of the invention. Examples of such components or compounds are detailed along the description.

[0067] The composition of the invention may be formulated for pharmaceutical administration, i.e., forming part of pharmaceutical products to be administered to the subject by any means of administration; and / or for food administration, i.e., forming part of the foods, feeds or nutritional supplements consumed in a subject’s diet. Thus, in some embodiments, the composition of the invention is a pharmaceutical composition (“pharmaceutical composition of the invention”) and / or a nutritional composition (“nutritional composition of the invention”).

[0068] Additionally, the composition of the invention may further comprises (i) one or more components or compounds having any biological, pharmacological and / or veterinary useful activity, for the purpose of the present invention (i.e. exerting fat reduction effects, improvement of the lipid profile or any other effect disclosed in the Example section for the CTE and strain of the invention combination; and / or useful activity for any of the uses of the invention disclosed in the present patent application); and / or (ii) one or more components that, upon administration to a subject, may increase, enhance and / or promote the activity of the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and / or the extract of the invention. As understood by those skilled in the art, the additional components or compounds must be compatible with the effects exerted by the combination of the strain of the invention and the extract of the invention.

[0069] The term "pharmaceutical composition" also encompasses veterinary compositions.

[0070] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. The term "excipient" refers to a substance that helps the absorption of any components or compounds of the composition of the invention, namely, the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and the extract of the invention, or stabilizes the components or compounds and / or assists the preparation of the pharmaceutical composition in the sense of giving it consistency or flavours to make it more pleasant. Thus, the excipients may have the function, by way of example but not limited thereto, of binding the components (for example, starches, sugars or cellulose), sweetening, colouring, protecting an active ingredient (for example, to insulate it from air and / or moisture), filling a pill, a capsule or any other presentation or having a disintegrating function to facilitate dissolution of the components, without excluding other excipients not listed in this paragraph. Therefore, the term "excipient" is defined as that material that, included in the galenic forms, is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical and chemical properties of a pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of components or compounds of the pharmaceutical composition, that is, be compatible with the effect exerted by the combination of the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and the extract of the invention.

[0071] The "galenic form" or "pharmaceutic form" is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or a drug. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

[0072] The "vehicle" or "carrier" is preferably an inert substance. Carrier functions are to facilitate the incorporation of other components or compounds, allow better dosage and administration and / or give consistency and form to the pharmaceutical composition. Therefore, a carrier is a substance used in the drug to dilute any of the components or compounds of the pharmaceutical composition of the present invention to a given volume or weight; or even without diluting these components or compounds, it is able to allow better dosage and administration and / or give consistency and form to the drug. When the presentation is liquid, the pharmaceutically acceptable carrier is the diluent. The carrier can be natural or unnatural. Examples of pharmaceutically acceptable carriers include, without being limited thereto, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatine, lactose, starch, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0073] Furthermore, the excipient and the carrier must be pharmacologically acceptable, i.e. , the excipient and the carrier are permitted and evaluated so as not to cause damage to the subject to whom it is administered.

[0074] In the event that the composition of the invention is a nutritional composition, i.e., the nutritional composition of the invention, said nutritional composition may be a food or feed, or be incorporated into a food, feed or food product intended for human and / or animal consumption.

[0075] Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the nutritional composition of the invention is a food, feed or a nutritional supplement.

[0076] In some embodiments, the nutritional composition of the invention is a nutritional or dietary supplement.

[0077] In the present invention, the term "nutritional composition" refers to any food, feed, which, regardless of providing nutrients to the subject who consumes it, it beneficially affects one or more functions of the body, so as to leverage and / or provide better health and wellness.

[0078] The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement", "food supplement", or "alimentary supplement" or “alimentary complement”, refers to products or preparations whose purpose is to supplement the normal diet of a subject consisting of sources of concentrated nutrients or other substances with a nutritional or physiological effect. In the present invention, the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and the extract of the invention are the "substances" with a nutritional and / or physiological effect on the subject.

[0079] The food supplement may be in single or combined form and be marketed in dosage forms, i.e., in capsules, pills, tablets and other similar forms, sachets of powder, ampoules of liquids and drop dispensing bottles and other similar forms of liquids and powders designed to be taken in a single amount.

[0080] There is a wide range of nutrients and other elements that may be present in alimentary complements including, among others, vitamins, minerals, amino acids, essential fatty acids, fibre, enzymes, plants and plant extracts. Since their role is to complement the supply of nutrients in a diet, they should not be used as a substitute for a balanced diet, and intake should not exceed the daily dose expressly recommended by a doctor or a nutritionist, or evaluated in specific clinical trials. The composition of the invention can also be part of the so-called "food for special groups", i.e., foods that are intended to meet specific nutritional needs.

[0081] Examples of foods that may comprise the composition of the invention include, but are not limited to, food (including food and feed for animal nutrition), dairy products, vegetable products, meat products, snacks, chocolates, beverages, baby food, cereals, fried foods, industrial bakery products, confectionary, biscuits, kibbles, baked treats and wet canned food. Examples of dairy products include, but are not limited to, products derived from fermented milk (for example, but not limited to, yogurt or cheese) or non-fermented milk (for example, but not limited to, ice cream, butter, margarine or whey). The vegetable product is, for example, but not limited to, a cereal in any form of presentation, a fermented product (for example, soy yogurt, oat yogurt, etc.), an unfermented product or a snack. The beverages include, but are not limited to, a non-fermented milk or a vegetable beverage.

[0082] In a preferred embodiment, alone or in combination with other preferred embodiments, the food product or food is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, cereals, baked goods, milk-based products (such as milkshakes or smoothies), meat products and beverages.

[0083] In a preferred embodiment, alone or in combination with other preferred embodiments, the strain of the invention (and / or strains and / or cellular components derived therefrom) and the extract of the invention, are present in an effective amount, more preferably in a therapeutically effective amount, in order to exert their effects, such as the effects disclosed in the Example section, or along the description wherein the several uses are specifically explained.

[0084] Within the context of the present invention, an “effective amount” is any amount of the component or compound of the composition of the invention which, when administered to a subject, is sufficient to produce the desired effect. Said component or compound of the composition of the invention, including the nutritional or pharmaceutical composition, refers to the strain of the invention (and / or strains and / or cellular components derived therefrom) and / or the extract of the invention.

[0085] The effective amount may vary depending on, for example, the age, body weight, general health, sex and diet of the subject, as well as on the mode and time of administration, the excretion rate or any potential co-treatment with other drugs.

[0086] In a preferred embodiment, alone or in combination with other preferred embodiments, the strain of the invention is present in the composition in an amount ranging from 105to 1012cfu or cells per serving. More preferably, the strain of the invention is present in an amount ranging from 107to 1011cfu or cells.

[0087] In a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention can be recommended “as average” in humans within the ranges of a minimum daily dose of 250 mg / day, and a maximum daily dose of 800 mg / day for at least 3 months. The maximum daily dose in this embodiment is based on the caffeine content; for the concrete sample concentrated into 6.55% of caffeine, the maximum daily is 600 mg / day, avoiding the maximum caffeine daily intake established for 400 mg / day. Therefore, in a more preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention is administered in an amount of 400 mg / day.

[0088] In a preferred embodiment, alone or in combination with other preferred embodiments, the composition comprising the extract of the invention and the strain of the invention, i.e. the composition of the invention, is a combination preparation (or kit of parts), for simultaneous or sequential use.

[0089] As used herein, combined preparation (or kit of parts) means that the components of the preparation, the strain and the extract of the invention, do not need to be in a physical combination (for instance, they do not need to be in one dosing unit or in one single entity) in order to be available for sequential (separate) application. Thus, it implies that it does not necessarily result in a true combination, in view of the physical separation of the components.

[0090] In some embodiments, alone or in combination with other preferred embodiments, the strain of the invention and the extract of the invention are administered sequentially.

[0091] In a preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention is administered at a dose of 3.6 to 114.3 mg / kg / day (both ends included). Preferably, the extract of the invention is administered at a dose of 5 to 50 mg / kg / day (both ends included).

[0092] In another preferred embodiment, alone or in combination with other preferred embodiments, the extract of the invention is administered at a dose of ranging from 732 mg / kg / day to 1830 mg / kg / day (both ends included).

[0093] As used herein, mg / kg / day is the dose, in this case of the extract of the invention, given in mg of the extract of the invention per kg of the subject per day.

[0094] In another preferred embodiment, alone or in combination with other preferred embodiments, the strain of the invention is administered at a dose of 105to 1012cfu or cells per day (both ends included). More preferably, at a dose of 107to 1011cfu or cells (both ends included). In a more preferred embodiment, the extract of the invention and the strain of the invention are administered during, at least, 3 months.

[0095] In some embodiments, the composition of the invention may comprise other microorganisms in addition to the strain of the invention. In a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention further comprises a microorganism, and / or their components, selected from the group consisting of Bacillus sp., Lactobacillus sp., Lacticaseibacillus, Lactiplantibacillus sp., Levilactobacillus sp., Levilactobacillus sp., Ugilactobacillus sp., Umosilactobacillus sp., Streptococcus sp., Bifidobacterium sp., Saccharomyces sp., Kluyveromyces sp., and combinations thereof. More preferably, the composition of the invention further comprises a microorganism, and / or their components, selected from the group consisting of L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. kefir, L. brevis, L. casei, L. plantarum, L. fermentum, L. paracasei, L. acidophilus, L. paraplantarum, L. reuteri, Streptococcus thermophilus, B. longum, B. breve, B. bifidum, B. catenulatum, B. adolescentis, B. pseudocatenulatum, Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces lactis, Kluyveromyces marxianus, and any combination thereof.

[0096] In some specific embodiments, the nutritional composition of the invention is a food, feed or a nutritional supplement for farm animals and / or pets.

[0097] In some specific embodiments, the nutritional composition of the invention is a food or a nutritional supplement for humans.

[0098] The strain and the extract of the invention combination, preferably the composition of the invention, can be used in different food matrixes and / or in nutritional supplements such as capsules, tablets and melting tablets, soft gels, sachets, gummies, food formulations (soft chew, baked treats, kibbles, wet canned..), dairy products, vegetable products, plant-based products, meat products, snacks, chocolates, beverages, baby food, cereals, protein bars, fried foods, industrial bakery products, biscuits, oils, etc. Uses of the invention

[0099] As stated above, the present application provides data demonstrating that the combination of the strain of the invention and the extract of the invention exerts unexpected and surprisingly enhanced metabolic health effects, including conditions, symptoms and disorders related to metabolic syndrome.

[0100] Thus, in other aspect, the present invention refers to the composition of the invention for use as a medicament.

[0101] The term “medicament”, as used herein, refers to any composition / substance used for the prevention, diagnosis, alleviation, treatment or cure of disease in a subject or which may be administered to a subject for the purpose of restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action in its body.

[0102] In other aspect, the present invention relates to the composition of the invention, for use in improving metabolic health in a subject, preferably wherein the improvement of metabolic health is measured by one or more of the following indicators: i) improved blood lipid profile, ii) lowering of plasma LDL cholesterol, iii) improved glycemic response, iv) visceral and / or subcutaneous adiposity reduction, v) leptin levels reduction, vi) insulin levels reduction, vii) blood glucose levels reduction, viii) insulin resistance reduction, as measured by HOMA-IR index, or ix) any combination of the above.

[0103] Above-mentioned indicators can be assessed by tests or methods widely known in the state of the art. In other aspect, the present invention relates to the composition of the invention, for use in the treatment, prevention and / or improvement of metabolic syndrome, and / or related conditions, disorders or symptoms, in a subject.

[0104] As used herein, the term "to treat" or "treatment" comprises: Inhibiting the disease, disorder or pathological condition, i.e., stopping its development; relieving the disease, disorder or pathological condition, i.e., causing regression of the disease, disorder or pathological condition; stabilizing the disease, disorder or pathological condition in a subject; ameliorating the symptoms associated to the disease, disorder or pathological condition in a subject; and / or slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Thus, the term "to treat" or "treatment" does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders. The treatment of a disorder, pathological condition or disease may, for example, lead to a halt in the progression of the disease, disorder or pathological condition (e.g., no deterioration caused by any of the symptoms or no increase of symptoms) or a delay in the progression of the disease, disorder or pathological condition (in case the halt in progression is of a transient nature). The "treatment" of a disorder, disease or pathological condition may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject suffering from the disease, disorder or pathological condition. Accordingly, the "treatment" may also refer to an amelioration of the disorder, pathological condition, or disease, which may, e.g., lead to a halt in the progression of disease, disorder or pathological condition, or a delay in the progression of the disease, disorder or pathological condition.

