Method of using a b-hexosyltransferase enzyme to make an n-acetyllactosamine (lacnac)-enriched galactooligosaccharide (GOS) composition

Abstract

Provided herein are methods for producing galactooligosaccharide (GOS) compositions from a liquid milk product using a cell-free β-hexosyltransferase, or active fragment thereof. The methods may comprise adding N-acetylglucosamine (GlcNAc) to aid in production of a GOS composition comprising the human milk oligosaccharide (HMO) N- acetyllactosamine (LacNAc) and GOS with LacNAc. Also disclosed are compositions useful for producing GOS compositions with and without LacNAc and food and beverage products comprising the same.

Description

[0001] METHOD OF USING A B-HEXOSYLTRANSFERASE ENZYME TO MAKE AN N- ACETYLLACTOSAMINE (LACNAC)-ENRICHED

[0002] GALACTOOLIGOSACCHARIDE (GOS) COMPOSITION

[0003] FIELD OF THE INVENTION

[0004] The present disclosure relates generally to the fields of galactooligosaccharide (GOS) compositions and methods of their preparation from liquid milk products. More specifically, the present disclosure provides methods and compositions for converting lactose in unprocessed, liquid milk into GOS using a P-hexosyltransferase. In particular, this disclosure provides methods and compositions related to production of GOS compositions with and without the human milk oligosaccharide (HMO) N-acetyllactosamine (LacNAc).

[0005] BACKGROUND OF THE INVENTION

[0006] A subject of the present disclosure is the preparation of galactooligosaccharide(s) (GOS) compositions from milk products. The milk products are preferably liquid milk products that have not undergone a processing step, such as unprocessed, liquid milk, and contain natural levels of lactose that can be converted to GOS by directly utilizing the liquid milk product in unprocessed form. The methods disclosed in the present application involve the use of P-hexosyltransferase (BHT) to convert lactose from milk products into GOS, optionally, in the presence of GlcNAc to produce LacNAc, such as GOS with LacNAc.

[0007] GOS are considered one of the preferred choices of prebiotics and in the gastrointestinal tract. GOS are resistant to enzymes and transit though the small intestine without being digested, but in the large intestine GOS are fermented and can activate growth of intestinal bifidobacteria such as Lactobacillus acidophilus and L. casei, hence acting as a prebiotic.

[0008] GOS are non-digestible oligosaccharides owing to the conformation of their anomeric C atom (Cl or C2), which allows their glycosidic bonds to evade hydrolysis by digestive enzymes in the stomach or small intestine. Free oligosaccharides are found in the milk of all placental mammals, providing a natural example of prebiotic feeding during infancy. According to the latest definition by the International Scientific Association for Probiotics and Prebiotics (ISAPP) “a dietary prebiotic is a selectively fermented ingredient that results in specific changes in the composition and / or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health.” The composition of human milk oligosaccharides (HMO) is very complex, which makes it unlikely to find alternative sources containing oligosaccharides of analogous composition. Improved colonic health among breastfed infants has been attributed to the presence of GOS in the mother's milk. In fact, infant formula with added GOS replicated the bifidogenic effect of the human milk with respect to metabolic activity of colonic microbiota and bacterial numbers. Among non-milk oligosaccharides, GOS are of special interest as their structure resembles the core molecules of HMOs. However, GOS concentration and composition vary with the method and the enzyme utilized for their generation, which in turn may influence their prebiotic effects and the proliferation of colonic probiotic strains. Traditionally, GOS have been produced using p-galactosidases from mesophilic microorganisms. Mesophilic P-galactosidases require high initial concentrations of lactose to drive the reaction away from lactose hydrolysis and towards GOS synthesis. Since lactose is more soluble at elevated temperatures, thermostable P-galactosidases exhibiting high initial velocities and increased halflives have been utilized to reach a favorable equilibrium for the transgalactosylation reaction. However, competitive inhibition by glucose and / or galactose is another obstacle that remains which may be overcome by incorporating cells in the reaction.

