Prebiotic composition

Abstract

To provide a composition for use as a prebiotic, the composition comprising black leaf tea particles having a particle size of less than 250 μm, in order to utilize black leaf tea particles for delivering health benefits associated with consumption of prebiotics, such as treatment or prevention of a gastrointestinal condition, and / or treatment or prevention of a condition associated with sub-optimal immunity, based on the identification that black leaf tea divided into very fine particles has improved prebiotic effects compared with black tea infusion and that, even where the infusion comprise at least the same amount of black tea flavonoids, the particles still exhibit improved prebiotic effects.SOLUTION: The present invention relates a composition for use as a prebiotic, the composition comprising black leaf tea particles having a particle size of less than 250 μm.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for use as a prebiotic, in particular a composition for use as a prebiotic comprising black leaf tea particles. [Background technology]

[0002] Gastrointestinal health is a major concern of consumers in both developed and developing countries, both in terms of daily gastrointestinal comfort and also in terms of resistance to or prevention of acute and chronic diseases, such as infectious diarrhea and inflammatory bowel disease. There is a demand for products that can be consumed daily, which are able to provide such benefits.

[0003] Currently available strategies to address these concerns include probiotic products, in which a liquid (typically an everyday beverage) containing one or more types of beneficial bacteria is consumed to elicit benefits, such as improved gut health and / or improved natural immunity. This approach is limited because it requires the product to be refrigerated (to maintain the viability of the bacteria that provide the benefit), and efficacy remains a challenge because the bacteria must pass the harsh acidic environment of the stomach to reach the colon in sufficient numbers to provide the intended effect. Moreover, such considerations mean that probiotic products are typically expensive for consumers and require complex supply chains to deliver them effectively.

[0004] An alternative strategy is the use of prebiotics, which are dietary components that have beneficial health effects through selective metabolism of the dietary components in the intestinal tract. Typical targets for such an approach are bifidobacteria and lactobacilli (i.e. common probiotic species), because they are generally recognized as safe, are commonly found in large numbers in breast-fed infants, modify the intestinal environment through the production of short-chain fatty acids, meaning that pathogenic bacteria cannot colonize, produce antibacterial components that target pathogenic bacteria, improve the immune status of the host, and reduce colonic inflammation associated with unhealthy modern diets.

[0005] Currently available prebiotics include fructans, such as inulin and fructooligosaccharides, which have been shown to significantly increase bifidobacteria in humans.

[0006] Prebiotics do not suffer from the same problem of their viability as probiotics, and they can be made into many product formats without losing viability.However, in some aspects, current prebiotics are difficult to make into common food products due to their impact on the flavor (fructans can be slightly sweet) and structure (they can change the mouthfeel) of the product.In addition, products often need to be made with more than the effective amount of prebiotics due to the fact that fructans can suffer from degradation during pasteurization, baking, sterilization and similar processes.These considerations mean that processed foods containing prebiotics are still expensive, and therefore limited to only a few customers due to price.

[0007] Thus, there is a demand from customers for affordable solutions that can provide benefits in the areas of gut health, resistance to infectious diseases and / or prevention of chronic gastrointestinal disorders.

[0008] Tea has been consumed in some form for over 4000 years and is popular in both developed and developing countries. Tea is popular for many reasons: it is generally perceived to be healthy, it can be a safe alternative to untreated water, it is associated with benefits such as relaxation, mental alertness, and has a widely accepted flavor. The low cost of tea also means that it can be consumed by consumers in all socio-economic groups.

[0009] Some literature suggests that consumption of black tea and / or its components can have a positive effect on the health of consumers. For example, US 2008 / 119545 and US 2008 / 075795 (both in the name of C. HENSLEY & S. PYO) disclose methods and compositions for preventing and treating avian influenza utilizing an effective amount of ingredients having a composition selected from the group consisting of theaflavin, theaflavin-3,3'-digallate, theaflavin-3-monogallate, theaflavin-3 gallate, theaflavin-3'-gallate, thearubigin, gallic acid, tannic acid, (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (+)-epicatechin (EC), (-)-gallocatechin gallate (GCG), and catechin.

[0010] It has also been proposed that black tea and / or its components can have a positive effect on intestinal function and / or health. For example, K. Jafari et al. (Medical Hypotheses, 2006, 67(2), p.419) disclose that black tea extract and its main polyphenol pigments can reverse gastrointestinal damage in irritable bowel syndrome; L. Chaudhuri et al. (Life Sciences, 2000, 66(9), pp. 847-854) disclose that black tea hot water extract significantly accelerates gastrointestinal transit (GIT) in vivo in mice; and SE Besra et al. (Phytotherapy Research, 2003, 17, pp. 380-384) disclose the antidiarrheal activity of black tea hot water extract.

[0011] Compositions containing black tea ingredients in combination with conventional prebiotics are also known. For example, WO 2007 / 056432 (PERQUE INC) discloses a composition containing one or more prebiotics (e.g., one or more dietary fibers) in combination with selenium compounds, flavonoids and / or flavonols, and phosphatides; and US 2008 / 085349 (ZY CHEN) discloses a beverage containing tea ingredients and a non-digestible health sweetener (VitaSugar® IMO).

[0012] It is also recognised that black tea and / or its components can have a positive effect on the intestinal microflora.

[0013] US Patent No. 5,071,653 (ITOEN LTD) discloses a substantially flavorless extract from the leaves of C. sinensis that promotes the growth of bifidobacteria. The composition is provided by extracting water- or ethanol-soluble solids from the leaves of C. sinensis with a polar organic solvent that is not miscible with water. There is no evidence in US Patent No. 5,071,653 that the extract disclosed therein has a true prebiotic effect (i.e., the promotion of the growth of bifidobacteria takes precedence over the promotion of the growth of pathogenic bacteria, such as Clostridium). Furthermore, the extract disclosed therein is substantially flavorless and therefore likely lacks the flavonoids (e.g. thearubigins) that give black tea its unique flavor.