[0105] As used herein, the term "prevention" refers to the avoidance of occurrence of the disease, disorder or pathological condition in a subject, particularly when the subject has predisposition for the pathological condition but has not yet been diagnosed, or refers to the minimization or hampering of the occurrence of the disease or pathological condition in a subject. It may also comprise reducing the risk of developing a disease, disorder or pathological condition in a subject. Metabolic syndrome is a collection of health disorders / diseases, or a condition, characterized by the clustering of alterations in glucose, lipid metabolism, and / or blood pressure, that may increase the chance of developing diabetes, preferably type- 2 diabetes, and cardiovascular diseases among others. Particularly, metabolic syndrome is often characterized by any of a number of metabolic disorders or conditions, which are generally considered to most typify metabolic syndrome when more than one are present in a single individual. These related conditions or disorders associated with metabolic syndrome include: Obesity, dyslipidemia (this include a family of blood fat disorders including, e.g ., high triglycerides, low HDL cholesterol, and high LDL cholesterol), high blood pressure, insulin resistance, glucose intolerance (the inability to properly use insulin or blood sugar), a chronic prothrombotic state (e.g., characterized by high fibrinogen or plasminogen activator inhibitor levels in the blood), and a chronic proinflammatory state (e.g., characterized by higher than normal levels of high-sensitivity C-reactive protein in the blood).

[0106] In a preferred embodiment, alone or in combination with other preferred embodiments, the related conditions, disorders, or symptoms of metabolic syndrome are selected from the list consisting of obesity, overweight, insulin resistance, glucose intolerance, hyperglycemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension, a chronic prothrombotic state, a chronic proinflammatory state, and any combination thereof. In a more preferred embodiment, alone or in combination with other preferred embodiments, the metabolic syndrome related condition, disorder or symptom is, at least, one, two, three, four, or more, selected from the list consisting of obesity, overweight, insulin resistance, glucose intolerance, hyperglycemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension, a chronic prothrombotic state, a chronic proinflammatory state, and any combination thereof.

[0107] As explained above, the combination of the strain of the invention and the extract of the invention has a fat reducing enhanced effect, at an unexpected degree, since as it is shown for instance in C. elegans assays (Example 3), the percentage of fat reduction associated to the mentioned combination is higher than the sum of the fat reduction percentage associated to the individual ingredients of said combination (i.e. , the Tea Complex Extract and HT-BPL1 , or viable BPL1 , separately administered). Thus, a more preferred embodiment provides the composition of the invention for use in the treatment, prevention and / or improvement of obesity, overweight or related diseases and / or disorders in a subject

[0108] The term "obesity" in general refers to an abnormal or excessive fat accumulation that presents a risk to health, being “overweight” a level of obesity. A crude population measure of obesity in adults is the body mass index (BMI), a person's weight (in kilograms) divided by the square of his or her height (in meters). The term "obesity" is herein adopted to describe a condition characterized by, preferably, a BMI > 30 kg / m2while the term “overweight” is herein adopted to describe a condition characterized by, preferably, a BMI > 25 kg / m2but < 30 kg / m2. For adolescents, "obesity" refers to a condition characterized by two standard deviations body mass index for age and sex from the World Health Organization (WHO) growth reference for school-aged children and adolescents. The terms "obesity" thus imply a medical indication for treatment.

[0109] For the purposes of the present invention, the terms "related or associated diseases / disorders" and "diseases / disorders caused by overweight and / or obesity" comprise: Diabetes, metabolic syndrome, hypertension, hyperglycemia, inflammation, type-2 diabetes, cardiovascular disease, hypercholesterolemia, hormonal disorders, infertility, etc. and those diseases or disorders which obesity or overweight is a risk factor of suffering them.

[0110] As it has been previously mentioned, metabolic syndrome and related conditions or disorders associated with it may increase the chance of developing diabetes, particularly type 2 diabetes mellitus and cardiovascular diseases. Thus, another aspect of the present invention refers to the composition of the invention for use in the treatment, prevention and / or improvement of diabetes, preferably type-2 diabetes, and / or a cardiovascular disease or disorder, preferably wherein the cardiovascular disease or disorder is selected from the list consisting of hypertension, atherosclerosis, coronary artery disease, peripheral artery disease, aortic disease, cerebrovascular disease, heart failure and stroke, in a subject. Within the context of the present invention, the term "subject" relates to any animal from any species. Examples of subjects include, but are not limited to: Animals of commercial interest such as birds (hens, ostriches, chicks, geese, partridges, etc.), rabbits, hares, pets (dogs, cats, etc.), sheep, goat cattle (goats, etc.), swine (boars, pigs, etc.), equine livestock (horses, ponies, etc.), and cattle (bulls, cows, oxen, etc.); animals of hunting interest, such as stags, deer, reindeer, etc.; and humans.

[0111] In some embodiments, the subject is a mammal; in some preferred embodiments, the mammal is a human being of any race, sex or age.

[0112] In some other preferred embodiments, the mammal is a farm animal, including but not limited to, cows, horses, goats, llamas, sheep, pigs, chickens, or any animal raised or bred for commercial purposes. In some other preferred embodiments, the mammal is a pet, including but not limited to, dogs, cats or rabbits.

[0113] In some embodiments, alone or in combination with other preferred embodiments, the composition of the invention is administered to a subject through the diet.

[0114] In some embodiments of the invention, alone or in combination with other preferred embodiments, the administration regime of the composition of the invention is, at least, once daily; twice daily; or three times a day, one with each main food intake (breakfast and / or lunch and / or dinner).

[0115] In another aspect, the present invention relates to a method of improving metabolic health in a subject, preferably a subject in need of improving the metabolic health, comprising administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0116] In another aspect, the present invention relates to a method for the treatment, prevention and / or improvement of metabolic syndrome, and / or related conditions, disorders or symptoms, in a subject (“method of metabolic syndrome treatment or prevention of the invention”), preferably a mammal, more preferably a human, comprising administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0117] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament (or a pharmaceutical composition) for improving metabolic health in a subject. The improvement of metabolic health can be measured, for instance, by one or more of the indicators mentioned above in a previous aspect.

[0118] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament (or a pharmaceutical composition) for the treatment, prevention and / or improvement of metabolic syndrome, and / or related conditions, disorders or symptoms, in a subject (“metabolic syndrome use of the invention”), preferably a mammal, more preferably a human.

[0119] All the preferred embodiments and explanations of the terms used in the method of metabolic syndrome treatment or prevention of the invention and in the metabolic syndrome use of the invention have been explained above. All particular terms, definitions and embodiments of previous aspects of the invention are applicable to the method of metabolic syndrome treatment and metabolic syndrome use of the invention, including the term and preferred embodiments for “metabolic syndrome”, as well related conditions, disorders or symptoms.

[0120] Other aspect of the invention relates to a method of improving metabolic health in a subject, preferably a mammal, more preferably a human, comprising administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0121] Other aspect of the invention relates to a method for the treatment, prevention and / or improvement of diabetes, preferably type-2 diabetes and / or a cardiovascular disease or disorder, in a subject, preferably a mammal, more preferably a human, comprising administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0122] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament (or a pharmaceutical composition) for the treatment, prevention and / or improvement diabetes, preferably type-2 diabetes and / or a cardiovascular disease or disorder, in a subject, preferably a mammal, more preferably a human.

[0123] Other aspect of the invention relates to a method for the treatment, prevention and / or improvement of obesity, overweight or related diseases and / or disorders in a subject, preferably a mammal, more preferably a human, comprising administering to the subject the composition of the invention or an effective amount of the strain of the invention and the effective amount of the extract of the invention.

[0124] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament (or a pharmaceutical composition) for the treatment, prevention and / or improvement of obesity, overweight or related diseases and / or disorders in a subject, preferably a mammal, more preferably a human.

[0125] As previously mentioned, the inventors have also demonstrated that Tea Complex Extract, HT-BPL1 and the combination of both modulates gut microbiota in the context of HFHS highlighting the capacity of the blend to increase bacterial richness and beneficial bacteria like Bifidobacterium species, while preventing the increase of potential opportunistic bacteria like F. butyricus (Example 5).

[0126] Thus, other aspect of the invention relates to the composition of the invention for use in the modulation of the gut microbiota in a subject.

[0127] Another aspect of the invention relates to the use of the composition of the invention for the modulation of the gut microbiota in a subject.

[0128] Another aspect of the invention relates to a method for modulating the gut microbiota in a subject which comprises administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0129] In a preferred embodiment, the modulation of the gut microbiota comprises, or consists of, one or more of the following: (i) an increase of the abundance of gut Muricomes, Prevotellaceae UCG 001 and Enterorhabdus, more preferably wherein said increased is compared to when a subject is subjected to a HFHS diet;

[0130] (ii) an increase of the abundance of gut Bifidobacterium species;

[0131] (iii) a reduction of the abundance of Adlercreutzia;-,

[0132] (iv) a reduction of the abundance of Romboutsia;

[0133] (v) an increase of the abundance of Lachnospiraceae;

[0134] (vi) an increase of the abundance of Papilibacter;

[0135] (vii) an increase and / or restoration of Oscillospiraceae levels;

[0136] (viii) a decrease and / or prevention of the increase of Flintibacter butyricus levels; and / or

[0137] (ix) an increase and / or prevention of the decrease of Bifidobacterium pseudoIongum.

[0138] Methodology for assessing the modulation of gut microbiota, including the variation of bacteria as stated in (i) to (ix) above is shown for instance in Example 5.

[0139] All particular terms, definitions and embodiments of previous aspects of the invention are also applicable to the present methods and uses of the invention.

[0140] Non-therapeutic use of the invention

[0141] The present invention also encompasses non-therapeutic uses of the composition of the invention, and of all its particular embodiments alone or in combination with each other. Thus, in another aspect, the present invention relates to the non-therapeutic use of the composition of the invention for body fat reduction and / or maintenance of body weight. The particular embodiments relating to the composition of the invention are applicable to the present inventive aspect. The composition of the invention can also be used for non-therapeutic fat reduction in non-obese subjects, i.e. in “normalweight” subjects having a BMI < 25 kg / m2or "overweight" subjects , subjects having a BMI > 25 kg / m2but < 30 kg / m2, as defined above, and without any obesity- associated health implications. Such use is exclusively for aesthetic or cosmetic reasons (cosmetic use) and not based on a medical indication. Such use may also involve feed or nutritional purposes, not based on a medical indication. Also, body fat reduction and / or maintenance of body weight in non-obese subjects might involve body weight reduction and / or maintenance. The cosmetic product will uniquely have a cosmetic effect in the subject who uses it related to body fat reduction and / or maintenance of body weight. The term “cosmetic effect” is explained below. Analogously, the present invention relates to a non-therapeutic method for body fat reduction comprising administering the composition of the invention (comprising all its particular embodiments alone or in combination with each other), or an effective amount of the strain of the invention and an effective amount of the extract of the invention, to non-obese subjects.

[0142] “Non-therapeutic” as used herein, means that the composition does not cure a specific pathological disorder.

[0143] The cosmetic product will uniquely have a cosmetic effect in the subject who uses it related to body fat reduction.

[0144] The term "cosmetic effect" as used herein refers to the desired advantageous impact of composition of the invention, with regard to appearance, which is associated with loss or maintenance of body fat, and preferably an enhancement of body shape and definition. It is to be understood that the non-therapeutic cosmetic treatment of a subject with the composition of the invention is for aesthetic reasons only and is exclusively accomplished in subjects that do not exhibit an amount of body fat that significantly shows or increases health risks. Non-therapeutic, cosmetic treatment with the composition of the invention may also induce loss of weight and / or serve for weight control in order to prevent a (non-pathological) body fat accumulation and / or gain of weight.

[0145] In another aspect, the present invention relates to the use of the invention, or the composition of the invention, for the elaboration of a food or feed product. The terms “food” and “feed product” have been defined previously herein and are applicable to the present inventive aspect. The process and methods for elaborating foods or feed products are widely known in the state of the art.

[0146] Another aspect of the present invention refers to a method of reducing fat accumulation in a subject, preferably mammal, more preferably human, comprising administering to the subject the composition of the invention, or an effective amount of the strain of the invention and an effective amount of the extract of the invention.

[0147] DESCRIPTION OF THE DRAWINGS

[0148] Fig. 1. Chromatographic profile of Tea Complex Powdered Extract standardized to flavan-3-ols (3. Gallocatechin, 5. Epigallocatechin, 6. Catechin, 8. Epicatechin, 9. Epigallocatechin gallate, 10. Gallocatechin-3-gallate, 11. Epicatechin-3-gallate, 12. Catechin-3-gallate, 13. Theaflavin, 14. Theaflavin-3-monogallate, 15. Theaflavin-3'- monogallate, 16. Theaflavin-3,3'-gallate) and xanthines (2. Theobromine, 4. Theophylline & 7. Caffeine) analyzed by High Performance Liquid Chromatography coupled to photo diode Array at 275 nm (A), UV spectra of chromatographic peak 1 (B), UV spectra of chromatographic peaks 2, 4 and 7 (C), UV spectra of chromatographic peaks 3, 5, 6, 8, 9, 10, 11 and 12 (D), and UV spectra of chromatographic peaks 13, 14, 15 and 16 (E).

[0149] Fig. 2. Volatile profile of Tea Complex Extract analyzed by gas chromatography.