[0009] The prebiotic LacNAc is regarded as one of the most important building blocks for higher order HMO generation. However, feasible industrial production routes by chemical synthesis suffer from low yields, thus favoring the biocatalysis of LacNAc. The major difference between other biosynthesis routes and the biological synthesis of LacNAc with the enzyme BHT, is lower cost and higher purity. Embodiments of the present disclosure demonstrate that GOS and LacNAc production by BHT as described herein is more suitable for industrial scale when compared with other processes. LacNAc can be generated by mixing lactose, including lactose contained in a milk product, such as fresh, unprocessed milk, or processed milk, and GlcNAc with BHT. The present disclosure provides methods of using cell-free BHT for producing LacNAc, LacNAc -enriched GOS, and related GOS compositions without LacNAc. The methods and compositions disclosed herein for producing GOS compositions, including GOS compositions comprising the HMO LacNAc, using cell- free BHT in milk, such as unprocessed, whole milk, provides a much needed and improved method for producing GOS and LacNAc without the requirement of using a milk product that has been processed. As BHT optimally performs in the presence of wide ranging lactose concentrations - minimal and high lactose concentrations - the milk products do not require any processing prior to being used in the methods disclosed herein. As such, the methods and compositions disclosed herein provide a more efficient and effective method over those currently known in the art of producing GOS compositions with and without the HMO LacNAc from dairy products, such as milk. The improved methods and compositions disclosed herein are useful for generating food and beverage products comprising GOS with and with LacNAc, including fermented or unfermented dairy products. Also disclosed herein are food and beverage products comprising GOS compositions described in the present application, including dairy products such as yogurt, yogurt-based, and / or yogurt-containing products.

[0010] SUMMARY OF THE INVENTION

[0011] Provided herein are methods of generating a galactooligosaccharide (GOS) composition from a milk product, said method comprising contacting said milk product with a starter culture composition comprising a plurality of microbial cells, and / or cell constituents, contacting said milk product with a cell-free p-hexosyltransferase, or active fragment thereof, and heating said milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition.

[0012] In some instances, the method further comprises the step of contacting said milk product with N-acetylglucosamine (GlcNAc) prior to heating said milk product.

[0013] In some instances of the methods disclosed herein, said GOS composition comprises N- acetyllactosamine (LacNAc).

[0014] In some instances, said milk product is non-human, mammal milk, such as cow milk or goat milk.

[0015] In some instances, wherein said milk is unprocessed, raw milk. In some instances, said milk is modified and / or processed milk. In some instances, said milk is pasteurized milk. In some instances, said milk is skimmed or semi-skimmed milk. In some instances, said milk product comprises between 2% and 50% (w / v) lactose.

[0016] In some instances, said plurality of microbial cells comprises at least one lactic acid bacteria.

[0017] In some instances, said P-hexosyltransferase enzyme, or active fragment thereof, catalyzes the addition of galactose to GlcNAc.

[0018] In some instances, said P-hexosyltransferase, or active fragment thereof, is added to said milk product at a concentration of between 0.5 U / g and 5.0 U / g of lactose. In some instances, said heating step results in fermentation of said milk product. In some instances, the pH of the milk product prior to heating in step (c) is between pH 6.5 and pH 7.0.

[0019] In some instances, said milk product is maintained at a temperature between 40°C-44°C until the pH of the GOS composition is at or below pH 4.8, or at or below pH 4.6.

[0020] In some instances, said milk product is maintained at a temperature between 40°C-44°C until lactose content in said GOS composition is reduced between 20% and 99% compared to lactose content in said milk product.

[0021] In some instances, the method further comprises the step of cooling the GOS composition to a temperature of about 4°C.

[0022] In some instances, the method further comprises the step of contacting said milk product with a lactose hydrolyzing enzyme, or active fragment thereof.

[0023] In some instances of the methods disclosed herein, said lactose hydrolyzing enzyme, or active fragment thereof, is contacted with said milk product prior to heating in step (c).

[0024] In some instances, said lactose hydrolyzing enzyme, or active fragment thereof, comprises a P-galactosidase, or active fragment thereof.