[0014] WO 2004 / 056205 (UNILEVER) discloses the use of a prepared food product comprising black tea leaves, black tea extracts or mixtures thereof, wherein the black tea leaves or black tea extracts are present in an unbound state, for maintaining or improving the balance of the microflora and / or for treating or preventing diarrhea in a subject consuming the composition.

[0015] More recently, the prebiotic effects of the flavonoid components of black tea (and in particular thearubigins) have been reported.

[0016] WO 2010 / 133404 (UNILEVER) discloses the use of compositions comprising black tea flavonoids as prebiotics and / or for the treatment or prevention of conditions associated with poor gut health or low immunity. The flavonoids include thearubigins in an amount of at least 82% by weight of the tea flavonoids. Summary of the Invention [Problem to be solved by the invention]

[0017] We have now identified that black leaf tea divided into very fine particles has an improved prebiotic effect compared to black tea infusions, even when the infusions contain at least equivalent amounts of black tea flavonoids. Thus, black tea particles can be expected to be particularly effective in providing health benefits associated with prebiotic consumption, such as the treatment or prevention of gastrointestinal conditions and / or conditions associated with suboptimal immunity. [Means for solving the problem]

[0018] Tests and Definitions PREBIOTIC As used herein, the term "prebiotic" refers to a substance that is orally consumed by an individual to beneficially affect the individual by selectively stimulating the growth and / or activity of one or more of a limited number of bacteria in the individual's colon. Preferred prebiotics are those that selectively stimulate the growth and / or activity of Bifidobacteria and / or Lactobacillus. Even more preferred are those that stimulate the growth and / or activity of Bifidobacteria and / or Lactobacillus in preference to pathogenic bacteria, such as Clostridium.

[0019] The prebiotic effect in the gut can be assessed based on the growth of health-promoting bacteria, such as Lactobacillus and Bifidobacteria, the reduction of gut pathogens and / or the increase or decrease in the production of health-related bacterial metabolites, the latter including, for example, short-chain fatty acids (acetate, propionate and butyrate), which are generally considered positive for colon health, whereas ammonia and branched short-chain fatty acids are recognized as risk factors for colon carcinogenesis.

[0020] Tea As used herein, the term "tea" refers to material derived from the leaves and / or stems of Camellia sinensis var. sinensis and / or Camellia sinensis var. assamica. "Black tea" refers to tea in which the leaves and / or stems have undergone a so-called "fermentation" process in which they are oxidized by certain endogenous enzymes. This oxidation can be further supplemented by the activity of exogenous enzymes, such as oxidases, laccases and peroxidases.

[0021] "Leaf tea" for the purposes of this invention means tea leaves and / or stems in unsteeped form and dried to a moisture content of less than 30% by weight, typically having a moisture content in the range of 1 to 10% by weight.

[0022] PARTICLE SIZE When referring to leaf tea particle size, this refers to the size of the tea particles that can pass (in terms of the largest particle size) or be retained (in terms of the smallest particle size) through a sieve with an opening of a reference opening size. For example, tea leaf particles with a particle size of less than 250 μm can pass through a sieve having a Tyler mesh size of 60 mesh; and tea leaf particles having a particle size of at least 90 μm are retained by a sieve having a Tyler mesh size of 170 mesh.

[0023] FLAVONOIDS, THEARUBIGINS AND CATECHINS Definitions and analytical methods for determining flavonoids and thearubigins can be found in Christiane Lakenbrink, Svenja Lapczynski, Beate Maiwald and Ulrich H. Engelhardt. J. Agric. Food Chem., 2000, 48 (7), 2848-2852. Briefly, the total flavonoids and thearubigins in a composition can be calculated according to the following relationships (1) and (2): Total flavonoids (wt%) = TP - (GA + TG + CQA) (1) Thearubigins (wt%) = Total flavonoids - (C + TF + FOG + FAG) (2) (TP is the weight percent of total phenolics in the composition; GA is the weight percent of gallic acid in the composition; TG is the weight percent of theogallin in the composition; CQA is the weight percent of chlorogenic acid in the composition; C is the weight percent of catechin in the composition; TF is the weight percent of theaflavin in the composition; FOG is the weight percent of flavonol glycosides (expressed as aglycones) in the composition; and FAG is the weight percent of flavone glycosides (expressed as aglycones) in the composition). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Summary of the Invention In a first aspect, the present invention provides the use of a composition comprising black tea leaf particles as a prebiotic, wherein the black tea leaf particles have a particle size of less than 250 μm.

[0025] Surprisingly, the inventors have discovered that black tea leaf particles having a particle size of less than 250 μm have an improved prebiotic effect compared to black tea infusion solids. - a composition for use as a prebiotic comprising black tea leaf particles, the black tea leaf particles having a particle size of less than 250 μm; - a method of producing a prebiotic effect in an individual, the method comprising administering to the individual an effective amount of a composition comprising black tea leaf particles, wherein the black tea leaf particles have a particle size of less than 250 μm; - a composition for use as a medicine comprising black tea leaf particles, the black tea leaf particles having a particle size of less than 250 μm; - a composition for use as a medicament in the treatment or prevention of a gastrointestinal condition comprising black tea leaf particles, wherein the black tea leaf particles have a particle size of less than 250 μm; - a composition for use as a medicament in the treatment or prevention of a suboptimal immune related condition comprising black tea leaf particles, wherein the black tea leaf particles have a particle size of less than 250 μm; - a composition for use as a medicament for the removal or reduction of visceral pain comprising black tea leaf particles, the black tea leaf particles having a particle size of less than 250 μm; - use of a composition comprising black tea leaf particles having a particle size of less than 250 μm in the manufacture of a medicament for the treatment or prevention of a gastrointestinal condition; - use of a composition comprising black tea leaf particles having a particle size of less than 250 μm in the manufacture of a medicament for the treatment or prevention of a suboptimal immune related condition; - Use of a composition comprising black tea leaf particles having a particle size of less than 250 μm in the manufacture of a medicament for the relief or alleviation of visceral pain. It can also be said to be relating to.