[0150] Fig. 3. Relative proportion of volatile components from Tea Complex.

[0151] Fig. 4. HPLC analysis of Tea Complex Extract as control (A), and digested samples at salivary (B), gastric (C).

[0152] Fig. 5. HPLC analysis of Tea Complex Extract at intestinal stages.

[0153] Fig. 6. Effect of blend Tea Complex Extract and HT BPL1 on C. elegans body fat reduction. Data are represented as the mean ± SD. ***p < 0.001 vs NG; ****p < 0.0001 vs NG; ### p < 0.001 vs “Tea Complex Extract + HT-BPL1”. NG, nematode growth media.

[0154] Fig. 7. Fat content as % of fluorescence produced by Nile red staining vs NG in nematodes fed with E. coli OP50 and / or orlistat and different botanical extracts together with HT-BPL1. Data are represented as the mean ± SD. * p < 0.05 vs NG; ** p < 0.01 vs NG. NG, nematode growth media. Fig. 8. Fat content as % of fluorescence produced by Nile red staining vs NG in nematodes fed with E. coli OP50 and / or orlistat and different B. animalis subsp. lactis strains together with Tea Complex Extract. Data are represented as the mean ± SD.

[0155] * p < 0.05 vs NG; ** p < 0.01 vs NG. NG, nematode growth media.

[0156] Fig.9. Effect of blend Tea Complex Extract and BPL1 on C. elegans body fat reduction. Data are represented as the mean ± SD. * p < 0.05 vs NG; *** p < 0.001 vs NG; ## p < 0.01 vs “Tea Complex Extract + BPL1”. NG, nematode growth media.

[0157] Fig. 10. Body weight increase (A), total food intake (B), total caloric intake (C) and total liquid intake (D) of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $$ p < 0.01 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1.

[0158] Fig. 11. Glycemia during the oral glucose tolerance test (A) and its area under the curve (B) in mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; $$ p < 0.01 vs HFHS + TC; && p < 0.01 vs HFHS + HT-BPL1.

[0159] Fig. 12. Blood glucose (A) and Insulin (B), and HOMA-IR index (C) in mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM.

[0160] * p < 0.05 vs Control; ** p < 0.01 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; $ p < 0.05 vs HFHS + TC; $$ p < 0.01 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1. Fig. 13. Plasmatic levels of total cholesterol (A), LDL-cholesterol (B), HDL-cholesterol (C), triglycerides (D), leptin (E) and adiponectin (F) in mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $ p < 0.05 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1.

[0161] Fig. 14. Adipocyte area of epididymal white adipose tissue of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. *** p < 0.001 vs Control; ### p < 0.001 vs HFHS; $$$ p < 0.001 vs HFHS + TC; &&& p < 0.001 vs HFHS + HT-BPL1.

[0162] Fig. 15. mRNA levels of lipoprotein lipase (LPL), hormone-sensitive lipase (LI PE), adrenergic receptor 3p (AdR3p), fatty acid synthase (FASn), uncoupling protein 1 (LICP1), leptin receptor (LEPr), peroxisome proliferator activated receptor y (PPARy) and PPARy coactivator- 1a (PGC-1a) (A), and of interleukin-6 (IL-6), interleukin-1 p (IL-1P), tumoral necrosis factor-a (TNF-a), monocyte chemoattractant protein-1 (MCP-1), NADPH oxidase 4 (NOX-4), superoxide dismutase 1 (SOD-1), glutathione peroxidase 3 (GPX-3) and glutathione reductase (GSR) (B) in epididymal white adipose tissue of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT- BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $ p < 0.05 vs HFHS + TC; $$ p < 0.01 vs HFHS + TC; & p < 0.05 vs HFHS + HT- BPL1.

[0163] Fig. 16. pAkt / Akt ratio of liver (A) and gastrocnemius (B) explants incubated or not with insulin (10'6M) of mice fed with a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control incubation; *** p < 0.001 vs Control incubation; # p < 0.05 vs HFHS-lnsulin; ## p < 0.01 vs HFHS-lnsulin; ### p < 0.001 vs HFHS-lnsulin.

[0164] Fig. 17. Triglyceride content (mg / dL) (A) and mRNA levels of interleukin-6 (IL-6), interleukin-i p (IL-1 p), tumoral necrosis factor-a (TNF-a), monocyte chemoattractant protein-1 (MCP-1), NADPH oxidase 4 (NOX-4), superoxide dismutase 1 (SOD-1), glutathione peroxidase 3 (GPX-3) and glutathione reductase (GSR) (B) in the liver of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT- BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; & p < 0.05 vs HFHS + HT-BPL1.

[0165] Fig. 18. mRNA levels of interleukin-6 (IL-6), interleukin-1 p (IL-1 p), interleukin-10 (IL- 10), tumoral necrosis factor-a (TNF-a) and monocyte chemoattractant protein-1 (MCP-1) (A), and of NADPH oxidase 1 (NOX-1), superoxide dismutase 1 (SOD-1), glutathione peroxidase 3 (GPX-3) and glutathione reductase (GSR) (B) in gastrocnemius muscle of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $ p < 0.05 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1.

[0166] Fig. 19. Heart rate (A) and diastolic (B) and systolic (C) blood pressure of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; *** p < 0.001 vs Control; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; & p < 0.05 vs HFHS + HT-BPL1 . Fig. 20. Contraction response to endothelin-1 (ET-1) as % of KCI contraction (A) and its EC50 (B), contraction response to angiotensin-ll (Ang-ll) as % of KCI contraction (C) and its EC50 (D), and Emax of norepinephrine (NA) contraction response in presence or absence of L-NAME (E) of abdominal aortic segments of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; $ p < 0.05 vs HFHS + TC; + p < 0.05 vs vehicle.

[0167] Fig. 21. Emax of acetylcholine (ACh) vasodilation response in presence or absence of apocynin (A), nitrates and nitrites release (B), vasodilation response to sodium nitroprusside (NTP) as % of basal tone (C), EC50 of insulin vasodilation response (D), and pAkt / Akt ratio in presence or absence of insulin 10'6M (E) of thoracic aortic segments of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; ** p < 0.01 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; $ p < 0.05 vs HFHS + TC; + p < 0.05 vs vehicle.

[0168] Fig. 22. mRNA levels of interleukin-6 (IL-6), interleukin-1 p (IL-1 P), tumoral necrosis factor-a (TNF-a) and monocyte chemoattractant protein-1 (MCP-1) (A), of NADPH oxidase 4 (NOX-4), superoxide dismutase 1 (SOD-1), glutathione peroxidase 3 (GPX- 3) and glutathione reductase (GSR) (B), and of angiotensin 1a (AT1a) and 2 (AT2) receptors (C) in the aorta of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM. * p < 0.05 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $ p < 0.05 vs HFHS + TC; $$ p < 0.01 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1 .

[0169] Fig. 23. p-eNOS / eNOS ratio in the aorta of mice fed a standard chow (Chow), mice fed a high-fat diet / sucrose diet (HFHS), mice fed a high-fat diet / sucrose diet supplemented with Complex Tea Extract (HFHS+CTE), mice fed a high-fat diet / sucrose diet supplemented with BPL1® HT (HFHS+ BPL1® HT) and mice fed a high-fat diet / sucrose diet supplemented with Complex Tea Extract and BPL1 ™ HT (HFHS+CTE+ BPL1® HT). Values are represented as mean value ± SEM; n=8-10 mice / group. * p < 0.05 vs. Chow; ** p < 0.01 vs. Chow; *** p < 0.001 vs. Chow; # p < 0.05 vs. HFHS; ## p < 0.01 vs. HFHS; ### p < 0.001 vs. HFHS; $ p < 0.05 vs. HFHS+CTE; $$ p < 0.01 vs. HFHS+CTE; & p < 0.05 vs. HFHS+ BPL1® HT.

[0170] Fig. 24. Boxplots of alpha diversity (Richness, Shannon, and Simpson indexes) of mice fed a standard chow (Chow), mice fed a high-fat diet / sucrose diet (HFHS), mice fed a high-fat diet / sucrose diet supplemented with Complex Tea Extract (HFHS+CTE), mice fed a high-fat diet / sucrose diet supplemented with BPL1® HT (HFHS+BPL1® HT) and mice fed a high-fat diet / sucrose diet supplemented with Complex Tea Extract and BPL1® HT (HFHS+CTE+ BPL1® HT) for 23 weeks. An analysis of variance (ANO A) or a Kruskal-Wallis test was performed for parametric or non-parametric data. Student's t-test was performed as ANOVA post-hoc analysis. * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 between groups.

[0171] Fig. 25a-b. Association heatmaps showing the Maaslin2 Coefficient (Coeff) between clinical variables, gene expression and protein expression and gut genera abundances. Left heatmap: dark gray color means the feature is directly associated with taxa abundance, while white color is inversely associated. Right heatmap: dark gray means that the taxon is over-represented in the first group of the comparison (bottom), while white means that the taxon is under-represented in the first group (bottom). Black boxes with an arrow highlight taxa associated with several clinical parameters of metabolic syndrome and altered by high-fat diet / sucrose diet (HFHS) compared to standard chow (Chow); black boxes with a circle highlight the taxa that were altered by HFHS compared to Chow and modulated by the supplementation with Complex Tea Extract (HFHS+CTE), BPL1® HT (HFHS+ BPL1® HT) or the blend Complex Tea Extract and BPL1® HT (HFHS+CTE+ BPL1® HT). Black boxes with a square highlight the taxa that were modulated only by the blend, and that are associated negatively with clinical parameters of metabolic syndrome. * adj. p < 0.05 in left heatmap means the feature is significantly associated with taxa abundance. * adj. p < 0.05 in right heatmap means the differential abundance between groups and the taxon is present in at least 50% of samples of one of the compared groups. BaseMean bar plots show the mean abundances of each genera. Coeff means the coefficient of association between clinical variables, gene expression and protein expression and gut genera abundances. LogFC means the log fold-change of genera abundance between the compared groups.

[0172] Fig. 26. Abundance of bacterial genera altered by a high-fat diet / sucrose diet (HFHS) and modulated by the supplementation of Complex Tea Extract (HFHS+CTE), BPL1® HT (HFHS+ BPL1® HT) or blend (HFHS+CTE+ BPL1® HT). The figure show boxplots of abundance (logarithm) of bacteria genera in each group. * in boxplots mean differential abundance in taxa between groups calculated by DESeq2, corrected by FDR and only if taxa are present in at least 50% of the samples of one of the compared groups.

[0173] Fig. 27. Abundance of bacterial genera modulated only by the supplementation of a blend of Complex Tea Extract and BPL1® HT during high-fat diet / sucrose diet (HFHS + CTE + BPL1® HT) associated with clinical parameters of metabolic syndrome. The figure shows boxplots of abundance (logarithm) of bacteria genera in each group. * in boxplots mean differential abundance in taxa between groups calculated by DESeq2, corrected by FDR and only if taxa are present in at least 50% of the samples of one of the compared groups.

[0174] Fig. 28a-b. Association heatmaps showing the Maaslin2 Coefficient (Coeff) between clinical variables, gene expression and protein expression and gut ASV abundances. Left heatmap: dark gray color means the feature is directly associated with taxa abundance, while white color is inversely associated. Right heatmap: dark gray means that the taxon is over-represented in the first group of the comparison (bottom), while white means that the taxon is under-represented in the first group (bottom). Black boxes with an arrow highlight taxa associated with several clinical parameters of metabolic syndrome and altered by high-fat diet / sucrose diet (HFHS) compared to standard chow (Chow); black boxes highlight the taxa that were altered by HFHS compared to Chow and modulated by the supplementation with Complex Tea Extract (HFHS+CTE), BPL1® HT (HFHS+ BPL1® HT) or the blend Complex Tea Extract and BPL1® HT (HFHS+CTE+ BPL1® HT). * adj. p < 0.05 in left heatmap means the feature is significantly associated with taxa abundance. * adj. p < 0.05 in right heatmap means the differential abundance between groups and the taxon is present in at least 50% of samples of one of the compared groups. BaseMean bar plots show the mean abundances of each ASV. Coeff means the coefficient of association between clinical variables, gene expression and protein expression and gut ASV abundances. LogFC means the log fold-change of ASV abundance between the compared groups.

[0175] Fig. 29. Abundance of bacterial ASV altered by a high-fat diet / sucrose diet (HFHS) and modulated by the supplementation of Complex Tea Extract (HFHS+CTE), BPL1® HT (HFHS+ BPL1® HT) or blend (HFHS+CTE+ BPL1® HT). The figure show boxplots of abundance (logarithm) of ASV in each group. * in boxplots mean differential abundance in taxa between groups calculated by DESeq2, corrected by FDR and only if taxa are present in at least 50% of the samples of one of the compared groups.

[0176] Examples

[0177] Example 1. Tea Complex Extract characterization

[0178] Different analytical techniques were used for the characterization of Tea Complex Extract, mostly liquid and gas chromatography, together with spectrophotometry.