[0025] In some instances, said method comprises the addition of a -hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, resulting in greater efficacy of lactose reduction compared to said method when performed without addition of a P-hexosyltransferase, or active fragment thereof.

[0026] In some instances, said method comprises the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, wherein GOS levels are decreased no more than 1% during said heating step.

[0027] In some instances, said method further comprises maintaining the GOS composition at a temperature of no higher than about 4°C for 1-60 days.

[0028] In some instances, the pH of said GOS composition is no higher than pH 4.6 during storage at 4°C; optionally, wherein the pH is maintained at between pH 3.5 and 4.6.

[0029] In some instances, said method comprises adding to said milk product and / or said GOS composition at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder.

[0030] In some instances, said GOS composition comprises a food or beverage product, such as a dairy-based food or a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula.

[0031] Also disclosed herein are milk-based compositions, wherein said composition comprises: a liquid milk product; a plurality of microbial cells, and / or cell constituents; and a cell-free P- hexosyltransferase enzyme, or active fragment thereof, wherein the pH of said composition is between pH 6.5 and pH 7.0.

[0032] In some instances, said composition further comprises N-acetylglucosamine (GlcNAc).

[0033] In some instances, said milk product is non-human, mammal milk, such as cow milk or goat milk.

[0034] In some instances, said milk is unprocessed, raw milk. In some instances, said milk is modified and / or processed milk. In some instances, said milk is a pasteurized milk. In some instances, said milk is skimmed or semi-skimmed milk.

[0035] In some instances, said plurality of microbial cells comprises at least one lactic acid bacteria.

[0036] In some instances, the milk-based composition further comprises a lactose hydrolyzing enzyme, such as a -galactosidase, or active fragment thereof.

[0037] In some instances, said -hexosyltransferase enzyme and / or said lactose hydrolyzing enzyme, or active fragment(s) thereof, is present at a concentration of 2.5 U / g or below.

[0038] In some instances, said composition comprises a galactooligosaccharide (GOS) content of 0.5% (w / v) or below.

[0039] Also disclosed herein are galactooligosaccharide (GOS) compositions, wherein said GOS composition comprises: N-acetyllactosamine (LacN Ac) -containing GOS, and / or GOS without LacNAc; and a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is between pH 3.4 and pH 7.0.

[0040] In some instances, said composition further comprises N-acetylglucosamine (GlcNAc).

[0041] In some instances, said plurality of microbial cells comprises at least one lactic acid bacteria.

[0042] In some instances, said composition comprises a P-hexosyltransferase, or active fragment thereof. In some instances, said composition comprises a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof.

[0043] In some instances, said composition comprises lactose in the range of between 0.5% and 10% (w / v). In some instances, said composition comprises glucose in the range of between 0.001% and 1% (w / v). In some instances, said composition comprises galactose in the range of between 0.001% and 1.5% (w / v). Also disclosed herein are galactooligosaccharide (GOS) compositions, wherein said GOS composition comprises: N-acetyllactosamine (LacNAc)-enriched GOS and / or GOS without LacNAc; and a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is pH 4.8 or less than pH 4.8.

[0044] In some instances, the pH of said composition is between pH 3.4 and pH 4.8.

[0045] In some instances, said plurality of microbial cells comprises at least one lactic acid bacteria.

[0046] In some instances, said composition comprises a -hexosyltransferase, or active fragment thereof. In some instances, said composition comprises a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof.

[0047] In some instances, said composition comprises lactose in the range of between 0.5% and 3% (w / v). In some instances, said composition comprises glucose in the range of between 0.6% and 1% (w / v). In some instances, said composition comprises galactose in the range of between 0.6% and 1.5% (w / v).

[0048] In some instances of the milk-based compositions or GOS compositions disclosed herein, said composition further comprises at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable. In some instances, said composition is in the form of a liquid, solid, semi-solid, or powder.

[0049] Also disclosed herein are food or beverage products comprising any of the milk-based compositions or GOS compositions disclosed herein.