[0026] In a further aspect, the present invention provides a beverage comprising black tea leaf particles dispersed in an aqueous liquid, wherein the black tea leaf particles have a particle size of less than 250 μm.

[0027] The beverage is suitable for application in the uses listed above. Furthermore, the very fine particle size (i.e. particle size less than 250 μm) means that the tea leaf particles can remain suspended in the aqueous liquid for extended periods of time and / or do not result in an unpleasant non-smooth texture when the beverage is consumed.

[0028] The present invention relates to a method for producing a beverage comprising the steps of: (a) providing an aqueous liquid; and (b) dispersing black tea leaf particles in an aqueous liquid; Also provided is a method comprising:

[0029] In a further aspect, the present invention provides a packaged beverage ingredient suitable for use in a method, said ingredient comprising a composition comprising black tea leaf particles packaged in a container, wherein the amount of the composition packaged in the container is from 0.5 to 500 g, and the black tea leaf particles have a particle size of less than 250 μm.

[0030] Except in the examples, all numbers in this description expressing amounts of materials, periods of time, length scales, reaction conditions, physical properties and / or uses of materials can be optionally understood to be modified by the term "about."

[0031] It should be noted that when specifying any range of values, any particular upper limit value may be associated with any particular lower limit value.

[0032] For the avoidance of doubt, the term "comprising" is intended to mean "including" and not necessarily "consisting of" or "composed of." In other words, the listed steps or options are not necessarily all-inclusive.

[0033] The disclosure of the invention as understood herein should be considered to encompass all embodiments in which the claims are understood to be multiple dependent on one another, regardless of whether the claims have multiple dependencies or overlaps.

[0034] Where features are disclosed with respect to a particular embodiment of the invention (e.g., method of use), such disclosure is also construed to apply mutatis mutandis to any other embodiment of the invention (e.g., composition).

[0035] Detailed Description Prebiotic and immunomodulatory uses and methods In one embodiment of the invention, a composition comprising black tea leaf particles is administered to an individual with the aim of producing a prebiotic effect in the individual. The resulting prebiotic effect, i.e. the selective stimulation of the growth and / or activity of one or more of a limited number of bacteria, can beneficially affect the health of the individual in one or more ways.

[0036] For example, administration of the composition can be used to treat or prevent conditions associated with suboptimal immunity, such as viral infections, chronic inflammation and / or allergies. The conditions associated with suboptimal immunity are preferably viral infections selected from influenza, colds and / or coronaviruses.

[0037] Additionally or alternatively, administration of the composition may treat or prevent a gastrointestinal condition, preferably selected from inflammatory bowel disease, irritable bowel syndrome, infectious diarrhea, environmental bowel disease, or a combination thereof.

[0038] The compositions may additionally or alternatively be administered to eliminate or alleviate visceral pain.

[0039] The composition is administered orally.

[0040] The composition comprises an effective amount of black tea leaf particles. In particular, the amount of black tea leaf particles administered to an individual daily is sufficient to produce a notable prebiotic effect in the intestinal microflora of the individual. Thus, the composition is preferably administered to an individual in an amount that provides at least 0.5g of black tea leaf particles per day, more preferably at least 1.0g of black tea leaf particles per day, and most preferably at least 1.5g of black tea leaf particles per day. Large amounts of black tea leaf particles do not need to be administered to achieve a prebiotic effect. Thus, the composition is preferably administered to an individual in an amount that provides at most 20g of black tea leaf particles per day, more preferably at most 15g of black tea leaf particles per day, even more preferably at most 10g of black tea leaf particles per day, and most preferably at most 6g of black tea leaf particles per day.

[0041] Black tea leaf particles (BLACK LEAF TEA PARTICLES) Black tea leaf particles for use in the compositions, uses, beverages, beverage ingredients and methods of the present invention have a particle size of less than 250 μm, preferably less than 200 μm, more preferably less than 150 μm and most preferably less than 100 μm. Although there is no particular limitation on the lower end of the particle size range, black tea leaf particles preferably have a particle size of greater than 5 μm, more preferably greater than 10 μm, even more preferably greater than 20 μm.

[0042] If tea particles are present in the composition but are outside a particular particle size, it is preferred that at least 85% by weight of the black tea leaf particles have this particle size, more preferably at least 90%, even more preferably at least 95%, and most preferably 99% to 100% by weight have this particle size. The weight percentage of particles having a particular particle size can be determined by separating into fractions according to particle size (e.g. by sieving) and subsequently weighing the fractions. When determining particle size, the tea leaf particles are in dry form and have a moisture content of less than 30% by weight (typically 1 to 10% by weight).

[0043] Tea leaves contain material with a range of particle sizes as a result of conventional manufacturing methods. In practice, tea leaves are typically classified into various grades (e.g., whole leaf, broken, fannings, and dust) before being sold at auction. One way to classify tea is according to particle size. For example, tea leaves can be passed through a series of vibrating sieves, and the various grades are retained and collected. Tea leaf particles with a particle size of less than 250 μm can be separated in this way. For example, leaf tea can be sieved through a sieve with a Tyler mesh size of 60 mesh.

[0044] Although it is possible to obtain black tea leaf particles from commercially available black tea leaves by sieving (described above), this is not very efficient as only a small proportion of the tea leaves are of the appropriate size. Therefore, particles are preferably provided by a process of grinding the black tea leaves to obtain black tea leaf particles, with the aim of increasing the proportion of tea leaves having the appropriate particle size. For example, the tea leaves can be ground using an impact mill, hammer mill, ball mill, jet mill, cone mill, roll mill, stone mill, centrifugal mill, etc. The ground tea leaves can optionally be passed through one or a series of sieves described above, with the aim of selecting a fraction of black tea leaf particles having a particular particle size.

