Improved prebiotic formulations

Abstract

The disclosed method comprises: reducing a fermentation broth comprising 2'-fucosyllactose to produce a treated fermentation broth comprising 2'-fucosyllactose and 2'-fucosyllactitol; separating a stream comprising 2'-fucosyllactose and 2'-fucosyllactitol from the treated fermentation broth; purifying the separated stream; concentrating the purified and separated stream; dehydrating the concentrated, purified and separated stream to form a prebiotic solid product comprising 2'-fucosyllactose and 0.2 to 6.0 wt.% of 2'-fucosyllactitol, the above content being wt.% based on the total weight of the prebiotic solid product, wherein the concentration step and the dehydration step can be performed in any order.

Description

Technical Field

[0001] The present invention relates to improved prebiotic formulations. More specifically, the present invention relates to prebiotic formulations comprising 2'-fucosyllactose and 2'-fucosyllactitol. Background Art

[0002] Breast milk contains a unique array of oligosaccharides called human milk oligosaccharides (HMOs), which are structurally diverse, non-conjugated polysaccharides. Although HMOs are the third most abundant solid component of breast milk (after lactose and fat), human infants are unable to digest HMOs. Instead, they function as prebiotics, helping to establish commensal bacteria. Prebiotics, or non-digestible carbohydrates, are used to enhance specific beneficial microbial species. For example, 2'-fucosyllactose (2'-FL), which occurs naturally in human breast milk, is a known prebiotic that stimulates the growth of a specific population of beneficial microorganisms of the genus Bifidobacterium. 2'-FL cannot be digested by endogenous human enzymes but is used as an energy source by commensal bacteria in the infant gut to selectively support the growth of beneficial bacteria (prebiotic action). It, along with potentially >200 similar human oligosaccharides, is secreted from the mammary epithelial cells of lactating women into their milk. The α1,2 / 3 / 4 fucosyl moieties of HMOs, including the α1,2-linked fucose of 2'-FL, tend to protect HMOs from digestion unless these bonds at the non-reducing end are cleaved first. Therefore, the prebiotic effects of 2'-FL depend on several activities of bacterial enzymes, such as α1,2-fucosidase activity that cleaves fucose and releases lactose; activity that metabolizes the released fucose to lactate and 1,2-propanediol; activity that metabolizes the proportion of lactose that is not used by human host cells; and several other activities. Supporting the development of a healthy digestive tract in infants helps with the development of their immune system, as much of the infant's immune system is found in the digestive tract. The occurrence and concentration of these complex oligosaccharides are unique to humans and are not found in significant amounts in the milk of other mammals, such as domesticated dairy animals. There have been efforts to develop improved systems for manufacturing HMOs, such as EP14827224.8, WO2019 / 003133, WO2019 / 003135, US2017 / 0304375 and WO2019 / 003136.

[0003] Therefore, it is known that a balanced microbiota composition is beneficial for overall health and well-being. Lactitol is a known prebiotic that enhances the microorganisms of the Lactobacillus (and to a lesser extent Bifidobacterium) genus in the intestine, as well as increasing the levels of short-chain fatty acids. A composition comprising 2'-fucosyllactitol (2'-FLol) will stimulate a wide range of beneficial microbiota, resulting in a better intestinal balance, because 2'-FLol is easily hydrolyzed by specific fucosidases produced by the intestinal microbiota (Bifidobacteria), releasing lactitol and fucose. In addition, lactitol is not digested by human digestive enzymes, while lactose can. Therefore, it is believed that a mixture of 2'-FL and 2'-FLol provides a more robust and versatile prebiotic system than 2'-FL alone, because their actions are complementary; for example, if 2'-FL is hydrolyzed too quickly during storage or meal preparation, 2'-FLol or lactitol will still be present in the mixed system. Furthermore, the caloric value of lactitol is 2 kcal / g, while the caloric value of lactose is 3.94 kcal / g.

[0004] 2'-FL is typically produced in microbial fermentation reactors; however, such conventional processes produce only low levels of 2'-FLOl as a byproduct. There is a need for methods of making compositions containing 2'-FL and 2'-FLOl in which 2'-FLOl is present at increased levels. Summary of the invention

[0005] Surprisingly, a method has been discovered for making a mixture containing 2'-FL and 2'-FLol in which 2'-FLol is present at increased levels, wherein 2'-FL is produced in a microbial fermentation subjected to reducing conditions to produce 2'-FLol.

[0006] In one embodiment, the present disclosure provides a method comprising: reducing a fermentation broth comprising 2'-fucosyllactose to produce a treated fermentation broth comprising 2'-fucosyllactose and 2'-fucosyllactitol; separating a stream comprising 2'-fucosyllactose and 2'-fucosyllactitol from the treated fermentation broth; purifying the separated stream; concentrating the purified and separated stream; dehydrating the concentrated, purified and separated stream to form a prebiotic solid product comprising 2'-fucosyllactose and 0.2 to 6.0 wt.% of 2'-fucosyllactitol based on the total wt.% of the prebiotic solid product, wherein the concentration step and the dehydration step can be performed in any order.