[0179] Materials and methods

[0180] Reagents, chemicals

[0181] The following standards were used for liquid chromatography analyses: xanthines (caffeine, theobromine & theophylline), monomeric flavan-3-ols [(+)-catechin, catechin gallate, (-)-Epicatechin, Epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate (EGCg), green tea catechin mix], theaflavins [theaflavin, theaflavins mix (tea extract from Camellia sinensis (L.))] and gallic acid. They were purchased from Merck (Darmstadt, Germany) and Phytolab (Vestenbergsgreuth, Germany) for the identification and / or quantification of characteristic bioactive components from tea (Camellia sinensis (L.)). Trifluoroacetic acid, acetic acid, acetonitrile, dimethyl sulfoxide and water of chromatographic quality were purchased from VWR (Radnor, PA, USA).

[0182] For gas chromatography analyses, the standards used were: 1-penten-3-ol, n- hexanal, n-hexanol, Z-3-hexenal, E-2-hexenal, Z-3-hexenol, E-2-hexenol, and pentanol, and for Group II, linalool, linalool oxides, methyl salicylate, phenyl acetaldehyde, geraniol, benzyl alcohol, 2-phenylethanol, benzaldehyde, a-ionone, and p-ionone, were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol, acetic acid and furfural were obtained from VWR (Radnor, PA, USA). Folin- Ciocalteu’s reagent, catechin hydrate (98% purity), and anhydrous sodium carbonate (99% purity) from Merck (Dramstadt, Germany) were also employed.

[0183] High Performance Liquid Chromatography (HPLC)

[0184] Flavan-3-ols, methylxanthines, theaflavins and gallic acid analyses were performed according to Lee and Ong et al. (Lee and Ong, 2000). The HPLC equipment used for the analysis consisted of a Shimadzu NEXERA XR UHPLC 70MPa coupled to a photodiode array detector SPD-M40 model (Izasa Scientific, Spain). The chromatographic analyses were performed by an octadecyl silane column ZORBAX ECLIPSE PLUS C18 (250 mm, 4.6 mm, 5 p) together with its corresponding precolumn (Agilent Technologies; Santa Clara, CA, USA).

[0185] Detection was performed at 275 nm, the temperature of the oven was set at 32 °C, the work flow was maintained at 1.0 mL / min, and the injection volume 2 pL. Binary gradient system used for the chromatographic separation consisted on Phase (A) 5% (v / v) acetonitrile 0.035 (v / v) trifluoroacetic acid, and Phase (B) 50% (v / v) acetonitrile 0.025% (v / v) trifluoroacetic acid. The initial conditions were set with A-B (90:10), and the gradient slightly increased to 20% at 10 minutes, to 40% from 25 to 27 min. Finally, the column was again balanced to the initial gradient conditions for 3 minutes before the next injection.

[0186] Identification of monomeric and oligomeric flavan-3-ols was performed by comparing retention time and UV-Vis spectra of the corresponding standards. Quantification was performed by external calibration curve with at least 5 different concentration points (r2=0.99), the results were expressed in percentage (%, dry basis). The sum of monomeric flavan-3-ols, methylxanthines and theaflavins were respectively quantified as EGCg, caffeine and theaflavin equivalents.

[0187] Headspace SPME-GC-FID / MS Analysis

[0188] According to Inarejos-Garcia et al. (2021), each powdered tea sample dissolved in water (250 pg / mL) was placed into a 20-mL headspace vial, sealed with a silicone septum. Water blanks were analyzed in the beginning of the sequence and between samples. By exposing the SPME fiber (2 cm; divinylbenzol / carboxen / polydimethylsiloxane 50 pm / 30 pm for both latter coating; Supelco Sigma-Aldrich), headspace sampling was performed at 60 °C for 60 min. Then, the adsorbed volatile components were desorbed from the fiber at 250 °C during 60 s and transferred (nitrogen at 1.0 mL / min) by splitless injection (GC6890N+MS5973Network, Agilent, Waldbronn, Germany) on a 60-m HP-Innowax column (0.25 mm i.d., 0.25 pm polyethylenglycol film thickness, Agilent). The oven temperature ramp was as follows: 50 °C for 5 min, 3 °C / min up to 110 °C (0 min) and 5 °C / min up to 230 °C (31 min). The FID was set at 250 °C, the MSD transfer line at 280° C, the temperature of the ionization source at 230 °C, the quadrupole at 150 °C, the electron multiplier tube to 70 eV, and the scan to m / z 20-250 (acquisition rate of 10 spectral matches). The identification was performed by the NIST05 El Database (National Institute of Standards and Technology, Gaithersburg, MD, USA) and confirmed by authentic reference standards. The ratio of individual components was expressed as relative area percentages.

[0189] Total Polyphenols Content

[0190] The spectrophotometric method, described by the International Organization for Standardization ISO 14502-1 :2005, using a nano-spectrophotometer (SPECTROstarNano BMG LABTECH), expressing the total polyphenols content as catechin equivalents (percentage, dry basis).

[0191] Results As can be observed in Figure 1 , two main different groups of molecules, chiefly flavan-3-ols (monomeric flavanols, theaflavins) and methylxanthines, together with gallic acid, which were considered for quantification.

[0192] As average, the concentration of total flavan-3-ols was 19.46% (dry basis) and methylxanthines 7.02% (dry basis; Table 1). Total polyphenols results were over 30% according also to the specifications (41.2%, dry basis).

[0193] Table 1 . Bioactive composition (%, dry basis) of Tea Complex Extract (TCE) analyzed Control

[0194] Theobromine 0.44 ± 0.15

[0195] Gallocatechin 0.71 ± 0.54

[0196] Theophyllyne 0.05 ± 0.01

[0197] Epigallocatechin 2.05 ± 1.93

[0198] Catechin 0.41 ± 0.13

[0199] Caffeine 6.55 ± 0.51

[0200] Epicatechin 1 .30 ± 0.54

[0201] EGCg 9.29 ± 1.14

[0202] Gallocatechin-3-gallate 1 .14 ± 0.40

[0203] Epicatechin-3-gallate 2.96 ± 1.41

[0204] Catechin-3-gallate 1 .60 ± 2.00

[0205] Theaflavins 0.43 ± 0.12

[0206] Total flavan-3-ols 19.46 ± 3.92

[0207] Total xanthines 7.02 ± 0.60

[0208] The gas chromatography analysis of Tea Complex Extract showed a complete profile of volatile components (Figures 2 & 3); according to Inarejos-Garcia et al., 2021 , hexanal, hexanol, hex-3-en-1-ol are within Group I of characteristic volatiles from Camellia sinensis, whereas linalool, linalool oxides, methyl salicylate, geraniol, benzyl alcohol, phenethyl alcohol and benzaldehyde belong to Group II. Other components such as limonene are also characteristic to this botanical specie.

[0209] To sum up, Tea Complex powdered extract shows a complete profile of polar components characteristic from tea (Camellia sinensis (L.)), mostly flavan-3-ols and methylxanthines, together with the corresponding volatile components. References

[0210] Bee-Lan Lee, Choon-Nam Ong. Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis. Journal of Chromatography A, 2000, 881 (1-2), 439-447.

[0211] Inarejos-Garcia, AM., Helbig, I., Klette, P., Weber, S., Maeder, J., Morlock, G. Authentication of Commercial Powdered Tea Extracts (Camellia sinensis L.) by Gas Chromatography. ACS Food Science and Technology, 2021 , 1 , 4, 596-604.

[0212] ISO 14502-1 :2005: Determination of substances characteristic of green and black tea- Part 1 : Content of total polyphenols in tea-Colorimetric method using Folin-Ciocalteu reagent (2005).

[0213] Example 2. Tea Complex Extract Bioavailability: In vitro digestion of tea complex

[0214] Bioavailability is the part of the ingested component which is available for utilization in physiological functions, evaluated in vivo. The bioavailability is defined by digestibility and solubility of the component at gastrointestinal level, its absorption at intestinal level and finally transportation into the circulation. Furthermore, bioavailability includes bioaccessibility and bioactivity. Bioaccessibility is the fraction that is delivered from the component to the gastrointestinal tract and therefore, it is available for intestinal adsorption, which is usually evaluated in vitro (Etcheverry et al., 2012).

[0215] In the last decades different in vitro methods were developed trying to simulate mimic the physiological conditions that may occur during digestion. Static or biochemical methods normally include three digestion steps (oral, gastric, intestinal), keeping the components static in the reactor.

[0216] These methods simulate a limited number of parameters of physiological digestion (to be described below), and do not mimic physical processes such as shearing, mixing, hydration, changes in conditions over time, or peristalsis (Fernandez-Garcia et al., 2009).

[0217] Materials and methods

[0218] Phosphate buffer saline (PBS) pH 7.4, pepsin from porcine gastric mucosa (P6887- 250MG-5G), a-amylase-(A0521-100UN), pancreatin (P7545-25G) enzymes, bile powders (B8631-100G) and hydrogen chloride acid (HCI), Bicarbonate (NaHCOs) products were all purchased from Merck (Darmstadt, Germany). Methylxanthines (caffeine, theobromine & theophylline), monomeric flavan-3-ols [(+)-catechin, catechin gallate, (-)-Epicatechin, Epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate (EGCg), green tea catechin mix], theaflavins [theaflavin, theaflavins mix (tea extract from Camellia sinensis)] and gallic acid, were purchased from Merck (Darmstadt, Germany) and Phytolab (Vestenbergsgreuth, Germany). These standards were used for the identification and / or quantification of characteristic major bioactive components from tea (Camellia sinensis (L.)) and their digested fractions.

[0219] Trifluoroacetic acid, acetonitrile, methanol and water of chromatographic method solvents quality were purchased from VWR (Radnor, PA, USA).

[0220] In vitro digestion method

[0221] Static “in vitro" digestion was performed according to Hollebeeck et al. (2013), structured into three stages: oral, gastric, and intestinal digestion, and considering the nutritional facts of the Tea Complex Extract (Table 2). Briefly, 2 g of Tea Complex Extract was dissolved with 20 mL of PBS (volume of about 20-30 mL). The salivary step runs for 5-10 min in continuous shaking (model Grant bio PSU-10i; VWR, Spain) at pH 6.8-6.9 at 37°C under aerobic conditions with 3.9 units a-amylase / mL dissolved in PBS in centrifuge tubes of 50 mL. Afterwards, the pH of the media was set at 2-3 (37°C), and treated with 71.2 units pepsin / mL dissolved in 0.1 M HCI under anaerobic conditions in continuous shaking slow movement during 90-120 min to mimic the digestion conditions, with a final volume of 40-50 mL. Finally, the duodenal step was set for 150 min at pH 7-7.4 at 37°C, with 9.2 mg pancreatin / mL and 55.2 mg bile extract / mL (1 :6 ratio enzyme: bile) under anaerobic conditions, to a final volume of about 80-90 mL. Samples were heated (90°C) to stop the reaction on each phase, and centrifuged at 7000 g (Eppendorf 5804R VWR; Radnor, PA, USA) for 10 min at 4°C.

[0222] Supernatants were dried in a vacuum oven (Vaciotem-T Selecta, Spain) to dryness, and stored until further analyses.

[0223] Liquid chromatography analyses

[0224] The chromatographic analyses of digested fractions samples were performed according to Lee & Ong (2000). HPLC equipment used for the analysis consisted of a Shimadzu NEXERA XR UHPLC 70MPa coupled to a photodiode array detector SPD-M40 model (Izasa Scientific, Spain). The chromatographic analyses were performed by an octadecyl silane column ZORBAX ECLIPSE PLUS C18 (250 mm, 4.6 mm, 5 p) together with the corresponding precolumn (Agilent Technologies; Santa Clara, CA, USA).

[0225] Tea Complex Extract (TCE) samples digested were prepared for analysis dissolved in 100% dimethyl sulfoxide (DMSO) in centrifuge tubes of 10 mL in a concentration around 50.000-100.000 ppm followed by shaking into the orbital shaker during 30 minutes at maximum shaking velocity, and finally filtered through syringe nylon filter (0.45 pm) previous to HPLC analysis.

[0226] For identification and quantification purposes, all standards were prepared in 10 mL flasks with 100% dimethyl sulfoxide (DMSO), and corresponding homogenization. Three working calibration standards solutions (EGCg, caffeine, theaflavin) were employed for the quantification of the main groups of components in the tea functional powdered extracts. The sum of monomeric flavan-3-ols was quantified as EGCg equivalents, the sum of xanthines as caffeine equivalents and the sum of theaflavins as theaflavin equivalents. Gallic acid was used to identification standard marker. External calibration curves were performed with at least 5 different concentration points (r2=0.99). Detection was performed at 275 nm, the temperature of the oven was set at 32°C, the work flow was maintained at 1.0 mL / min, and the injection volume was 2 pL. Binary gradient system used for the chromatographic separation consisted on Phase (A) 5% (v / v) acetonitrile 0.035 (v / v) trifluoroacetic acid, and Phase (B) 50% (v / v) acetonitrile 0.025% (v / v) trifluoroacetic acid. The initial conditions were set with A-B (90:10), and the gradient slightly increased to 20% at 10 minutes, to 40% from 25 to 27 min. Finally, the column was again balanced to the initial gradient conditions for 3 minutes before the next injection.