[0045] When the tea particles of the present invention are in uninfused form they will contain significant amounts of flavonoids. Preferably the black tea leaf particles contain at least 2% flavonoids by weight of the black tea leaf particle, more preferably at least 4%, and most preferably from 6 to 20% flavonoids by weight of the black tea leaf particle.

[0046] The black tea flavonoids for use in the compositions, uses, beverages, beverage ingredients and methods of the present invention are preferably rich in thearubigins.For example, the black tea flavonoids may contain thearubigins in an amount of at least 70% by weight of the flavonoids.More preferably, the black tea flavonoids contain thearubigins in an amount of at least 82% by weight of the tea flavonoids, and most preferably, at least 84% by weight of the tea flavonoids.

[0047] Typically, black tea flavonoids include non-thearubigin components, such as theaflavins and / or catechins. Thus, the amount of thearubigins in the black tea flavonoids may be less than 99.99% by weight of the black tea flavonoids, or even less than 98% by weight of the black tea flavonoids. It is particularly preferred that the amount of catechins in the black tea flavonoids is limited, since catechins are believed to reduce the prebiotic effect of thearubigins. Thus, it is preferred that the flavonoids contain catechins in an amount less than 12% by weight of the flavonoids, more preferably less than 10% by weight of the flavonoids, even more preferably less than 8% by weight, and most preferably 0.01 to 6% by weight. Additionally or alternatively, black tea flavonoids may contain less than 3% theaflavins by weight of the tea flavonoids, since theaflavins have little or no prebiotic effect. More preferably, the flavonoids of the black tea contain less than 1.5% theaflavins by weight of the flavonoids of the black tea, even more preferably less than 0.5% and most preferably from 0.0001 to 0.1% theaflavins by weight of the black tea flavonoids.

[0048] BEVERAGE AND BEVERAGE PRECURSOR In a preferred embodiment of the present invention, the composition is administered to an individual in the form of an edible product. The edible product can be a food product. For example, the product can be margarine, low-fat spread, confectionery product (e.g. chocolate), ice cream, yogurt, dressing, mayonnaise, sauce, bakery product, shortening, soup or cheese. However, the very small particle size of black tea leaf particles can make them particularly suitable for suspension in an aqueous medium to form a beverage. It is therefore particularly preferred that the edible product is a beverage, most preferably a tea-based beverage.

[0049] The beverage containing black tea leaf particles is dispersed in an aqueous liquid in sufficient quantity to provide that the prebiotic effective amount of particles can be achieved without having to consume a large amount of beverage in a day.Thus, the beverage preferably contains black tea leaf particles in a mass of at least 0.5g, more preferably more than 1.0g, and most preferably at least 1.5g.However, the product does not need to contain a large amount of black tea leaf particles.In particular, it is preferred that the mass of black tea leaf particles in the product is at most 20g, more preferably at most 15g, even more preferably at most 10g, and most preferably at most 5g.

[0050] The mass of the beverage is not so large as to require excessive amounts of the beverage to be consumed daily, therefore the beverage has a mass of less than 500g, preferably less than 450g, more preferably between 50 and 350g.

[0051] Because black tea leaf particles can affect the colour and / or texture of the beverage it is preferred to use the particles in a limited concentration range, in particular the concentration of black tea leaf particles is preferably from 0.15 to 10% by weight of the beverage, more preferably from 0.2 to 4% and most preferably from 0.5 to 3%.

[0052] The beverage may be produced in any suitable manner, however, in a preferred embodiment, the beverage is produced by: (a) providing an aqueous liquid; and (b) dispersing the black tea leaf particles in an aqueous liquid. The composition is produced using a method comprising:

[0053] Preferably, the aqueous liquid and tea particles are mixed in a weight ratio of at least 5:1 aqueous liquid to black tea leaf particles, more preferably at least 10:1, even more preferably at least 50:1, and most preferably at least 100:1. The weight ratio is preferably not too high otherwise an excess amount of liquid would need to be consumed to deliver an effective amount of tea particles. Preferably, the aqueous liquid and tea particles are mixed in a weight ratio of less than 1000:1 aqueous liquid to black tea leaf particles, more preferably at most 900:1, even more preferably at most 500:1, and most preferably at most 300:1.

[0054] This method allows the aqueous liquid and black tea leaf particles to be manipulated separately prior to combination. Additionally, a higher proportion of aqueous liquid than black tea leaf particles can minimize the effect of the particles on the texture and / or color of the liquid.

[0055] The liquid is an aqueous liquid, i.e., contains more than 50% water by weight of the liquid, hi some embodiments, the aqueous liquid can be in the form of an oil-in-water or water-in-oil emulsion, preferably an oil-in-water emulsion.

[0056] Preferred aqueous liquids are water, milk and vegetable milk analogs (e.g., almond milk, soy milk, oat milk, etc.) Most preferably, the aqueous liquid is water.

[0057] Prior to being dispersed in the aqueous liquid, the black tea leaf particles are preferably packaged. Thus, the beverage ingredient of the present invention preferably comprises a composition comprising black tea leaf particles packaged in a container.

[0058] The mass of the raw material composition packaged in the container is preferably sufficient to prepare at least one serving of beverage. For example, the amount can be at least 0.5g, more preferably at least 1g, and most preferably at least 1.5g. In some embodiments, the package can contain multiple servings, and the amount of the composition in the container can be, for example, up to 500g, more preferably up to 250g. However, in a particularly preferred embodiment, the packaged amount is the amount required for one serving. Thus, it is preferred that the amount is at most 5g, more preferably at most 4g, and most preferably at least 3.5g.