[0007] In another embodiment, the present disclosure provides a method comprising administering an effective amount of a prebiotic solid product composition to a subject in need thereof, thereby producing an in vivo supply of lactitol.

[0008] In yet another embodiment, the present disclosure provides a method of manufacturing a food, a growing-up milk, a dietary supplement, or a medicament, wherein the method comprises: manufacturing a prebiotic solid product; and mixing the prebiotic solid product with one or more ingredients suitable for the food, the growing-up milk, the dietary supplement, or the medicament.

[0009] In another embodiment, the present disclosure provides a method for manufacturing a food, a growing-up milk, a dietary supplement or a medicine, wherein the method comprises: manufacturing a prebiotic solid product; dissolving the prebiotic solid product in a solvent; and mixing the dissolved prebiotic solid product with one or more ingredients suitable for the food, the growing-up milk, the dietary supplement or the medicine.

[0010] In yet another embodiment, the present disclosure provides a method of manufacturing an infant formula, wherein the method comprises: manufacturing a prebiotic solid product, and mixing the prebiotic solid product with at least one infant formula ingredient.

[0011] In one embodiment, the present disclosure provides a method of making an infant formula, wherein the method comprises: making a prebiotic solid product, dissolving the prebiotic solid product in a solvent; and mixing the dissolved prebiotic solid product with at least one infant formula ingredient.

[0012] In another embodiment, the present disclosure provides a food, growing-up milk, dietary supplement or medicament obtained by the following method: the method includes manufacturing a prebiotic solid product; and mixing the prebiotic solid product with at least one infant formula ingredient, or first dissolving the prebiotic solid product in a solvent, and mixing the dissolved prebiotic solid product with at least one infant formula ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The subject matter of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0014] Figure 1 is a process flow diagram illustrating the separation and purification of a prebiotic solid stream.

[0015] Figure 2 It shows that in 125mM sodium phosphate buffer at 37°C and pH 6.5, composition 2 (47mM 2'-FL and 2mM 2'-FLol) was hydrolyzed into lactose and lactitol, respectively, by 12μg / ml fucosidase from Bifidobacterium longum. Solid line, 2'-FLol (solid circle) and its derivative lactitol (open circle); dotted line, 2'-FL (solid square) and its derivative lactose (open square).

[0016] Figure 3It shows that in 125mM sodium phosphate buffer at 37°C and pH 6.5, composition 2 (47mM 2'-FL and 2mM 2'-FLol) was hydrolyzed into lactose and lactitol, respectively, by 12μg / ml fucosidase from Bifidobacterium sp. Solid line, 2'-FLol (solid circle) and its derivative lactitol (open circle); dotted line, 2'-FL (solid square) and its derivative lactose (open square).

[0017] Figure 4 It shows that in 125mM sodium phosphate buffer at 37°C and pH 6.5, composition 2 (47mM 2'-FL and 2mM 2'-FLol) was hydrolyzed into lactose and lactitol, respectively, by 2μg / ml fucosidase from Bifidobacterium longum. Solid line, 2'-FLol (solid circle) and its derivative lactitol (open circle); dotted line, 2'-FL (solid square) and its derivative lactose (open square).

[0018] Figure 5 It shows that in 125mM sodium phosphate buffer at 37°C and pH 6.5, composition 2 (47mM 2'-FL and 2mM 2'-FLol) was hydrolyzed into lactose and lactitol, respectively, by 2μg / ml fucosidase from Bifidobacterium sp. Solid line, 2'-FLol (solid circle) and its derivative lactitol (open circle); dotted line, 2'-FL (solid square) and its derivative lactose (open square).

[0019] Figure 6 Illustration of incubation of composition 2 (47 mM 2'-FL and 2 mM 2'-FLol) with a no-enzyme control of water in 125 mM sodium phosphate buffer at 37°C, pH 6.5. Solid line, 2'-FLol (filled circles) and its derivative lactitol (open circles); dotted line, 2'-FL (filled squares) and its derivative lactose (open squares). DETAILED DESCRIPTION

[0020] The present disclosure features methods for producing, isolating and purifying improved prebiotic solid products. In one embodiment, the present disclosure provides a method comprising: reducing a fermentation broth comprising 2'-FL to produce a treated fermentation broth comprising 2'-FL and 2'FLol; separating a stream comprising 2'-FL and 2'FLol from the treated fermentation broth; purifying the separated stream; concentrating the purified and separated stream; dehydrating the concentrated, purified and separated stream to form a prebiotic solid product comprising 2'-fucosyllactose and 0.2 to 6.0 wt.% of 2'-fucosyllactitol based on the total wt.% of the prebiotic solid product, wherein the concentration step and the dehydration step can be performed in any order. For the purposes of this specification, the term prebiotic refers to a selectively fermented ingredient that causes specific changes in the composition and / or activity of the gastrointestinal microbiota, thereby providing benefits to the health of the host. Characteristics of a prebiotic include: (i) it should be resistant to the acidic pH of the stomach, (ii) it should not be hydrolyzed by mammalian enzymes, (iii) it should not be absorbed from the gastrointestinal tract, (iv) it can be fermented by the intestinal microbiota, and (iii) the growth and / or activity of intestinal bacteria can be selectively stimulated by this compound, and this process can improve the health of the host.