[0227] Table 2. Nutritional values of Tea Complex Extract (g / 100g)

[0228] Average values (per 100g) Value Unit

[0229] Energy 1394 Kj

[0230] Energy 325 Kcal

[0231] Fat 0.5 g of which saturates 0.1 g

[0232] Carbohydrates 58.9 g

[0233] Of which sugars 12.0 g

[0234] Fiber 6.2 g

[0235] Protein 19.1 g

[0236] Salt 0.6 g

[0237] Sodium 0.2 g

[0238] Results

[0239] As can be observed in Figure 4A, the complete chromatographic profile of Tea Complex Extract before digestion is characterized for a high concentration of total flavan-3-ols (19.46%, dry basis) and methylxanthines (7.02%, dry basis; Table 3).

[0240] Table 3. Bioactive composition (%, dry basis) of Tea Complex Extract (TCE) throughout different “in vitro" digestion steps analyzed by HPLC.

[0241] Bioactive Components . -r^r- Decrease

[0242] / o / j • » TCE Contro Digested TCE ,, . . .

[0243] (%, dry basis) (folds)

[0244] Theobromine 0.44 ± 0.15 0.12 ± 0.07 3.67

[0245] Gallocatechin 0.71 ± 0.54 0.04 ± 0.03 17.75

[0246] Theophylline 0.05 ± 0.01 0.02 ± 0.00 2.50

[0247] Epigallocatechin 2.05 ± 1.93 0.04 ± 0.04 51.25

[0248] Catechin 0.41 ± 0.13 0.14 ± 0.11 2.93

[0249] Caffeine 6.55 ± 0.51 1.81 ± 0.66 3.62 Epicatechin 1.30 ± 0.54 0.31 ± 0.20 4.19

[0250] EGCg 9.29 ± 1.14 0.11 ± 0.17 84.45

[0251] Gallocatechin-3-gallate 1.14 ± 0.40 0.06 ± 0.07 19.00

[0252] Epicatechin-3-gallate 2.96 ± 1.41 0.50 ± 0.46 5.92

[0253] Catechin-3-gallate 1.60 ± 2.00 0.09 ± 0.06 17.78

[0254] Theaflavins 0.43 ± 0.12 0.12 ± 0.01 3.58

[0255] Total flavan-3-ols 19.46 ± 3.92 1.23 ± 0.98 15.82

[0256] Total xanthines 7.02 ± 0.60 1.93 ± 0.73 3.64

[0257] After digestion process, the Tea Complex Extract samples showed a significant reduction of flavan-3-ols and methylxanthines concentration throughout the complete process (salivary, gastric and intestinal steps) as in Table 3. In fact, total flavan-3-ols showed about 5 folds higher decrease after digestion process (Figure 5) compared to total xanthines (respectively 15.82 and 3.64 folds; Table 3). Regarding individual composition, as expected EGCg showed the highest decreased after digestion (84.45 folds less concentrated, Table 3), however, theophylline showed lowest decrease after digestion (2.50 folds, Table 3).

[0258] Based on the results, major bioactive components were reduced, mostly flavan-3-ols, and within them, chiefly EGCg (Figure 4A) which, according to literature, shows lower oxidative stability compared to the rest of flavan-3-ols as follows: EGCg>Epigallocatechin>Epicatechin gallate>Epicatechin (Yoshioka et al., 1991), which was in line with the results obtained after the digestion regarding decrease: EGCg (84 folds), Epigallocatechin (51 folds), Epicatechin-3-gallate (6 folds) and Epicatechin (4 folds). Apart from the oxidative stability, other factors such as the presence of other nutrients may interact with these polar components (Foegeding et al., 2017; Renard et a!., 2017).

[0259] References

[0260] Etcheverry, P., Grusako, MA., Fleige, LE. Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc, and vitamins B6, B12, D, and E. Frontiers in Physiology 2012, 3, 1-22. Fernandez-Garcia, E., Carvajal-Lerida, I., Perez-Galvez, A. In vitro bioaccessibility assessment as a prediction tool of nutritional efficiency. Nutrition Research 2009, 29, 751-760.

[0261] Hollebeeck, S., Borlon, F., Schneider, Y-J., Larondelle, Y., Rogez, H. Development of a standardised human in vitro digestion protocol based on macronutrient digestion using response surface methodology. Food Chemistry 2013, 138 (2-3), 1936-1944.

[0262] Bee-Lan Lee, Choon-Nam Ong. Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis. Journal of Chromatography A, 2000, 881 (1-2), 439-447.

[0263] Yoshioka, H., Sugiura, K., Kawahara, R., Fujita, T., Making, M., Kamiya, M., Tsuyumu, S. Formation of Radicals and Chemiluminescence during the Autoxidation of Tea Catechins. Agricultural and Biological Chemistry 1991 , 55 (11), 2717-2723.

[0264] Foegeding, EA., Plundrich, N., Schneider, M., Campbell, C., Lila, MA. Proteinpolyphenol particles for delivering structural and health functionality. Food Hydrocolloids 2017, 72, 163-173.

[0265] Renard, C., Watrelot, A., Le Bourvellec, C. Interactions between polyphenols and polysaccharides: Mechanisms and consequences in food processing and digestion. Trends in Food Science and Technology, 2017, 60, 43-51.

[0266] Example 3. A blend with tea complex and heat-treated BPL1 with enhanced effect on fat deposition reduction in a C. elegans model

[0267] The example aims to summarize the main findings regarding the combination of Tea Complex Extract and HT-BPL1 in the reduction of fat deposition.

[0268] First, different analysis in the organism model of C. elegans were performed, showing an enhanced fat reducing effect of the blend when compared with the single ingredients. Furthermore, 1) a comparative analysis with combinations of HT-BPL1 and different botanical extracts, and 2) a comparative analysis with combinations of Tea Complex Extract and other heat-treated B. animalis strains (different than BPL1) were made. An assay with the alive BPL1 was also performed to confirm that the effect is conserved in a blend with the viable version of the strain.

[0269] Material and methods

[0270] Tested ingredients

[0271] First, Tea Complex Extract was combined with HT-BPL1 and evaluated in fat reduction model in order to search for an enhancement of fat reduction activity when administered together to the nematode.

[0272] In addition, alternative botanical extracts, tea cream, yerba mate, black tea, green tea, white tea and mint leaves, were evaluated in combination with HT-BPL1 in the C.elegans fat reduction model. Besides BPL1 , six strains belonging to Bifidobacterium animalis (BPL6, BPL21 , BPL27, BPL30, B420 and BB12), were tested in combination with Tea Complex Extract in C. elegans fat reduction model.

[0273] BPL6, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from dairy origin;

[0274] BPL21 , obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from faeces from breastmilk fed baby;

[0275] BPL27, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from breastmilk;

[0276] BPL30, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from infant faeces;

[0277] BB-12 (®; Jungersen M., The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12®. Microorganisms. 2014 Mar 28;2(2):92-110) (Benchmark) originates from Chr. Hansen’s collection of dairy cultures; B420™ (Benchmark, Howaru; Uusitupa HM, et al., Bifidobacterium animalis subsp. lactis 420 for Metabolic Health: Review of the Research. Nutrients. 2020 Mar 25; 12(4): 892).

[0278] Fat reduction model in C. elegans

[0279] Fat reduction was assessed in C. elegans (wild type strain N2) in vivo model using the Nile red staining method and further fluorescence quantification by spectrofluorometry. Briefly, the different combinations (botanical extracts and heat- treated B. animalis strains) were added to the surface of standard NGM plates already seeded with Escherichia coli OP50 and the fluorescent dye Nile red (0.05 pg / mL) (Sigma Aldrich; St. Louis, MO, USA). In the case of BPL1 , both heat-treated and alive strain were tested in combination with Tea Complex Extract. The age-synchronized worms were incubated in these plates until reaching young adult stage, and the fluorescence quantification was performed in a spectrofluorometer. Orlistat, a well- known drug to treat obesity, was included as a positive control. Experiments were carried out in duplicate.

[0280] Results

[0281] 3.1. The blend Tea Complex + HT-BPL1 exerts enhanced fat reduction effect in the model of C. elegans.

[0282] In a first step, the combination of Tea Complex Extract with HT BPL1 was tested in the model of C. elegans as described elsewhere ([Martorell et al., 2016] J Agric Food Chem. 64, 3462-3472).

[0283] Results indicate that the blend Tea Complex Extract + HT BPL1 strain provided 27.14 % of fat reduction, showing an enhanced fat reducing effect compared with the single components of the blend (Figure 6 and Table 4).

[0284] Table 4. Evaluation of synergies on fat reduction in C. elegans.

[0285] Ingredient _ % Fat reduction vs Control

[0286] Orlistat 38.11

[0287] BPL1 HT 9.64 Complex Tea Extract 10.10

[0288] Complex Tea Extract + BPL1 HT 27.14

[0289] 3.2. The enhanced effect of the blend Tea Complex + HT BPL1 is surprising and higher than other combinations.

[0290] In order to study that the enhanced effect was specific of the combination of Tea Complex Extract + HT-BPL1 , further experiments were performed by combining HT- BPL1 with alternative botanical extracts. Six different botanical extracts were evaluated in combination with HT-BPL1 but any combination produced an enhancement in fat reduction activity, and besides fat reduction effect was lower than obtained with the blend of Tea Complex Extract with HT-BPL1 (Figure 7).

[0291] Finally, Tea Complex extract was combined with alternative B. animalis strains and evaluated on C. elegans fat reduction model. The combination of Tea complex Extract with HT-BPL1 provided a significant 27.14% fat reduction effect, higher than other combinations including alternative B. animalis strains (Figure 8).

[0292] 3.3. The fat reduction enhanced effect in C. elegans is also observed with viable BPL1

[0293] In order to confirm that the viable strain also exerts an enhanced effect in C. elegans fat reduction when combined with Tea Complex Extract, the blend of viable BPL1 and Tea Complex Extract was tested in the model of C. elegans fat reduction.

[0294] Results indicate that the blend Tea Complex Extract + BPL1 strain showed also an enhanced fat reducing effect compared with the single components of the blend (Figure 9).

[0295] Conclusions

[0296] These results indicate that the blend of Tea Complex Extract with HT-BPL1 has an enhanced fat reduction effect compared with blends of the extract with alternative B. animalis strains or with other botanical extracts. The blend of Tea Complex Extract with HT-BPL1 exerts a surprising effect, not obvious, and its effect is particularly dependent on the ingredients used (Tea complex and HT-BPL1 B. animalis strain). The enhanced effect is also observed with viable BPL1 .

[0297] Example 4. In vivo effects of supplementation with Tea Complex and the postbiotic HT-BPL1 on the cardiometabolic alterations associated with metabolic syndrome in mice

[0298] The objective of this Example is to assess the beneficial effects of a black and green tea extract (Tea Complex Extract; TC) and the postbiotic HT-BPL1 administered alone or in combination, on the metabolic and cardiovascular alterations associated with metabolic syndrome in a murine model of metabolic syndrome.

[0299] Material and methods

[0300] The study was performed with 16-week-old male C57BL / 6J mice, that were housed two per cage and maintained with a 12 h light cycle under controlled conditions of humidity (50-60%) and temperature (22-24 °C). All the experiments were conducted according to the European Union Legislation and with the approval of the Animal Care and Ethical Committee of the Community of Madrid (Madrid, Spain) (PROEX 133.4 / 22).

[0301] Mice were fed ad libitum and divided into five experimental groups (n=8-9 per group) for 20 weeks: (I) mice fed with a standard diet (Control), (II) mice fed with a high fat / high sucrose (HFHS) diet containing 58% kcal from fat with sucrose (HFHS), (III) mice fed with a HFHS diet supplemented with 1.6% of Tea Complex Extract (HFHS + TC), (IV) mice fed with a HFHS diet and supplemented daily with HT-BPL1 (1x1010cells / mice / day) in the drinking water, (V) mice fed with a HFHS diet supplemented with 1.6% of Tea Complex Extract and with HT-BPL1 (1010cells / mice / day) in the drinking water.

[0302] Body weight and solid and liquid intake were monitored weekly during treatment. After treatment period, rats were sacrificed by an overdose of sodium pentobarbital (100 mg / kg). To obtain plasma, truncal blood was collected after euthanasia in tubes with EDTA (1 .5 mg / mL) and centrifuged at 3000 rpm for 20 min. Liver, spleen and adipose tissue depots (epididymal, retroperitoneal, subcutaneous, brown and PVAT) were dissected, weighed and stored at -80 °C for further analysis.