[0059] Where the prebiotic effect is produced by whole leaf tea particles, it is preferred that the container in which the raw compositions are packaged is suitable for introducing the compositions into aqueous liquids (i.e. as in a tea bag) rather than containing them during infusion. It is therefore particularly preferred that the container is a water resistant (as opposed to water permeable) container such as a sachet, bottle, tub, carton or pouch.

[0060] The invention will now be described, by way of example only, with reference to the following examples. EXAMPLES

[0061] Tea material Powdered soluble black tea solids (hereafter referred to as "Black Infusion Powder") were prepared as follows: Kenyan BOP (Broken Orange Pekoe) grade black tea was added to water (tea leaves to water ratio of 1:10 by weight) at 90°C and infused for 20 minutes. The tea leaves were then removed and the resulting infusion was freeze-dried.

[0062] Micronized black leaf tea (hereafter referred to as "Black Micronized Leaf Powder") was prepared as follows: Kenyan BOP (Broken Orange Pekoe) grade black tea was powdered using an ultra-centrifugal mill (Model ZM100 from Retsch GmbH & Co. KG). The mill was operated with a load control of up to 10, a speed of 14000 rpm and an internal filter / sieve size of 80 μm (ring filter). This starting BOP material had a particle size of about 1.4 mm, while the micronized black leaf powder had a particle size of less than 80 μm.

[0063] Measuring prebiotic effects Overall microbial fermentation (pH and gas production) and microbial metabolic activity with respect to the production of glycolytic end products (short-chain fatty acids and lactate) and proteolytic end products (ammonium) were studied in vitro in three healthy adult donors. The effect of tea materials on the microbial community structure was assessed through 16S targeted Illumina sequencing.

[0064] Short-term colonic incubation The short-term screening assay consisted of colonic incubation of a single dose of tea material under conditions representative of the proximal colonic region of an adult human, using a representative bacterial inoculum.

[0065] At the start of the short-term colonic incubation, black tea infusion powder was added at a concentration of 1% (w / v) and micronized black tea leaf powder at a concentration of 1.5% (w / v) to a sugar-depleted nutrient medium containing basic nutrients present in the colon (e.g., host-derived glycans, e.g., mucins). A blank containing only the sugar-depleted nutrient background medium was also included to assess the background activity of the microbial community. The reason for using less black tea infusion powder is that the material consists of a relatively higher content of polyphenols (including thearubigins) compared to the micronized black tea leaf powder.

[0066] As a source of colonic microbiota, fecal inocula of three donors were added (healthy adults who do not drink tea). Incubations were performed at 37 °C for 48 h under shaking (90 rpm) and anaerobic conditions. The incubations were performed in reactors of sufficiently high capacity and completely independent volume to allow not only robust microbial fermentation but also the collection of a large number of samples over time. The sample collection allows the evaluation of the production of metabolites and allows understanding of the complex microbial interactions that occur. Each condition was performed in triplicate to account for biological variations, resulting in 27 independent incubations (two treatments and one blank, tested in triplicate in three donors).

[0067] End point of short-term incubation -The entire microbial fermentation pH: The degree of acidification during the experiment is one of the measures that represents the intensity of the microbial metabolism of the test product. The pH of the incubation was determined at 0, 6, 24 and 48 h after the start of the incubation and thus gives a rough indication of the speed of fermentation of the different tea materials.

[0068] Gas production: Colonic incubations were carried out in a closed incubation system. This allows to evaluate the accumulation of gas in the headspace, which can be measured using a manometer. Gas production is one of the measures of microbial activity and therefore of the fermentation speed of the tea material. Gas production during incubation was determined at 0, 6, 24 and 48 h after the start of the incubation.

[0069] -Changes in microbial activity The following analyses were used to compare the different tea materials:

[0070] Short Chain Fatty Acids (SCFA): This is an assessment of the microbial carbohydrate (acetate, propionate and butyrate) or protein metabolism (branched SCFAs) and can be compared with the typical fermentation pattern of normal GI microbiota. Samples for SCFA analysis were analyzed after 0, 6, 24 and 48 h of incubation.

[0071] Lactate: The human intestine harbors both lactate-producing and lactate-utilizing bacteria. Lactate is produced by lactic acid bacteria and reduces the pH of the environment, thereby also acting as an antibacterial agent. After penetrating the microbial cell, protonated lactic acid can dissociate and release intracellular protons, resulting in acidification and microbial cell death. It can also be rapidly converted by other microorganisms, particularly to butyrate. Samples for lactate analysis are analyzed after 0, 6, 24 and 48 h of incubation.

[0072] Ammonium: It is a product of protein degradation, which leads to the production of potentially toxic or carcinogenic compounds, such as p-cresol and p-phenol. It can be used as an indirect marker for the effectiveness of low substrates. Since it is only produced towards the end of incubation, it was measured after 24 and 48 h of incubation, but not after 6 h.

[0073] -Changes in microbial community composition (Illumina) Two techniques were combined by Vandeputte et al. [(2017) “Quantitative microbiome profiling links gut community variation to microbial load”. Nature: 551, 507-511] to roughly map the changes in communities induced by different tea materials: 16S targeted Illumina sequencing and flow cytometry.

[0074] 16S targeted Illumina sequencing: This is a PCR-based method in which microbial sequences are amplified to saturation, thus providing the relative abundance of different taxa at different phylogenetic levels (microbial phylum, family and OTU levels). Illumina was performed at the beginning of incubation and after 24h and 48h. The methodology required primers spanning two hypervariable regions (V3-V4) of the 16S rRNA gene. Using a paired-end sequencing approach, sequencing of 2x250bp results in an amplification product of 424bp. Such fragments are more taxonomically informative than smaller fragments.