[0021] Fermentation steps

[0022] Fermentation can be carried out in any suitable fermentation medium, such as a chemically defined fermentation medium. The fermentation medium can vary based on the microbial organism used.

[0023] Preferably, in the fermentation step, sugar, lactose and fermentation nutrients are supplied to a microbial culture genetically modified to produce 2'-FL, and then fermented to produce 2'-FL and other carbohydrates such as difucosyllactose, 2'-fucosyl-d-lactulose, lactose / isolactose, 3'-fucosyllactose, fucosylgalactose. The fermentation medium can be supplemented with trace minerals and vitamins, such as calcium pantothenate, biotin, niacin, myo-inositol, thiamine hydrochloride, para-aminobenzoic acid. Food-grade processing aids such as defoamers and pH control agents can also be used. The temperature of the fermentation step is generally 10 to 50°C, preferably 25 to 35°C, more preferably 28 to 32°C. The pH of the fermentation step is generally 3 to 8, preferably 5 to 7, more preferably 5.5 to 6.5. At the end of the fermentation step, the fermentation broth can be subjected to a heat treatment step to inactivate the microorganisms before the downstream processing step.

[0024] Preferably, the microbial organism is a genetically modified yeast, such as a Saccharomyces strain, a Candida strain, a Hansenula strain, a Kluyveromyces strain, a Pichia strain, a Schizosaccharomyces strain, a Schwanniomyces strain, a Torulaspora strain, a Yarrowia strain, or a Zygosaccharomyces strain. More preferably, the yeast is Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii or Zygosaccharomyces bailii.

[0025] Preferably, the microbial organism can also be selected from the bacterial genera Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium, Bacterioides, Prevotella, Escherichia or Propionibacterium.More preferably, the bacterial species is Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, B. longum; Enterococcus faecium; Escherichia coli; Lactobacillus acidophilus. acidophilus), Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakel, Lactococcus lactis (including but not limited to subspecies lactis, subspecies cremoris and subspecies diacetyl lactic acid); Leuconostoc mesenteroides (including but not limited to subspecies mesenteroides); Pedicococcus acidilactici (including but not limited to subspecies mesenteroides); acidilactici), P. pentosaceus; Propionibacterium acidipropionici, P. freudenreichiis sp. shermanii; Staphylococcus carnosus; and Streptococcus thermophilus. The fermentation broth may also include other products of the fermentation process, such as cells (biomass), fermentation medium, residual substrate material, and any molecules / byproducts produced during the fermentation process.

[0026] Optionally, the fermentation broth may be subjected to a heat treatment step to kill bacteria or other undesirable microorganisms that may be present to any significant extent.

[0027] Restore steps

[0028] The fermentation broth comprising 2'-fucosyllactose is reduced to produce a treated fermentation broth comprising 2'-fucosyllactose and 2'-fucosyllactitol. During the reduction process, 2'-fucosyllactitol is formed by at least one of the following two pathways:

[0029] Path #1

[0030] -Lactose + 2H + +2e - → Lactitol

[0031] -Lactitol + GDP-fucose → 2'-fucosyllactitol + GDP pathway #2

[0032] -Lactose + GDP-fucose → 2'-fucosyllactose + GDP

[0033] 2'-Salvinosyl lactose + 2H + +2e - → Salicyllactitol

[0034] In these pathways, the term “2H + +2e - " stands for reducing equivalents, usually in the form of NAD(H), NADP(H), FAD(H) or intermediates. In both pathways, the reactions are catalyzed by microbial enzymes and these reactions will occur inside the microorganism. These pathways involve reduction reactions and therefore require reducing equivalents (H + +e - ). The extent of 2'-fucosyllactitol production can be affected by factors such as oxygen flow, where reduced oxygen concentrations enhance reducing conditions; the relative flow of lactose, which is directly reduced to lactitol in pathway #1; the relative flow of sucrose as a carbon source and electron carrier; or changes in the export of 2'-fucosyllactose from the microbial cells. Alternatively, reduction occurs spontaneously during fermentation. The temperature and pressure of the reduction step are the same as those of the fermentation step.