[0303] Glucose tolerance test

[0304] To perform the glucose tolerance test, mice were administered intraperitoneally a bolus of 2 mg / kg of glucose after an overnight fasting. Glycemia was measured 5 min before (basal glycaemia) and 30, 60, 120, and 150 min after glucose injection by venous tail puncture using Glucocard™ G (Arkray Factory Inc.; Koji Konan-cho, Japan). The total area under the curve (AUG) for the glucose response was calculated with the following formula: 25 x (fasting value) + 0.5 x (30 min value) + 0.75 x (1 h value) + 0.5 x (2 h value).

[0305] Homeostatic Model Assessment of Insulin Resistance (HOMA-IR)

[0306] Fasting glycemia was measured by venous tail puncture using Glucocard™ G (Arkray Factory Inc.; Koji Konan-cho, Japan) prior to sacrifice and after overnight fasting. Insulin was measured in plasma by an ELISA commercial kit from Merck Millipore (Darmstadt, Germany). The HOMA-IR index was calculated through the following formula: fasting glucose (mg / dL) x (fasting insulin (ng / nL) / 405).

[0307] Plasma measurements

[0308] Spin React S.A.U. (Sant Esteve de Bas, Gerona, Spain) commercial kits were used to measure plasmatic levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) following the manufacturer’s instructions. Leptin and adiponectin were measured in the plasma by ELISA kits from Merck Millipore (Darmstadt, Germany) following manufacturer’s instructions.

[0309] Measurement of malondialdehyde (MDA) in plasma To measure MDA in plasma, first, plasma samples were derivatized. Briefly, 25 pL of each standard and / or sample together with 500 pL of 2 M AcONa Buffer pH 3.5 + 0.2% thiobarbituric acid (Sigma Aldrich; St. Louis, MO, USA) were incubated at 95 °C for 1 h to hydrolyse lipid peroxides and to release MDA, forming MDA-TBA2 adducts. Then, 500 pL of the 50 mM KH2PO4 Buffer pH 6.8 was added to each standard / sample and finally mixed. All tubes were centrifuged (1000 g, 5 min at 4 °C), and the supernatant (200 pL) were recovered in another tube with 200 pL of the 50 mM KH2PO4 Buffer pH 3.5 and mixed again before HPLC analysis.

[0310] An HPLC equipment Shimadzu Nexera XR HPLC 70MPa coupled to a photodiode array detector SPD-M40 model (Izasa Scientific; Madrid, Spain) was used for the chromatographic analysis, employing an octadecyl silane column Zorbax Eclipse Plus C18 column (250 mm, 4.6 mm, 5 p), together with the corresponding precolumn (Agilent Technologies, Spain), at a wavelength of 532 nm.

[0311] Plasmatic antioxidant activity

[0312] Flavan-3-ols, flavonols and methylxanthines are major bioactive components in tea with antioxidant. The evaluation of the antioxidant capacity of these components in plasma samples is fundamental to better understand how their presence may balance different oxidative stress situations.

[0313] Plasma samples were deconjugated with 500 U / sample of p-glucuronidase (from E. coli type IX-A; VWR; Radnor, PA, USA) during 1h at 37 °C. Then, 1 mL of acetonitrile was added to extract the polar components from tea. The mixture was sonicated for 5 minutes and centrifuged at 3000 g for 10 minutes, and the supernatant collected, repeating the extraction step twice. Pooled supernatant was evaporated to dryness under vacuum, and dissolved in 500 pL of methanol 50% (v / v) before antioxidant capacity analyses. A nano-spectrophotometer (SPECTROstarNano BMG LABTECH) was used to analyze the antioxidant capacity, expressing the results as pM Trolox equivalents / pL plasma.

[0314] Adipocyte size Epididymal white adipose tissue explants were fixed overnight in 4% paraformaldehyde and embedded in paraffin wax. Then, 5 pm microtome cut sections were mounted into slides and stained with Harris Haematoxylin and Eosin. A Leica light microscope with a 10* objective (Wetzlar, Germany) was used to acquire images. To determine the adipocyte size, the area of each adipocyte was measured using FIJI for Windows 36 bit (NIG; Bethesda, MA, USA).

[0315] Liver, gastrocnemius muscle and aortic insulin sensitivity

[0316] To measure the insulin response as Akt phosphorylation, 100 mg of liver and gastrocnemius muscle explants and thoracic aortic segments were incubated at 37 °C in a 95% O2 and 5% CO2 incubator in Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 medium (DMEM / F-12) from Gibco (1 :1 mix; Invitrogen; Carlsbad, CA, USA) supplemented with penicillin (100 U / mL) and streptomycin (100 pg / mL) in the presence / absence of insulin (10'6M) (Sigma Aldrich; St. Louis, MO, USA). After 15 minutes of incubation, tissue explants were collected and stored at -80 °C for further analysis.

[0317] For protein analysis, tissue explants were homogenized in 500 pL of RIPA buffer and centrifugated at 14000 rpm for 20 min at 4 °C. The supernatant was collected and the Bradford method (Sigma Aldrich; St. Louis, MO, USA) was used to measure total protein content in the samples. For each assay, 10 pL of protein was loaded into each well of 10% acrylamide SDS gels (Bio-Rad; Hercules, CA, USA) and separated by electrophoresis. Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad; Hercules, CA, USA). Ponceau red dyeing (Sigma Aldrich; St. Louis, CA, USA) was used to determine transfer efficiency. Transferred membranes were blocked with tris-buffered saline (TBS) containing either 5% (w / v) non-fat dried milk or 5% BSA for non-phosphorylated and phosphorylated proteins, respectively. Then, membranes were incubated with the appropriate primary antibody for Akt (1 :1000; # 04-796, Merk Millipore; Darmstadt, Germany) or p-Akt (Ser 473) (1 :500; #9271 , Cell Signalling Technology; Danvers, MA, USA) at 4 °C overnight. Subsequently, membranes were washed and incubated with the appropriate secondary antibody conjugated with peroxidase (1 :2000; Pierce; Rockford, IL, USA). Peroxidase activity was visualized by chemiluminescence and quantified by densitometry using BioRad Molecular Imager ChemiDoc XRS System (Hercules, CA, USA). Values from mice fed with control diet were used to relativize protein expression levels for each sample.

[0318] Hepatic triglyceride content

[0319] 100 mg of hepatic tissue were homogenized in 300 pL of phosphate-buffer saline (PBS) and centrifuged at 4 °C for 10 min. Triglycerides were measured in the supernatant using a commercial kit (Spin React S.A.U.; Gerona, Spain) according to the manufacturer’s instructions.

[0320] Gene expression analysis by qPCR

[0321] Total RNA was extracted from 100 mg of epididymal adipose tissue, liver, gastrocnemius muscle, and aorta using the Tri-Reagent protocol and quantified with Nanodrop 2000 (Thermo Fisher Scientific; Hampton, NH, USA). 1 pg of total RNA was used to synthesized cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems; Foster City, CA, USA).

[0322] Assay-on-demand kits from Applied Biosystems (Foster City, CA, USA) were used to perform quantitative real-time polymerase chain reaction (qPCR) assays. Amplification was performed using TaqMan universal PCR Master Mix (Applied Biosystems; Foster City, CA, USA) following manufacturer’s instructions in a Step One System (Applied Biosystems; Foster City, CA, USA). In the epididymal adipose tissue, liver, gastrocnemius muscle and aorta, the gene expression of interleukin-6 (IL-6), interleukin-1 p (IL-1 / 3), tumoral necrosis factor-a (Tnf-a), monocyte chemoattractant protein (Mcp-1), superoxide dismutase 1 (Sod-1), glutathione peroxidase 3 (Gpx-3) and glutathione reductase (Gsr) were measured. Gene expression of NADPH oxidase-1 (Nox-1) was measured in the gastrocnemius muscle, and of NADPH oxidase-4 (Nox-4) in the epididymal adipose tissue, liver and aorta. Angiotensin 1a (AT1a) and 2 (AT2) receptors gene expression were also measured in the aorta. The gene expression of lipoprotein lipase (Lpl), hormone-sensitive lipase (Lipe), adrenergic receptor 3 (AdR3 / 3), fatty acid synthase (FASn), uncoupling protein 1 (UCP1), leptin receptor (LEPr), peroxisome proliferator activated receptor y (PPARy) and of PPARy coactivator 1-a (PGC-1a) was measured in the epididymal white adipose tissue. The housekeeping gene Hypoxanthine Phosphoribosyl transferase 1 (Hprt-1') was used to normalize values. Relative expression levels were determined by the 2-AACTmethod. All data are expressed as percentages vs Control.

[0323] Mean arterial blood pressure measurement by the Tail-Cuff System

[0324] Mean arterial blood pressure was measured in each mouse before sacrifice by tailcuff plethysmography using a Niprem 645 blood pressure system (Cibertec; Madrid, Spain). First, mice were placed in a quiet area at 22 ± 2 °C and habituated to the experimental conditions for at least 7 days. An occlusion cuff and a sensor were placed at the base of the tail after warming the mice to 34 °C for 10-15 min. Five to six measurements were recorded in each mouse inflating the occlusion cuff to 250 mm Hg and deflating it over 20 s. An average value was calculated for each animal with the data for each day of measurement.

[0325] Vascular reactivity experiments

[0326] After sacrifice, the aorta was dissected and cut into 2 mm long segments in sterile cold saline solution (NaCI 9 g / L). To prepare aortic segments to isometric tensions recording, two fine steel wires of 100 pm were passed through the lumen of each segment. One wire was fixed to the organ bath while the other was connected to a strain gauge for isometric tension recording (Universal Transducing Cell UC3 and Statham Microscale Accessory UL5, Statham Instruments Inc.). A PowerLab data acquisition system (AD Instruments; Colorado Springs, CO, USA) was used to record changes in isometric force. The 4 mL organ bath contained modified Krebs-Henseleit solution at 37 °C (mM): NaCI, 115; KCI, 4.6; KH2PO4, 1.2, MgSO4, 1.2; CaCI2, 2.5; NaHCOs, 25; glucose 11. An optimal passive tension of 1 g was applied to the vascular segments during 60-90 minutes. After equilibration, contractility of smooth muscle was determined adding 100 mM potassium chloride (KCI). Segments that failed to contract at least 0.5 g to KCI were discarded.

[0327] Abdominal aortic segments were used to evaluate the vasoconstriction response to accumulative doses of endothelin-1 (ET-1) (10’1°— 10-7M), angiotensin-ll (Ang-ll) (10_11-10-6M) or norepinephrine (NA) (109— 104M). Results were expressed as percentage of the contraction to 100 mM KCI. Half maximal effective concentration 50 (EC50) of ET-1 and Ang-ll vasocontraction responses and maximum effect (Emax) of NA vasocontraction response were determined.

[0328] Thoracic aortic segments were used to evaluate the vasodilation response to cumulative dose-responses curves of acetylcholine (ACh) (109— 104M), sodium nitroprusside (NTP) (109— 10-5M) or insulin (1 O'8- 10'5M). Segments were previously precontracted with phenylephrine 10'75M. Relaxation response was expressed as percentage of the initial tone. Emax of Ach vasodilation response and EC50 of insulin vasodilation response were also determined.

[0329] Nitric oxide release of aortic segments

[0330] Nitrite and nitrate concentrations were measured in the culture medium from aorta segment incubations using a modified method of the Griess assay. 100 pL of culture medium were mixed with 100 pL of vanadium chloride (Sigma Aldrich; St. Louis, MO, USA) on a 96-well plate. Then, 100 pL of the Griess reagent (1 :1 mixture of 1 % sulphanilamide and 0.1 % naphthylethylenediamine dihydrochloride (Merck Millipore; Darmstadt, Germany)) were added to each well and incubated at 37 °C for 30 min. After incubation, absorbance at 540 nm was measured. To calculate nitrite and nitrate concentrations a NaNO2 standard curve was used.

[0331] Statistical analysis

[0332] One-way ANOVA followed by a Bonferroni post-hoc test was performed for the statistical data analysis using GraphPad Prism 8.0 (San Diego, CA, USA). All values are expressed as the mean ± standard error of the mean (SEM). A p-value < 0.05 was considered statistically significant.

[0333] Analysis of the p-eNOS / eNOS ratio in aortic tissue

[0334] The p-eNOS / eNOS ratio was measured by Western Blot as mentioned above using 1 :1000 of the primary antibody for endothelial oxide nitric synthase (eNOS) (BD Bioscience, San Jose, CA, USA) and 1 :500 of the primary antibody for phospho- endothelial oxide nitric synthase (p-eNOS) (1 :500; Merck Millipore, Darmstadt, Germany).

[0335] Results

[0336] Figure 10A shows that the ingredients produced a significant decrease in body weight gain induced by the diet, the greater effect being in the case of the co-administration of the ingredients statistically significant compared to the ingredients per separate. Those results were related with the enhanced effects on the reduction of the different adipose tissue depots achieved by the coadministration of ingredients, as shown at Table 5, achieving statistically significant improvements compared to the stand-alone administrations. Furthermore, this was related to a decrease in total food and caloric intake, although without greater effects of the co-administration of the ingredients (Figures 10B and 10C, respectively). Co-administration of both ingredients increased water consumption compared to HFHS-fed animals and the ingredients separately, with no impact on body weight (Figure 10D).