[0075] Quantification of total bacterial cells by flow cytometry: Samples analyzed by Illumina sequencing were also analyzed by flow cytometry to determine the number of total bacterial cells, so that the proportional values ​​obtained by Illumina can be converted to absolute quantities. Samples were analyzed on a BD Facsverse. Samples were analyzed on a BD Facsverse. Samples were run using a high flow rate. Bacterial cells were differentiated from media debris and signal noise by applying a threshold of 200 in the SYTO channel. Appropriate parent and daughter gates were set to determine all populations.

[0076] result Microbial metabolic activity pH: In addition to the decrease in pH typical of the product addition due to the slightly acidic nature of the tea material, both materials produced a significantly stronger decrease in pH than the blank during the first 6 h of incubation in each donor (p<0.05). This indicates that fermentation of the product began early in the incubation. The strongest initial pH decrease was obtained with the black tea infusion powder in Donor A; both materials produced similar pH decreases in Donors B and C (i.e., not significantly different from each other). Fermentation of the tea material consistently produced a further decrease in pH during the 6-24 h time frame in each of the three donors (significantly stronger than the blank (p<0.05)), indicating that the tea material had not yet deteriorated after 6 h. Whereas the pH remained relatively stable in the blank incubations during the final 24 h of incubation, a further significant decrease in pH was observed in both tea materials in Donors A and B, as well as the micronized black tea leaf powder in Donor C (p<0.05). Overall, the strongest pH reduction was consistently observed in the micronized black leaf powder.

[0077] Gas production: Both materials stimulated gas production during the first 6 h in all donors as observed by gas pressures significantly higher than the blank in the treatment incubations (p<0.05). These observations confirm that fermentation of the product began at an early stage of incubation. Fermentation of the tea material consistently stimulated gas production during both the 6-24 h and 24-48 h time frames in each of the three donors (gas pressures significantly higher than the blank in the tea incubations (p<0.05)), indicating that the product had not yet deteriorated after 6 h. Overall, the highest gas pressures after 48 h were consistently obtained with the micronized black tea leaf particles.

[0078] Total SCFA: Both tea materials yielded significantly higher total SCFA levels than the blank in each donor (p<0.05). In donor C, both materials stimulated SCFA production already during the first 6 h of incubation. After 6 h, similar SCFA concentrations were obtained for both materials in donors A and B. By the end of the 48 h incubation, micronized black leaf powder consistently yielded the highest SCFA concentrations.

[0079] Acetate: Fermentation of both tea materials began already during the first 6 h of incubation. At the end of incubation, both materials yielded significantly higher acetate concentrations than the blank (p<0.05). The highest acetate concentrations were consistently obtained with the micronized black tea leaf powder.

[0080] Propionate: Both materials stimulated propionate production during the first 6 h of incubation. By the end of incubation, both materials yielded significantly higher propionate concentrations than the blank (p<0.05). Inter-individual differences were observed in the sense that the highest propionate concentrations in Donors A and B were obtained with micronized black tea leaf powder, while for Donor C the highest propionate concentration resulted from fermentation of the black tea infusion powder.

[0081] Butyrate: Both materials significantly increased butyrate levels compared to the blank in each of the donors tested (p<0.05), indicating that fermentation of the tea material strongly contributes to the production of butyrate. The concentration of butyrate was low after 6 h of incubation, which was expected considering that butyrate is a secondary metabolite that requires the primary production of acetate and / or lactate. It is therefore typically produced at the later stage of incubation (between 6 and 24 h in this case). With regard to propionate, inter-individual differences were observed in the sense that the highest butyrate concentrations at the end of incubation were observed with the micronized black tea leaf powder in donors A and C, but with the black tea infusion powder in donor B.

[0082] Branched SCFA: Overall, branched SCFA production occurred primarily during the 6-48 h time frame. Both materials yielded significantly lower branched SCFA concentrations than the blank in donors A and B (p<0.05). In donor C, branched SCFA concentrations were similar to the blank, although very low branched SCFA concentrations were observed for the blank and treatments in this particular donor.

[0083] Ammonium: Ammonium production occurred mainly during the first 24 h of incubation. In donors A and C, both materials yielded ammonium concentrations similar to the blank. However, in donor B, both materials were observed to produce significantly less ammonium compared to the blank (p<0.05).

[0084] Lactate: Both materials significantly increased lactate levels during the first 6 h compared to blank incubations (p<0.05). Lactate accumulation means that the rate of lactate production was higher than the rate of lactate consumption during the first 6 h. The highest lactate concentrations after 6 h were consistently obtained with micronized black leaf powder. Overall, lactate produced during the first 6 h was efficiently consumed after 24 h in all incubations, indicating efficient lactate conversion. Lactate consumption was consistently highest for micronized black leaf powder, which correlates with the higher lactate concentrations measured after 6 h.

[0085] Changes in microbial communities The changes in total and phylum levels are shown in Table 1 for 24 h incubation and in Table 2 for 48 h incubation. Values ​​are given as cells / ml.

[0086] For all three donors, the total number of microbial cells was increased most strongly with the micronized black tea leaf powder.

[0087] [Table 1]

[0088] [Table 2]

[0089] Analysis of the fecal inoculum showed that the three donors shared similarities in the most abundant microbial phyla occupying their gut microbiota, with significant differences observed at the microbial family and operational taxonomic unit (OUT) levels. For this reason, it is useful to describe the treatment effects at these levels separately for each donor.