[0035] After fermentation and reduction of the fermentation broth, the products of these steps are subjected to various steps to separate and purify the dry mixture of 2'-FL and 2'-FLO1. These steps are as follows Figure 1As shown. The separation step is used to separate 2'-FL and 2'-FLol from other materials contained in the fermentation broth. Such a separation step may include centrifugation, conventional filtration and microfiltration as described below, which produces a separated stream rich in 2'-FL and 2'-FLol. After the separation step, the separated stream is subjected to a purification step, which may include ultrafiltration, nanofiltration, anion exchange, cation exchange and decolorization to form a separated and purified stream. The separated and purified stream is then treated in a concentration step and a dehydration step. These steps may be performed in any order. In other words, the separated and purified stream may first be treated in a concentration step, and then the concentrated product may be sent to a dehydration step. Alternatively, the separated and purified stream may first be subjected to a dehydration step, and then the product of the dehydration step may be sent to a concentration step. The concentration step may include evaporation, reverse osmosis filtration and nanofiltration. The evaporation process may include, for example, falling film evaporation, rising film evaporation and rotary evaporation to form a separated, purified and concentrated stream. The dehydration step may include at least one of crystallization, drying, evaporation, and conventional filtration to form a prebiotic solid product.

[0036] The various unit operations used in the separation / purification process steps of the present disclosure are described below. Combinations of these unit operations can be used depending on the desired purity level. It is possible that some unit operations, such as centrifugation and conventional filtration, can also be used for both separation and concentration steps.

[0037] Separation and purification of fermentation broth

[0038] Conventional filtration

[0039] For the purposes of this specification, the term conventional filtration refers to methods using plate and frame filtration, recessed chamber filtration, belt filtration, vacuum filtration, horizontal metal leaf filtration, vertical metal-leaf filtration, stacked-disc filtration, rotary vacuum filtration, and combinations thereof.

[0040] Centrifugation

[0041] Centrifugation can be used to remove suspended matter, such as biomass. The desired prebiotic solid product is contained in the liquid not retained by centrifugation. Preferably, the centrifugation is operated continuously.

[0042] Microfiltration

[0043] A cross flow filtration process can be used as a guard filter to remove residual biomass that was not removed in the centrifugation step. Typically, microfiltration has a cut-off of 10 microns, preferably a cut-off of 5 microns, and more preferably a cut-off of 0.2 microns. The product stream is a liquid permeate.

[0044] Ultrafiltration

[0045] Cross-flow ultrafiltration can be used to remove proteins and other high molecular weight compounds, such as DNA and large polysaccharides, from the fermentation product. The pore size of the ultrafiltration membrane ranges from about 200 kD molecular weight cutoff ("MWCO") or less to about 1 kD MWCO. The product stream is the permeate. Preferably, after the ultrafiltration step, the yield of the desired prebiotic solids in the permeate is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%.

[0046] Nanofiltration

[0047] Cross-flow nanofiltration can be used to remove low molecular weight molecules, such as monosaccharides and disaccharides, peptides, small organic acids and salts, whose molecular weight is less than that of the desired prebiotic solids. The pore size of the nanofiltration membrane is from a molecular weight cutoff of about 500 Daltons (Da) or less to 200 Da MWCO or less. Preferably, the molecular weight cutoff is 500 Daltons (Da) or less, 450 Da MWCO, 400 Da MWCO, 350 Da MWCO, 300 Da MWCO, 250 Da MWCO or 200 Da MWCO or less. The product stream is the retentate. Preferably, after the nanofiltration step, the yield of the desired prebiotic solids in the retentate is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%.

[0048] Cation Exchange (CEX)

[0049] Ion exchange chromatography can be used to separate charged molecules from a stream containing prebiotic solids. CEX stationary phases typically contain aliphatic sulfonic acid groups that are negatively charged in aqueous solution, which bind tightly to any strongly basic analytes. This step removes positively charged components such as residual ammonia, metals, and peptides. The binding capacity of the resin used is generally 1.2 to 2.2 mmeq / L. Typical resins for cation exchange include Dow Dowex 88, Resindion JC series (such as JC603), and Resindion PK grades (such as PK216).

[0050] Decolorization

[0051] A decolorization step may be performed to remove color-containing components. This step may be performed using activated carbon such as Norit CA1 activated carbon, hydrophobic interaction chromatography (HIC) or another functionalizable adsorption resin such as Resindion Relite RAD / F. Decolorization may be performed before or after the anion exchange step.

[0052] Anion Exchange

[0053] The anion exchange step can be used to separate charged molecules. In an anion exchange column, the filler is positively charged, so negatively charged molecules are retained by Coulombic interactions. This step removes negatively charged components, such as sulfates, phosphates, organic acids, and negatively charged particles. The binding capacity of the resin used is generally 0.8 to 2.0 mmeq / L. Typical resins for anion exchange include Resindion Relite series such as RAM2, D182, JA100, and Diaion WA series such as WA20.