[0337] Table 5. Weights of organs of mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM.

[0338] Organ

[0339] HFHS + HT- HFHS + TC + weight Control HFHS HFHS + TC

[0340] BPL1 HT-BPL1

[0341] (mg / cm)

[0342] Heart 85.05 ± 3.1 113.23 ± 9.8* 89.20 ± 6.7* 82.99 ± 13.9* 63.75 ±

[0343] 12.3**$

[0344] Spleen 33.84 ± 1.8 65.60 ± 5.7™ 42.80 ± 42.49 ± 2.2*** 36.44 ±

[0345] 2 -] "* 2 4***$&

[0346] Liver 583.44 ± 20.7 920.52 ± 483.31 ± 575.62 ± 527.07 ±

[0347] 175.3* 49.3** 24.1* 50.9*

[0348] EWAT 364.00 ± 52.9 1460.96 ± 1138.00 ± 1226.73 ± 654.15 ±

[0349] 61.5™ 139.5™* 126.0™* 168.3***$& RWAT 168.81 ± 33.9 674.82 ± 481.60 ± 513.59 ± 247.19 ±

[0350] 72.3*** 64.8***# 54.1***# 92.0##$&

[0351] SWAT 122.80 ± 871.45 ± 488.60 ± 480.59 ± 237.50 ±

[0352] 17.12 106.3*** 74.1***## 57.4## 73.5###$&

[0353] BAT 58.08 ± 7.2 137.76 ± 85.50 ± 10.1*# 85.32 ± 7.2## 58.89 ±

[0354] 16.6*** Q -|###$&

[0355] PVAT 5.96 ± 0.7 12.27 ± 1.25*** 8.00 ± 0.1*## 8.70 ± 0.7**# 5.92 ±

[0356] BAT, interscapular brown adipose tissue; EWAT, epididymal white adipose tissue; PVAT, perivascular adipose tissue; RWAT, retroperitoneal white adipose tissue; SWAT, lumbar subcutaneous white adipose tissue. * p < 0.05 vs Control; ** p < 0.01 vs Control; *** p < 0.001 vs Control; # p < 0.05 vs HFHS; ## p < 0.01 vs HFHS; ### p < 0.001 vs HFHS; $ p < 0.05 vs HFHS + TC; & p < 0.05 vs HFHS + HT-BPL1 ; && p < 0.01 vs HFHS + HT-BPL1.

[0357] Similarly, Figure 11 shows that the co-administration of the ingredients significantly decreases the area under the curve (AUG) of blood glucose levels during the oral glucose tolerance test compared to the effects achieved by stand-alone administrations. Those results were in line with the HOMA-IR index. As shown in Figure 12, the co-administration significantly improved the effects on the reduction of HOMA-IR accomplished by the ingredients alone compared to HFHS fed animals.

[0358] No differences were found with the co-administration compared to the effects exerted by the stand-alone ingredients on the circulating levels of total cholesterol and HDL- cholesterol. Nevertheless, the combination significantly reduced the circulating levels of LDL-cholesterol, an effect not obtained with the ingredients. Finally, statistically significant enhanced effects were found with the co-administration in reducing the circulating levels of leptin, an hormone whose levels are directly correlated with the amount of body fat, and also in increasing the levels of adiponectin, an antiinflammatory adipokine related with improvements in insulin sensitivity (Figure 13).

[0359] As shown in Table 6, all the administrations reduced the circulating malondialdehyde levels which were found increased in the HFHS fed animals, but the differences were not statistically significant. In contrast, all ingredients statistically reversed the HFHS- diet-induced changes in plasmatic antioxidant capacity. Table 6. Malondialdehyde (MDA) concentration and antioxidant capacity in plasma samples obtained from mice fed a standard diet (Control), a high fat / sucrose diet (HFHS) or a high fat / sucrose diet supplemented with Tea Complex Extract (HFHS + TC), HT-BPL1 (HFHS + HT-BPL1) or both ingredients (HFHS + TC + HT-BPL1). Values are represented as mean ± SEM.

[0360] Control HFHS HFHS + TC HFHS + HT- HFHS + TC

[0361] BPL1 + HT-BPL1

[0362] MDA (nmol / mL) 0.58 ± 0.16a0.93 ± 0.04b0.63 ± 0.17ab0.65 ± 0.03ab0.76 ± 0.10ab

[0363] Antioxidant Capacity0.92 ± 0.05a3.30 ± 0.38c0.76 ± 0.00a0.86 ± 0.10a1.46 ± 0.19

[0364] (pM / pL pl3sma)b

[0365] Different Latin letters (a-c) indicate significant differences (p < 0.05).

[0366] The decrease in the fat mass shown previously in Table 5 was related with a statistically significant enhanced decrease in the adipocyte hypertrophy in the epididymal white adipose tissue produced by the co-administration compared to the independent administration of ingredients (Figure 14). The decrease in fat mass and adipocyte hypertrophy matched with an increase in the PGC-1a gene expression produced only by the co-administration in this adipose depot, a key factor in adipocyte differentiation and energy metabolism. In addition, only co-administration significantly increased the HFHS-induced decrease in leptin receptor gene expression in the epididymal white adipose tissue, maybe explaining the reduction on circulating leptin levels (Figure 15). Nevertheless, although the co-administration decreased the gene expression of proinflammatory cytokines and prooxidative enzymes in the epididymal white adipose tissue compared to HFHS fed animals, it did not enhance the effects achieved by the stand-alone interventions (except for MCP-1).

[0367] Since a proinflammatory adipose tissue is related to insulin resistance, after confirming a decrease in fat-derived inflammation, fat mass and an increase in circulatory adiponectin, the next step was to confirm if these effects improved the insulin sensitivity in peripheral tissues as the liver and the gastrocnemius skeletal muscle. As shown in Figure 16, the co-administration increased the activation of the PI3K / Akt pathway through Akt phosphorylation compared to HFHS fed animals, an intracellular pathway activated by insulin. Nevertheless, the effect was not higher than the one achieved by Tea Complex administration in both tissues. Improvements of liver insulin sensitivity exerted by the treatments were related with decreases in the hepatic triglyceride content and in the gene expression of the proinflam matory cytokines IL-6, I L-1 p, TNFa and MCP-1 and NOX-4 and glutathione reductase enzymes. Nevertheless, any enhanced effect was found with the combination of the ingredients compared to the stand-alone administrations (Figure 17).

[0368] In the gastrocnemius skeletal muscle, the improvement in insulin sensitivity was accompanied by a decrease in the gene expression of the proinflammatory cytokines IL-6, I L-1 p, TNFa and MCP-1 and an increase in the anti-inflammatory IL-10 by the different administrations. In fact, the co-administration decreased TNFa and increased IL-10 gene expressions in a statistically significant greater extent than stand-alone administrations (Figure 18). Any enhanced effect of the co-administration compared to the single administrations were found in the gene expression of prooxidative and antioxidant enzymes.

[0369] Since lipid profile and fat mass are risk factors for the development of cardiovascular diseases, and blood pressure is one of the parameters included in metabolic syndrome, the effects of the ingredients on cardiovascular parameters were also studied. Although no effects were found with the ingredients on heart rate and diastolic arterial pressure, all the administrations prevented the HFHS-diet-induced increase in systolic arterial pressure (with no enhanced effect of the co-administration) (Figure 19).

[0370] The reduction in systolic arterial blood pressure could be explained by (i) the decrease in the vasoconstriction response to endothelin-1 exerted by HT-BPL1 (Figures 20A and 20B), (ii) by the decrease in the vasoconstriction response to angiotensin-ll exerted by Tea Complex Extract (Figures 20C and 20D), and (iii) by the increase in the vasodilatory response to acetylcholine exerted by Tea Complex Extract due to its antioxidant properties in aortic segments at vascular reactivity experiments (Figure 21A). The improvement in the vasodilatory response to acetylcholine was related to a reduction in the HFHS-induced endothelial disfunction. On one side, blocking internal nitric oxide production with L-NAME increased the vasoconstriction response to norepinephrine in aortic segments of control and treated animals, suggesting that nitric oxide was reducing vasoconstriction response (Figure 20E). On the other side, aortic segments from HFHS-fed animals showed a reduction in nitrates release (Figure 21 B), without changes in the vasodilatory response induced by the nitrate donor sodium nitroprusside (Figure 21C). Although improvements in the vasodilatory response to acetylcholine and insulin have been observed in peripheral tissues, improvements in the HFHS diet-induced decrease in vasodilation in response to insulin were only observed with Tea Complex (Figure 21 D). Nevertheless, aortic segments from all administered with the ingredients HFHS-fed animals shown an activation of the PI3K / Akt pathways when segments were exposed to insulin (10'6M) (Figure 21 E).

[0371] The improvements observed in blood pressure and vascular response could be produced by a reduction of the gene expression of the proinflammatory cytokines IL- 6, IL-ip, TNFa and MCP-1 and the prooxidative enzyme NOX-4 induced by the ingredients at the aortic tissue (Figures 22A and 22B). In fact, the co-administration of the ingredients exerted a statistically significant enhanced effect in reducing the gene expression of TNFa and MCP-1 at the aorta. Also, the reduction in the vasoconstriction response to angiotensin-ll were related to a reduction in the gene expression of the vasoconstrictor receptor AT1a and an increase in the gene expression of the vasodilator receptor AT2 in the aorta of administered animals (Figure 22C). Furthermore, the altered endothelial function induced by metabolic syndrome was associated with a significant downregulation in the expression of the activated endothelial nitric oxide synthase (eNOS) in arterial tissue (p<0.05; Figure 23) and with a significant decrease in the release of nitric oxide (NO) that were prevented by the treatments (Fig. 21 B).

[0372] Conclusions

[0373] The administration of Tea Complex Extract or HT-BPL1 separately shows effects on metabolic and cardiovascular health in mice with metabolic syndrome, reducing the adiposity, insulin resistance and hypertension. However, the co-administration of both ingredients shows additional benefits in HFHS fed mice, producing greater loss of weight and fat mass, a decrease in blood glucose and a decrease in LDL-cholesterol levels. These results are not observed when administering both ingredients separately. For this reason, the co-administration of both compounds at the same time could constitute an interesting alternative to the use of traditional drugs for the management of metabolic health, including metabolic and cardiovascular alterations, as well as metabolic syndrome.

[0374] Example 5. Modulation of the gut microbiota by CTE and BPL1® HT supplementation.

[0375] Material and methods - Analysis of gut microbiome composition

[0376] Fecal samples were collected at the end of the study. Microbial DNA was isolated from stool samples using the QIAsymphony PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany). The V3-V4 hypervariable region of the 16S rRNA gene was amplified from genomic DNA using the well-known in the art 341 F / 805R primer pair. The 16S rRNA libraries were quantified via fluorometry using the Quant-iT™ Picofreen™ dsDNA Assay Kit (Thermofisher, Waltham, MA, USA). Prior to sequencing on the MiSeq platform (Illumina, San Diego, CA, USA) with a paired-end read configuration of 300 cycles, the libraries were pooled. The size and quantity of the pooled libraries were evaluated using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and the Library Quantification Kit for Illumina (Kapa Biosciences, Oslo, Norway), respectively. The PhiX Control library (v3) (Illumina) was added to the amplicon library. Image analysis, base calling, and quality assessment of the data were conducted using the MiSeq instrument and its MiSeq Control Software (MCS v2.6.2.1). Forward and reverse reads were merged using the BBMerge tool from the BBMap V.39 software. To minimize bias during annotation, amplification primers were trimmed with Cutadapt v4.9.

[0377] A quality filtering step was applied to exclude low-quality sequences using the Reformat tool from BBMap V.39 software. Sequences shorter than 200 nucleotides were excluded from the analysis, and bases at the ends of sequences with a Phred score below Q20 were removed. Sequences with an average quality score below Q20 across their entire length were also discarded. The reads were processed using the DADA2 algorithm with the ‘denoise-single’ command [Callahan, B. et al., DADA2: High-resolution sample inference from Illumina amplicon data. Nature methods 2016, 13, (7), 581-3.]. Error rates were calculated from a subset of reads using the ‘learnErrors’ function, and sample inference was performed with the ‘dada’ function to generate Amplicon Sequence Variants (ASVs). Chimeric ASVs were removed using the ‘removeChimeraDenovo’ function. Taxonomic annotation of ASVs was carried out using the NCBI 16S rRNA database version 2024 and the blastn tool (version 2.2.29+) [Altschul, S. F.; et al., Basic local alignment search tool. Journal of molecular biology 1990, 215, (3), 403-10], ASVs with less than 97% identity were reclassified using the NBAYES algorithm [Bokulich, N. A.; et al., Optimizing taxonomic classification of marker-gene amplicon sequences with QI I ME 2's q2-feature-classifier plugin. Microbiome 2018, 6, (1), 90] within the QIIME2 platform v2024.5 [Bolyen, E.; Rideout, et al Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature biotechnology 2019, 37, (8), 852-857], The NBAYES classifier was trained on the V3-V4 regions of the 16S rRNA gene using the SILVA v.138 database [Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Replies, J.; Glockner, F. O., The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic acids research 2013, 41 , (Database issue), D590-6], ASVs classified within the bacterial kingdom and present at a relative frequency of at least 0.001 % in a minimum of 3 samples were retained for subsequent bioinformatics analysis.