[0090] Donor A: Both materials resulted in an increase in Bifidobacteriaceae between 0 and 24 h. Higher levels than the blank were maintained during the 24-48 h time frame and contributed to OTU3 and OTU14, identified as closely related to Bifidobacterium adolescentis / faecale and B. longum subsp. longum / null, respectively. Each material stimulated at least one species of the family Bacteroidaceae. Overall, the micronized black leaf powder produced an immediate stimulatory effect (i.e., between 0 and 24 h), whereas the black tea infusion powder tended to stimulate Bacteroidaceae in the later stages of incubation (24-48 h). Furthermore, the black tea infusion powder rather decreased the overall population of Bacteroidaceae, whereas the micronized black leaf powder strongly increased the overall population of this family. The following observations were made: black tea infusion powder stimulated OTU4 (Bacteroides vulgatus) between 24 and 48 h. Micronized black tea leaf powder stimulated OTU4 (Bacteroides vulgatus), OTU17 (Bacteroides eggerthii), and OTU32 (Bacteroides massiliensis) between 0 and 24 h, and these stimulatory effects were maintained until the end of incubation. OTU18 (Bacteroides ovatus) was stimulated by micronized black tea leaf powder during the later stage of incubation (i.e., between 24 and 48 h). Both materials had an immediate stimulatory effect on Enterococcaceae that continued until the end of incubation. Micronized black tea leaf powder stimulated Erysipelotrichaceae throughout the 48-h incubation period, and this stimulatory effect was attributed to OTU24, identified as closely related to Holdemanella.Each material stimulated different members of the Lachnospiraceae family: black tea infusion powder stimulated OTU12 (Fusicatenibacter saccharivorans) and OTU21 (Butyrate-producing bacterium / Eubacterium rectale) throughout the 48-h incubation period, and stimulated OTU72 (undefined identity) in the first 24 h of incubation. Micronized black tea leaf powder stimulated OTU12 (Fusicatenibacter saccharivorans), OTU21 (Butyrate-producing bacterium / Eubacterium rectale), OTU26 (Blautia faecis), and OTU73 (Member of Clostridium cluster XIVb) throughout the 48-h incubation period, and stimulated OTU72 (undefined identity) between 0 and 24 h. Both materials stimulated Ruminococcaceae throughout the 48-h incubation period, with stimulatory effects on OTU9 and OTU10, both identified as closely related to Faecalibacterium prausnitzii, and OTU29, with undetermined identity. Micronized black tea leaf powder stimulated species of the Burkholderiaceae throughout the 48-h incubation period and stimulated Enterobacteriaceae during the later stages of incubation (24-48 h). The stimulatory effect on Enterobacteriaceae was consistently attributed to OTU1, identified as closely related to Escherichia coli / Shigella sonnei.

[0091] Donor B: Micronized black tea leaf powder led to an increase in Bifidobacteriaceae throughout 48 h of incubation. Black tea infusion powder stimulated Bifidobacteriaceae during the later stages of incubation (24-48 h). The stimulatory effect was contributed to OTU3, which is closely related to Bifidobacterium adolescentis / faecale. Micronized black tea leaf powder additionally stimulated OTU27, which was identified as closely related to Bifidobacterium bifidum. Micronized black tea leaf powder stimulated Atopobiaceae and mildly stimulated Coriobacteriaceae throughout 48 h of incubation. Micronized black tea leaf powder also had a stimulatory effect on Eggerthellaceae throughout the 48 h incubation period, which was contributed to OTU8, which was identified as closely related to Senegalimassilia. Micronized black tea leaf powder also stimulated OTU4, identified as closely related to members of the Bacteroidaceae family, more specifically Bacteroides vulgatus, and had a mild stimulatory effect on members of the Barnesiellaceae family at the late stage of incubation (24-48 h). Micronized black tea leaf powder led to an increase in members of the Prevotellaceae family already in the first 24 h of incubation, and this increase was maintained until the end of incubation, contributed by OTU60 (Prevotella sp.). Black tea infusion powder stimulated Prevotellaceae at the late stage of incubation (between 24-48 h). Black tea infusion powder had an immediate stimulatory effect on Enterococcaceae, which persisted until the end of incubation. The stimulatory effect of micronized black tea leaf powder on Enterococcaceae occurred at the late stage of fermentation (24-48 h). The stimulatory effect on Enterococcus was contributed by OTU2, which is closely related to Enterococcus durans / hirae / faecium / azikeevi / villorum.Micronized black tea leaf powder stimulated Erysipelotrichaceae during 24-48 h of incubation, and the stimulatory effect was attributed to OTU24, identified as closely related to Holdemanella. Both materials stimulated different species of the Lachnospiraceae family, with the following observations: both stimulated OTU7 (Dorea longicatena), OTU30 (Blautia obeum), OTU43 (Coprococcus eutactus / Butyrate-producing bacterium), and OTU45 (Blautia obeum). Black tea infusion powder both increased OTU25 (Ruminococcus faecius / R. torques) throughout the 48 h incubation period. Micronized black tea leaf powder co-stimulated OTU12 (Fusicatenibacter saccharivorans), OTU25 (Ruminococcus faecalis / R. torques), OTU46 (Roseburia) and OTU48 (Eubacterium eligens) throughout 48 h of incubation. Micronized black tea leaf powder moderately stimulated Lactobacillaceae. Each material stimulated different species of the Ruminococcaceae family: both materials stimulated OTU9 and OTU10, both closely related to Faecalibacterium prausnitzii, at levels significantly higher than the blank between 0 and 24 h, which was maintained during the 24-48 h time frame of incubation with micronized black tea leaf powder. Black tea infusion powder costimulated OTU11 (Gemmiger formicilis) during 0-24 h at levels significantly higher than the blank that were maintained during the 24-48 h time frame. Micronized black tea leaf powder costimulated OTU11 (Gemmiger formicilis) and OTU37 (Flavonifractor) during 0-24 h at levels significantly higher than the blank that were maintained during the 24-48 h time frame, and OTU28 (Faecalibacterium prausnitzii) both increased during 24-48 h.Both materials stimulated species of the family Streptococcaceae, resulting in significantly higher populations than the blank throughout incubation. Micronized black tea leaf powder stimulated members of the family Burkholderiaceae throughout the 48-h incubation period. Black tea infusion powder stimulated Burkholderiaceae at the later stages of incubation (24–48 h).