[0054] concentrate

[0055] The concentration step can be used to economically remove large amounts of liquid from a stream containing prebiotic solids using evaporation, reverse osmosis filtration, and nanofiltration. The evaporation process can include, for example, falling film evaporation, rising film evaporation, and rotary evaporation. The solid concentration entering the process is about 5 to 30 wt%. The solid concentration leaving the process is typically greater than 30 wt%. Preferably greater than 50 wt%. More preferably, the solid concentration leaving the dehydration operation is 60 to 80 wt%. The solid portion of the recovered material is greater than 80 wt.% prebiotic solids, with the remaining impurities being primarily alditols or disaccharides or trisaccharides.

[0056] Heat Treatment

[0057] The heat treatment step may be used to kill bacteria or other undesirable microorganisms that may be present to any significant extent and is essentially a pasteurization process. Any acceptable pasteurization conditions are possible, for example from 62.8°C for 30 minutes to 72°C for 20 seconds to 100°C for 0.01 seconds.

[0058] dry

[0059] Drying can be carried out by an indirect dryer in an indirect drying process, or by a direct dryer in a direct drying process. For the purpose of this specification, the term indirect dryer includes those devices that do not utilize direct contact of the material to be dried with the process gas for heating, but rely on heat transfer through the walls of the dryer (e.g., in the case of a drum dryer, through the shell wall) or alternatively through the walls of the hollow paddles of a paddle dryer, because they rotate through the solid while the heat transfer medium circulates in the hollow interior of the paddle. Other examples of indirect dryers include contact dryers and vacuum drum dryers. The heat transfer medium is preferably steam or heat transfer oil. More preferably, the heat transfer medium is steam. The term direct dryer includes a process in which the hot process gas moves through a container, directly contacting the circulating solid, such as a spray dryer. Flash dryers or fluidized bed dryers are other examples of such direct drying methods. Preferably, when direct drying is used, it is carried out by spray drying in a spray dryer. Preferably, the drying method is indirect drying. Preferably, the indirect drying method is drum drying. In drum drying, the heating surface is the shell of a rotating horizontal metal cylinder. The drum is preferably heated by steam condensing inside, so that the temperature of the drum wall reaches 110-155°C. Preferably, the residence time of the prebiotic solid material on the dryer is less than 3 minutes. The wet material is applied to the drum surface as a relatively thin layer. The dried product is removed from the drum with the help of a blade or string. Subsequently, grinding and sieving steps can be used to obtain the desired particle size range.

[0060] Grinding

[0061] If a grinding step is required, generally any grinding method suitable for the type of solid and the target particle size can be used. Such grinding equipment can include, for example, ball mills, hammer mills, SAG mills, rod mills, Raymond mills, and vertical mills.

[0062] crystallization

[0063] The crystallization process can be used to dehydrate the purified prebiotic solid material by first forming a prebiotic solid core in a supersaturated solution and then growing crystals. Such a process can be batch or continuous. Typical conditions for crystallization are shown in WO 2018 / 164937, the disclosure of which is incorporated herein by reference. The process involves concentrating the starting solution containing the prebiotic solid to a supersaturated state, then precipitating the prebiotic solid crystals from the supersaturated solution, while preferably placing the supersaturated solution at a temperature of at least 55°C. Preferably, the solution has a prebiotic solid content of at least 60wt.% and less than about 98wt.%. As the percentage of prebiotic solid crystals increases, crystallization is generally limited by viscosity, preferably with a typical endpoint of about 30wt.% crystals.

[0064] Prebiotic solid products

[0065] The prebiotic solid product contains 0.2 to 6.0 wt.% 2'-FLoI, 86.0 to 94.0 wt.% 2'-FL and 0 to 13.8 wt.% carbohydrate byproducts. For the purpose of this specification, the term "carbohydrate byproducts" includes difucosyllactose, 2'-fucosyl-d-lactulose, lactose / allolactose, 3'-fucosyllactose, fucosylgalactose, glucose / galactose and fructose. The amounts of these materials were determined using ion chromatography pulsed amperometric detection using a Dionex Carbopac PA1 column.

[0066] Preferably, the prebiotic solid contains 0.5 to 6.0 wt.%, more preferably 0.5 to 3.5 wt.% 2'-FLol, with corresponding levels of other carbohydrates being 0 to 13.5 wt.% and 2.5 to 13.5 wt.%, respectively. In both cases, 2'-FL is present in an amount of 86.0 to 94.0 wt.%.

[0067] In another embodiment, the prebiotic solid contains 2.0 to 4.5 wt.%, more preferably 2.0 to 4.0 wt.% 2'-FLol, with corresponding levels of carbohydrate byproducts of 1.5 to 12.0 wt.% and 2.0 to 12.0 wt.%, respectively. In both cases, 2'-FL is present in an amount of 86.0 to 94.0 wt.%.

[0068] In another embodiment, the prebiotic solid contains 0.2 to 10.0 wt.% 2'-FLoI, 86.0 to 90.0 wt.% 2'-FL, and 0 to 13.8 wt.% carbohydrate byproducts.

[0069] Preferably, the ratio of 2'-FL / 2'-FloI is from 14.3 to 470.