[0378] For alpha diversity analysis, data were normalized using a rarefaction technique provided by the Phyloseq R package v1.46 [McMurdie, P. J.; Holmes, S., phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PloS one 2013, 8, (4), e61217.]. Metrics such as Shannon, Simpson, and Richness indexes were calculated using the vegan R package v2.6-6.1. An ANOVA followed by a t-test or Kruskal-Wallis test followed by the Wilcoxon test was performed to assess differences between groups for parametric or non-parametric data, respectively.

[0379] Differential abundance of taxa was analyzed using the DESeq2 R package v1.42.0 [Love, M. I.; Huber, W.; Anders, S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology 2014, 15, (12), 550.], with normalization based on the ‘Relative Log Expression’ method. Scaling factors were calculated using the ‘EstimateSizeFactors’ function, which applies the median ratio between taxa abundances and the geometric mean. The ‘PosCounts’ method was used to account for taxa with multiple zeros across most samples, a common occurrence in metagenomic data. Taxa were considered differentially abundant if they had a Benjamini-Hochberg (BH) adjusted p-value < 0.05 and were present in at least 50% of samples within one of the compared groups. The MaAslin2 R package v1.4 [Mallick, H.; et al., Multivariable association discovery in population-scale meta-omics studies. PLoS computational biology 2021, 17, (11), e1009442.] was utilized to explore the association between microbial abundances and clinical and experimental variables. A linear model test was applied to each variable, treating the variable as a fixed effect. Microbial taxa counts were normalized using the DESeq2 normalization method, followed by log transformation of the normalized counts. Only taxa present in more than 20% of the samples were included in the analysis.

[0380] Heatmaps were created to summarize bacteria taxa differential abundance and the association between taxa abundance and clinical and experimental variables using the ComplexHeatmap R package v2.11.1 [Gu, Z.; Eils, R.; Schlesner, M., Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 2016, 32, (18), 2847-9.].

[0381] Results

[0382] A total of 909 ASVs were annotated as bacteria and passed the prevalence filter. In total, 12% of these ASV were classified at genera level. To evaluate if CTE, BPL1® HT or a combination of both can modulate gut microbiota in a context of HFHS diet, bacterial a-diversity, differential abundance of bacteria and association between clinical / molecular and microbial features were assessed.

[0383] Regarding alpha diversity, three metrics were calculated. Mice in HFHS showed a decrease in bacterial richness compared to mice in Chow diet (Figure 24, p<0.05). Supplementation of CTE or BPL1® HT prevented richness reduction, and the combination of both ingredients not only prevented the loss of richness by HFHS, but also increased richness compared to mice in Chow diet. The same pattern was observed in Shannon (Figure 24) and Simpson (Figure 24) without reaching statistical significance. Mice supplemented with a blend of CTE + BPL1® HT showed a tendency of higher indexes of alpha diversity compared to mice in CTE or BPL1® HT alone (Figure 24).

[0384] Multivariate analysis using MaAsLin2 identified several genera with significantly correlation with clinical parameters in HFHS associated with metabolic syndrome (MetS) condition. Specifically, 10 bacterial genera (Lactococcus, Corynebacterium, Romboutsia, Faecalibaculum, Enterococcus, Staphylococcus, Bilophia, Streptococcus, Ligilactobacillus, Adlercreutzia), which abundance were increased by HFHS diet, showed a positive association with clinical parameters (Figure 25a, black boxes, column with an arrow). Conversely, the abundance of another 10 genera (Bifidobacterium, Negletibacter, Muricomes, Prevotellaceae UCG 001, Turicimonas, Parasutterella, Anaerotaenia, Muribaculum, Duncaniella, Turicibacter, Enterohabdus), which abundance were decreased by HFHS diet, was negatively correlated with these parameters (Figure 25b, black boxes, column with an arrow).

[0385] Supplementation with CTE, BPL1® HT or CTE + BPL1® HT modulated the abundance of 5 of these 20 genera (Figure 25a-b, taxa in black boxes with circles in the name).

[0386] Specifically, HFHS diet supplemented with CTE or CTE + BPL1® HT increased the abundance of Muricomes (Figure 26A), Prevotellaceae UCG 001 (Figure 26B) and Enterorhabdus (Figure 26C) compared to mice in HFHS alone, reaching the same values than those of mice fed with chow in the case of Muricomes (Figure 26A) and Enterorhabdus (Figure 26C).

[0387] For one side, Bifidobacterium, negatively associated with several clinical parameters, decreased with HFHS diet, while the ingredients increased it, showing the blend the highest increase (Figure 26D). On the other side, supplementation with TC or BPL1® HT reduced the abundance of Romboutsia (Figure 26E) and BPL1® HT and CTE + BPL1® HT reduced Adlercreutzia (Figure 26F), genera positively associated with MetS parameters and increased in HFHS diet.

[0388] Additionally, only the supplementation of CTE + BPL1® HT increased the abundance of Lachnospiraceae_N K4A136_group (Figure 27A) (associated negatively with brown adipose tissue) and Papilibacter (Figure 27B) (associated negatively with OGTT AUG).

[0389] At ASVs level, the same pattern was observed: compared to Chow mice, the HFHS diet increased the abundance of 14 ASVs positively associated with MetS parameters (Figure 28a, black box, column with an arrow) and reduced 18 ASVs negatively associated (Figure 28b, black box, column with an arrow). From negatively associated ASVs, GTE and GTE + BPL1® HT were able to restore the abundance comparable to Chow mice of ASV126 Enterorhabdus (that could be annotated up to genera level) (Figure 29A) and only GTE + BPL1® HT restored ASV78 Oscillospiraceae (that could be annotated up to family level) (Figure 29B). GTE and GTE + BPL1® HT prevented the severe decrease of ASV16 Bifidobacterium pseudoIongum (Figure 29C), similar trend as in genera. Moreover, the ASVs positively associated with MetS, only the blend completely prevented the increase in ASV83 Flintibacter butyricus (Figure 29D) and GTE prevented the increase in ASV95 Romboutsia timonensis caused by HFHS diet (Figure 29E).

[0390] The presents experiments included in Example 5 were performed to investigate whether supplementation with the ingredients exerts any effects on modulating the gut microbiome.

[0391] Our results show that both GTE and BPL1® HT, alone and in combination, prevented the reduction of bacterial richness induced by HFHS diet. However, only mice supplemented with the blend reached levels of bacterial richness higher than mice fed with chow diet, highlighting the synergist effect of GTE and BPL1® HT. The lack of differences between the blend and the individual ingredients could be explained by the tendency of both GTE and BPL1® HT to increase bacterial richness compared to chow. High-calorie diets can cause gut dysbiosis, leading to reduced microbial diversity, greater energy extraction, impaired gut barrier function, and chronic low- grade inflammation. This triggers metabolic endotoxemia, oxidative stress, and abnormal regulation of genes controlling lipid and glucose metabolism as well as inflammatory pathways, all of which contribute significantly to metabolic disorders. In fact, a study in a large human population found that higher alpha diversity was associated with lower insulin resistance and lower prevalence of type 2 diabetes [ Chen, Z.; et al., Association of Insulin Resistance and Type 2 Diabetes With Gut Microbial Diversity: A Microbiome-Wide Analysis From Population Studies. JAMA network open 2021, 4, (7), e2118811.].

[0392] Association and differential abundance analysis revealed that CTE, BPL1® HT and the combination of both were able to modulate the abundance of bacterial biomarkers associated with clinical parameters of MetS. In this regard, the blend showed a tendency to increase the abundance of beneficial bacteria such as Bifidobacterium compared to CTE or BPL1® HT alone. The synergist effect of CTE and BPL1® HT was clearer at ASV level were mice supplemented with the blend had significantly lower abundance of Flintibacter butyricus, higher abundance of ASV78 Oscillospiraceae and a tendency of higher Bifidobacterium pseudoIongum compared to ingredients alone. Bifidobacterium are commensal bacteria used as probiotics, with positive reports on improving metabolic diseases [ Yu, Y.; et al., Complete-genome sequence and in vitro probiotic characteristics analysis of Bifidobacterium pseudoIongum YY- 26. Journal of applied microbiology 2022, 133, (4), 2599-2617], Prebiotics such as mannan-oligosaccharides and compounds such as glycerol monolaurate have been reported to improve obesity and metabolic disorders which were associated with the increased abundance of B. pseudoIongum [ Wang, H.; et al Mannan-oligosaccharide modulates the obesity and gut microbiota in high-fat diet-fed mice. Food & function 2018, 9, (7), 3916-3929.., Zhao, M.; et al, Modulation of the Gut Microbiota during High-Dose Glycerol Monolaurate-Mediated Amelioration of Obesity in Mice Fed a High-Fat Diet. mBio 2020, 11 , (2).]. The blend modulate others Bifidobacterium species besides Bifidobacterium animalis sub. lactis, the strain from BPL1® HT derived from. Regarding F. butyricus, a bacterium that in our study correlated positively with several MetS clinical parameters, only the blend completely prevented the increase of this bacteria produced by HFHS. This is in line with other study where it was observed that F. butyricus increase during lard- and bile acid-fed mice [Zhao, M.; et al. 2020],

[0393] The clearer tendency of synergies between CTE and BPL1® HT was observed on the alpha diversity analysis, which is done at ASV level, independent of the level of taxonomic annotation. Only 12% of the identified ASV could be annotated to genera level with this fact possibly limiting the biomarker analysis (clinical associations and differential abundance).

[0394] Thus, this study demonstrates that CTE, BPL1® HT and the combination of both modulates gut microbiota in the context of HFHS highlighting the capacity of the blend to increase bacterial richness and beneficial bacteria like Bifidobacterium species, while preventing the increase of potential opportunistic bacteria like F. butyricus.

Claims

CLAIMS1. Composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145 and an extract obtained from a blend of green tea and black tea leaves.

2. Composition according to claim 1 , wherein the Bifidobacterium animalis subsp. lactis strain is non-viable.

3. Composition according to claim 2, wherein the non-viable Bifidobacterium animalis subsp. lactis strain is heat-treated.

4. Composition according to any one of claims 1 to 3, wherein the extract is standardized to comprise a concentration of total flavan-3-ols of, at least, 10% by weight relative to the extract on a dry basis, and a concentration of total methylxanthines of, at least, 5% by weight relative to the extract on a dry basis, preferably wherein the extract is further standardized to comprise a concentration of total polyphenols of, at least, 30% by weight relative to the extract on a dry basis.

5. Composition according to any one of claims 1 to 4, wherein the composition is formulated in liquid form or in solid form.

6. Composition according to claim 5, wherein the solid formulation is selected from the group consisting of tablets, lozenges, sweets, gummies, candies, lollipops, chewable tablets, chewing gum, soft chews, capsules, sachets, powders, suppositories, gels, softgels, microspheres, granules, coated particles, coated tablets, tablets, g astro- resista nt tablets, gastro-resistant capsules, dispersible strips, films, dry kibble, baked treats, soft chews, and wet canned food.

7. Composition according to claim 5, wherein the liquid formulation is selected from the group consisting of oral solutions, suspensions, droplets, emulsions and syrups.

8. Composition according to any one of claims 1 to 7, wherein the composition is a pharmaceutical composition or a nutritional composition.

9. Composition according to claim 8, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or an excipient.

10. Composition according to claim 8, wherein the nutritional composition is a food, a feed or a nutritional supplement.

11. Composition according to anyone of claims 1 to 10, for use as a medicament.

12. Composition according to any one of claims 1 to 10, for use in improving metabolic health in a subject, preferably wherein the improvement of metabolic health is measured by one or more of the following indicators: i) improved blood lipid profile, ii) lowering of plasma LDL cholesterol, iii) improved glycemic response, iv) visceral and / or subcutaneous adiposity reduction, v) leptin levels reduction, vi) insulin levels reduction, vii) blood glucose levels reduction, viii) insulin resistance reduction, as measured by HOMA-IR index, or ix) any combination of the above.

13. Composition according to any one of claims 1 to 10 for use in the treatment, prevention and / or improvement of metabolic syndrome, and / or related conditions, disorders or symptoms, in a subject, preferably wherein the metabolic syndrome related condition, disorder or symptom is at least one selected from the list consisting of obesity, overweight, insulin resistance, glucose intolerance, hyperglycemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension, a chronic prothrombotic state, a chronic proinflammatory state, and any combination thereof.

14. Composition according to any one of claims 1 to 10 for use in the treatment, prevention and / or improvement of type-2 diabetes and / or a cardio-vascular disease or disorder, in a subject.

15. Non-therapeutic use of the composition according to any one of claims 1 to 10 for body fat reduction, and / or maintenance of body weight, in a subject.

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