[0092] Donor C: Both materials resulted in an increase in species of the family Bifidobacteriaceae, more specifically OTU3 (Bifidobacterium adolescentis / faecale), at levels higher than the blank during incubation with micronized black tea leaf powder from 0 to 24 h, which was maintained over the 24-48 h time frame of incubation with micronized black tea leaf powder. OTU14 (B. longum subsp. longum / null) was also increased during incubation with micronized black tea leaf powder from 24 to 48 h. Both materials stimulated Coriobacteriaceae at levels higher than the blank during incubation from 0 to 24 h, which was maintained until the end of incubation. While both materials stimulated certain species of the family Bacteroidaceae, overall, micronized black tea leaf powder stimulated members of the family Bacteroidaceae to the greatest extent. Both materials stimulated OTU4 (Bacteroides vulgatus) between 0 and 24 h at higher levels than the blank which was maintained until the end of incubation. Both materials co-increased OTU5 at several time points during incubation. Fermentation of micronized black tea leaf powder stimulated members of the family Tannerellaceae throughout incubation. Clostridiaceae was mildly stimulated by micronized black tea leaf powder between 0 and 24 h. Black tea infusion powder stimulated Erysipelotrichaceae between 0 and 24 h. Micronized black tea leaf powder stimulated Erysipelotrichaceae during the later stages of incubation (between 24 and 48 h). Both materials stimulated different members of the Lachnospiraceae family with the following observations: both stimulated OTU34 (Anaerostipes) and OTU41 (Clostridium glycyrrhizinilyticum) throughout the 48 h incubation period. Micronized black tea leaf powder increased both OTU49 (Clostridium citroniae / clostridioforme) and OTU54 (Butyrate-producing bacterium) between 0 and 24 h.Both materials stimulated members of the Ruminococcaceae family, which were attributed to a stimulatory effect on OTU9 (Faecalibacterium prausnitzii), OTU11 (Gemmiger formicilis), and OTU57 (Butyricicoccus). Both materials stimulated Streptococcaceae, which were mainly attributed to OTU6, identified as closely related to Streptococcus salivarius subs. Null. Micronized black tea leaf powder stimulated Veillonellaceae during 24-48 h, which were attributed to OTU13, identified as closely related to Veillonella dispar. Micronized black tea leaf powder stimulated Burkholderiaceae during the later stages of incubation (24-48 h), with a stimulatory effect on OTU44, identified as closely related to Sutterella wadsworthensis.

[0093] conclusion Short-term colonic incubation demonstrated that both tea materials were efficiently fermented by the gut microbiota of the three adult donors under study, resulting in enhanced SCFA (acetate, propionate and butyrate) and lactate levels, although small interindividual differences were observed regarding metabolite production.

[0094] Micronized black tea leaf powder produced a stronger pH reduction, higher gas production, and stronger overall SCFA production than black tea infusion powder. Acetate concentrations were also consistently higher with micronized black tea powder, and it was the micronized black tea leaf powder that stimulated the greatest lactate production.

[0095] The production of acetate and lactate indicates the action of lactic acid bacteria, while the production of propionate indicates that Bacteroidetes spp., typically capable of degrading complex molecules, may also have been involved in the breakdown of the substrate. The Illumina data confirmed this hypothesis, in that both materials consistently enriched for species of the family Bifidobacteriaceae in each of the three studied donors.

[0096] For all three donors, the total number of bacterial cells was increased most strongly with the micronized black tea leaf powder.

[0097] Therefore, it can be concluded that micronised black leaf tea powder has a significantly better prebiotic effect than black tea infusion powder.

Claims

1. 13. Use of a composition comprising black tea leaf particles as a prebiotic, wherein the black tea leaf particles have a particle size of less than 250 μm.

2. 1. A composition for pharmaceutical use comprising black tea leaf particles, wherein the black tea leaf particles have a particle size of less than 250 μm.

3. The composition of claim 2 in the treatment or prevention of conditions associated with suboptimal immunity.

4. The composition of claim 3, wherein the condition is a viral infection, chronic inflammation and / or allergy.

5. The composition of claim 4, wherein the viral infection is influenza, a cold and / or a coronavirus infection.

6. 3. The composition of claim 2 in the treatment or prevention of a gastrointestinal condition.

7. 7. The composition of claim 6, wherein the condition is selected from inflammatory bowel disease, irritable bowel syndrome, environmental bowel disease and infectious diarrhea.

8. The composition according to claim 2 for the relief or alleviation of visceral pain.

9. 9. The use according to claim 1 or the composition according to any one of claims 2 to 8, wherein the black tea leaf particles comprise at least 2% flavonoids by weight of the black tea leaf particles.

10. 10. The use or composition according to claim 9, wherein the flavonoids comprise thearubigins in an amount of at least 70% by weight of the tea flavonoids, preferably at least 82% by weight of the tea flavonoids.

11. The use or composition according to any one of claims 1 to 10, wherein the composition is administered to an individual in an amount of at least 0.5 g of black tea leaf particles per day.

12. 1. A beverage comprising black tea leaf particles dispersed in an aqueous liquid, the black tea leaf particles having a particle size of less than 250 μm.

13. 13. The beverage of claim 12, wherein the beverage has a mass of less than 500g and contains black tea leaf particles in an amount of at least 0.5g.

14. The process is as follows: (a) providing an aqueous liquid; and (b) dispersing black tea leaf particles in an aqueous liquid; 14. A method for producing a beverage according to claim 12 or 13, comprising:

15. A packaged beverage ingredient comprising a composition comprising black tea leaf particles packaged in a container, wherein the amount of the composition packaged in the container is from 0.5 to 500 g, and the black tea leaf particles have a particle size of less than 250 μm.

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