[0070] In another embodiment, the present disclosure provides a method comprising administering an effective amount of a prebiotic solid product composition to a subject in need thereof, thereby producing an in vivo supply of lactitol. As described above, in the intestine, 2'-FLol is hydrolyzed into fucose and lactitol.

[0071] In another embodiment, the present disclosure provides a method for manufacturing a food, a growing-up milk, a dietary supplement or a medicine, wherein the method comprises: manufacturing a prebiotic solid product; mixing the prebiotic solid product with one or more ingredients suitable for the food, the growing-up milk, the dietary supplement or the medicine.

[0072] In yet another embodiment, the present disclosure provides a method for manufacturing a food, a growing-up milk, a dietary supplement, or a medicine, wherein the method comprises manufacturing a prebiotic solid product; dissolving the prebiotic solid product in a solvent; and mixing the dissolved prebiotic solid product with one or more ingredients suitable for the food, the growing-up milk, the dietary supplement, or the medicine.

[0073] In another embodiment, the present disclosure provides a method of making an infant formula, wherein the method comprises making a prebiotic solid product and mixing the prebiotic solid product with at least one infant formula ingredient.

[0074] In one embodiment, the present disclosure provides a method of manufacturing an infant formula, wherein the method comprises manufacturing a prebiotic solid product and dissolving the prebiotic solid product in a solvent; and mixing the dissolved prebiotic solid product with at least one infant formula ingredient.

[0075] In another embodiment, the present disclosure provides a food, a growing-up milk, a dietary supplement or a medicament obtained from the following method, wherein the method comprises: manufacturing a prebiotic solid product; and mixing the prebiotic solid product with at least one infant formula ingredient, or first dissolving the prebiotic solid product in a solvent and mixing the dissolved prebiotic solid product with at least one infant formula ingredient.

[0076] For the purposes of this specification, the term "infant formula" means an industrially formulated breast milk substitute that (i) meets all normal nutritional requirements of infants from birth to 4 to 6 months of age, adapted to their physiological characteristics and / or fed in addition to other foods until about 1 year of age and older, or (ii) meets the normal nutritional requirements of premature infants, in accordance with applicable Codex Alimentarius and U.S. Food and Drug Administration standards. The term "infant" means children from birth to about one year of age. The term "growing-up milk (GUM)" means a nutritionally fortified milk, plant, amino acid and / or soy milk-based liquid product, or a powdered product for reconstitution into a liquid product, in each case intended for use as a drink, marketed and intended for children from twelve (12) months to thirty-six (36) months of age, and for children up to and including six (6) years of age.

[0077] It is known that there are many different sources and types of carbohydrates, fats, proteins, minerals and vitamins which can be used as formula ingredients in the infant formula or GUM described in the present subject matter, provided that these nutrients are compatible with the additional ingredients in the selected formulation and are also suitable for use in infant formula.

[0078] Carbohydrates suitable for use in infant formula can be simple or complex, lactose-containing or lactose-free, or combinations thereof, non-limiting examples of which include hydrolyzed, intact, native and / or chemically modified corn starch, maltodextrins, glucose polymers, sucrose, corn syrup, corn syrup solids, carbohydrates from rice or potato sources, glucose, fructose, lactose, high fructose corn syrup, and indigestible oligosaccharides such as 3'-fucosyllactose, 2',3'-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose , lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose and lacto-N-neohexaose, sialyl-lacto-N-tetraose a, sialyl lacto-N-tetraose b, sialyl lacto-N-tetraose c, disialyl lacto-N-tetraose, 3' and 6' sialyl lactose, lacto-N-tetraose, difucosyllactose, lacto-N-neotetraose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), and combinations thereof.

[0079] Proteins suitable for infant formula milk powder include hydrolyzed, partially hydrolyzed and unhydrolyzed or complete proteins or protein sources, which can be derived from any known or other suitable sources, such as milk (e.g., casein, whey), animals (e.g., meat, fish), cereals (e.g., rice, corn), plants (e.g., soybeans), or combinations thereof. Proteins used herein can also include free amino acids known to be used for or otherwise suitable for infant formula milk powder, or are replaced in whole or in part by them, non-limiting examples of which include alanine, arginine, asparagine, carnitine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, taurine, tyrosine, valine, and combinations thereof. These amino acids are most typically used in their L-type, although corresponding D-type isomers can also be used when nutritionally superior. Racemic or isomeric mixtures can also be used.

[0080] Suitable fats for infant formula include coconut oil, soybean oil, corn oil, olive oil, safflower oil, high oleic safflower oil, algae oil, MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oil, palm olein, rapeseed oil, marine oil, cottonseed oil, and combinations thereof.

[0081] Vitamins and similar other ingredients suitable for use in the infant formula of the present invention include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, its salts and derivatives, and combinations thereof.

[0082] Minerals suitable for use in infant formula include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, chromium, iodine, sodium, potassium, chloride, and combinations thereof. Preferably, the above nutrients are incorporated into infant formula that complies with generally accepted guidelines for appropriate geographic and user demographics, for example, as described in 21 U.S.C. § 350a (2018).

[0083] Infant formula or GUM may further include other optional ingredients that may modify the physical, chemical, aesthetic or processing characteristics of the composition, or may be used as a medicament or additional nutritional ingredient when used for the target infant population. Non-limiting examples of such optional ingredients include preservatives, additional antioxidants, emulsifiers, buffers, colorants, flavorings, nucleotides and nucleosides, probiotics, prebiotics, lactoferrin and related derivatives, thickeners and stabilizers, etc.

[0084] Example

[0085] The following examples further detail and illustrate the performance of the process of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims.

[0086] Analytical methods

[0087] Samples and standards were analyzed using high performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) after appropriate dilution. Samples and standards (10uL) were injected on a Thermo Dionex ICS3000 system. Separation was performed on a Thermo Dionex Carbopac PA1 column (250x4mm) equipped with a Thermo Dionex Carbopac PA1 guard column (50x4 mm). The compounds of interest, namely lactitol, 2'-fucosyllactose (2'-FL), lactose, and 2'-fucosyllactitol (2'-FLol) were eluted with 100mM NaOH as the mobile phase at a flow rate of 1.5mL / min for 10 minutes. Quantification of lactitol, 2'-FL, and lactose was performed using external calibration using commercially available reference materials. In the absence of 2'-FLol reference material, 2'-FLol was quantified by external calibration based on the lactitol reference material, assuming that lactitol and 2'-FLol responded identically to PAD.

[0088] Comparative Example

[0089] The amount of 2'-fucosyllactitol present in several commercially available 2'-fucosyllactose products was analyzed using the analytical method and compared with the materials of the present invention represented by Composition 1 and Composition 2.

[0090] Table 1

[0091] sample 2'-Fucosyllactitol (wt%) Composition 1 2.2 Composition 2 3.86 Friesland Campina 0.19 Dupont Not Detected Glycom 0.01 Jennewein Biotechnologie GmbH 0.1

[0092] 2'-Fucosyllactitol is enzymatically hydrolyzed by beneficial intestinal bacteria

[0093] In order to simulate the efficient hydrolysis of 2'-fucosyllactitol by intestinal bacteria, releasing lactitol, in vitro enzymatic hydrolysis experiments were performed in a total volume of 1 ml. Commercially available fucosidases from Bifidobacterium longum and Bifidobacterium were applied at 12 μg / ml for the hydrolysis of 49 mM 2-FL (control, Care4U, Dupont) or 47 mM 2'-FL and 2 mM 2'-FLoI (composition 2) in 800 μl 125 mM pH 6.5 sodium phosphate buffer, 200 μl of enzyme solution were added, and kept at 37°C and 1000 rpm (in a thermomixer from Eppendorf) for 24 hours.

[0094] Table 2

[0095]

[0096] After 2 hours, 125 μl samples were removed and immediately heated at 95°C for 5 minutes to inactivate the enzyme and then analyzed by HPAEC-PAD as described above.

[0097] Table 3

[0098] Substrate Fucosidase 2'-FL (wt%) 2'-FloI (wt%) Care4U Bifidobacterium longum 0 Not Detected Care4U Bifidobacterium sp. 3 Not Detected Care4U None (control reaction) 100 Not Detected Composition 2 of the present invention Bifidobacterium longum 0 0 Composition 2 of the present invention Bifidobacterium sp. 18 14 Composition 2 of the present invention None (control reaction) 99 97

[0099] The data demonstrate that fucosidases normally present in the mammalian digestive tract are capable of hydrolyzing 2'-FL and 2'-FLol.

[0100] Enzymatic hydrolysis of 2'-fucosyllactitol releases lactitol

[0101] To simulate the efficient hydrolysis of 2'-fucosyllactitol by intestinal bacteria and release of lactitol, in vitro enzymatic hydrolysis experiments were performed using commercially available fucosidases from Bifidobacterium longum and Bifidobacterium spp. at two different concentrations (2 and 12 μg / ml).

[0102] The hydrolysis of composition 2 (comprising 47 mM 2'-FL and 2 mM 2'-FLol) was carefully monitored for 24 hours at 37°C and 1000 rpm (in a thermomixer from Eppendorf). Composition 2 was dissolved in 125 mM sodium phosphate buffer at pH 6.5, and enzyme was added to 800 μl of the solution at a specified concentration. Time samples were collected after 10 minutes, 30 minutes, 2 hours, 8 hours and 24 hours. After taking out the time samples, the enzyme was immediately inactivated by heating at 95°C for 5 minutes and then analyzed by HPAEC-PAD as described above. The data were normalized to the maximum amount and expressed as a relative amount in percentage, with the maximum amount in the time series being set to 100%.

[0103] Table 4

[0104]

[0105]

[0106] By adding composition 2 and 12 μg / ml Bifidobacterium longum fucosidase ( Figure 2 ), 12 μg / ml Bifidobacterium fucosidase ( Figure 3 )、2μg / ml Bifidobacterium longum fucosidase ( Figure 4 ) or 2 μg / ml Bifidobacterium fucosidase ( Figure 5 ) were incubated together, the compositions of the present invention including 2'-FL and 2'-FLO1 were easily hydrolyzed to release lactose and lactitol, respectively. In contrast, the control without added enzyme was stable, and there was no hydrolysis of 2'-FL or 2'-FLO1 ( Figure 6 ). Thus, the data demonstrate that fucosidases, which are normally present in the mammalian digestive tract, can hydrolyze 2'-FL and 2'-FLol to release lactose and lactitol, respectively.

[0107] To those of ordinary skill, after reading the above disclosure, other features, advantages and embodiments of the present invention disclosed herein will be apparent. In this regard, although specific embodiments of the present invention have been described in considerable detail, these embodiments may be changed and modified without departing from the spirit and scope of the present invention as described and claimed.

Claims

1. A prebiotic solid product comprising 2'-fucosyllactose and 0.5 to 6.0 wt. % of 2'-fucosyllactitol based on the total wt. % of the prebiotic solid product. 2 . The prebiotic solid product according to claim 1 , wherein the 2′-fucosyllactitol is present in the prebiotic solid product in an amount of 0.5 to 3.5 wt. %. 3 . The prebiotic solid product according to claim 1 , wherein the 2′-fucosyllactitol is present in the prebiotic solid product in an amount of 2.0 to 4.5 wt. %. 4 . The prebiotic solid product according to claim 1 , wherein the 2′-fucosyllactitol is present in the prebiotic solid product in an amount of 2.0 to 4.0 wt. %.

5. The prebiotic solid product according to claim 1, wherein 2'-fucosyllactose is present in the prebiotic solid product in an amount of 86.0 to 94.0 wt.%, based on the total wt.% of the prebiotic solid product. 6 . The prebiotic solid product according to claim 5 , wherein the ratio of 2′-fucosyllactose / 2′-fucosyllactitol in the prebiotic solid product is 14.3 to 470.

7. The prebiotic solid product according to claim 1, comprising 0.5 to 6.0 wt.% of 2'-fucosyllactitol, 86.0 to 94.0 wt.% of 2'-fucosyllactose and 0 to 13.5 wt.% of carbohydrate by-products based on the total wt.% of the prebiotic solid product.

8. The prebiotic solid product according to claim 1, comprising 0.5 to 3.5 wt.% of 2'-fucosyllactitol, 86.0 to 94.0 wt.% of 2'-fucosyllactose and 2.5 to 13.5 wt.% of carbohydrate by-products based on the total wt.% of the prebiotic solid product.

9. The prebiotic solid product of claim 1, comprising 2.0 to 4.5 wt.% of 2'-fucosyllactitol, 86.0 to 94.0 wt.% of 2'-fucosyllactose and 1.5 to 12.0 wt.% of carbohydrate by-products based on the total wt.% of the prebiotic solid product.

10. The prebiotic solid product of claim 1, comprising 2.0 to 4.0 wt.% of 2'-fucosyllactitol, 86.0 to 94.0 wt.% of 2'-fucosyllactose and 2.0 to 12.0 wt.% of carbohydrate by-products based on the total wt.% of the prebiotic solid product.

11. The prebiotic solid product according to any one of claims 7 to 10, wherein the ratio of 2'-fucosyllactose / 2'-fucosyllactitol in the prebiotic solid product is 14.3 to 470.

12. A method for producing a food or a medicine, wherein the method comprises: a. Providing a prebiotic solid product according to any one of claims 1-11; and b. Mixing the prebiotic solid product with one or more ingredients suitable for food or medicine.

13. The method of claim 12, wherein the food product is a growing-up milk or a dietary supplement.

14. A method for producing a food or a medicine, wherein the method comprises: a. Providing a prebiotic solid product according to any one of claims 1-11; b. dissolving the prebiotic solid product in a solvent; and c. Mixing the dissolved prebiotic solid product with one or more ingredients suitable for manufacturing a food or a medicine.

15. The method of claim 14, wherein the food product is a growing-up milk or a dietary supplement.

16. A method for producing infant formula, wherein the method comprises: a. Providing a prebiotic solid product according to any one of claims 1-11; and b. Mixing the prebiotic solid product with at least one infant formula ingredient.

17. A method for producing infant formula, wherein the method comprises: a. Providing a prebiotic solid product according to any one of claims 1-11; and b. dissolving the prebiotic solid product in a solvent; and c. mixing the dissolved prebiotic solid product with at least one infant formula ingredient.

18. A food or medicine obtained by the method according to claim 12 or 14.

19. The food or drug according to claim 18, wherein the food is a growing-up milk or a dietary supplement.

20. An infant formula obtained by the method according to claim 16 or 17.

21. The method of claim 14 or 17, wherein the solvent is water.

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