[0050] In some instances, said food or beverage product comprises a dairy-based food, such as a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is a graph depicting GOS composition production (i.e., yogurt production) from an unprocessed, liquid milk product (i.e., whole milk) using a P-hexosyltransferase and a starter culture composition. Fiber accumulation, lactose reduction, pH reduction and glucose accumulation are shown for GOS production using multiple concentrations of BHT at 42°C for 24 hours, followed by a shift to 4°C. BHT concentration is presented as U / g lactose, as indicated.

[0053] FIG. 2 is a graph depicting GOS composition production (i.e., yogurt production) from an unprocessed, liquid milk product (i.e., whole milk) using a P-hexosyltransferase and P-galactosidase and a starter culture composition. Fiber accumulation, lactose reduction, galactose accumulation and glucose accumulation are shown for GOS production using multiple concentrations of BHT at 42°C for 8 hours, followed by a shift to 4°C. BHT concentration is presented as U / g lactose, as indicated.

[0054] FIG. 3 is a graph depicting GOS composition production (i.e., yogurt production) from an unprocessed, liquid milk product (i.e., whole milk) using P-galactosidase only with a starter culture composition or p-galactosidase and a P-hexosyltransferase and a starter culture composition. Fiber accumulation, lactose reduction, galactose accumulation and glucose accumulation are shown for GOS production at 42°C for 8 hours or 24 hours, followed by a shift to 4°C. Enzymes and time of incubation are as indicated.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

[0057] I. Definitions

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0059] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Further, the term “a plant” may include a plurality of plants.

[0060] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0061] The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range. The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0062] The term “cell-free”, for the purposes of this disclosure, refers to soluble, functional and / or secreted-form of a protein or polypeptide, such as a P- hexosyltransferase enzyme, or active fragment thereof. One of skill in the art would understand that a cell-free, soluble protein can include an N-terminal signal sequence, which is generally a hydrophobic sequence that mediates insertion of the protein through the membrane of the endoplasmic reticulum (ER) in a eukaryotic cell. A cell- free protein is capable of being expressed and purified and can be stably isolated outside of a host cell, while maintaining its functional capacity. In some embodiments, a cell-free P-hexosyltransferase, or active fragment thereof, can be expressed and isolated using a host cell system and subsequently purified for use in the methods disclosed herein. For example, a cell free P-hexosyltransferase, or active fragment thereof, can be added to a milk product in its isolated and functional form.

[0063] As used herein, the term “dairy products”, “dairy-based products” or “dairy compositions” are those comprising one or more milk components.

[0064] “Fermentation”, for the purposes of this disclosure, is a change in the chemical or physical composition of a substance through the use of microorganisms (e.g., a plurality of microbial cells). For instance, and without limitation, fermentation (also referred to in this disclosure as a “fermentation process” or “fermenting”) may include the breakdown of an organic substrate by or using microorganisms such as, but not limited to, bacteria or yeast. In some embodiments of the method disclosed herein, fermentation includes the use of lactic acid fermentation. For example, and without limitation, lactic acid fermentation may comprise microorganisms converting glucose, or other six-carbon sugars, into lactic acid. In some embodiments, fermentation comprises an aerobic process, wherein the aerobic process comprises a process occurring in the presence of oxygen. In some embodiments, fermentation comprises an anaerobic process, wherein the anaerobic process includes a process occurring in the absence of oxygen. In some embodiments, fermentation may include the use of a substrate, such as an organic substrate, such as a milk product, and one or more microorganisms, such as a plurality of microorganisms. In some embodiments, microorganisms used for fermentation include yeast, lactic acid bacteria, acetic acid bacteria, butyric acid bacteria, propionic acid bacteria, or combinations thereof.

[0065] “Fermented substrate”, for the purposes of this disclosure, is a substrate that has undergone the fermentation process through, inter alia, the introduction of a microorganism, or a plurality of microbial cells. In some embodiments, a microorganism, or a plurality of microbial cells, includes lactic acid bacteria, or any other bacteria or microorganisms described herein, which may be used to ferment a substrate, such as a milkbased substrate, including a milk product, such as milk, to create a fermented substrate, such as a fermented milk product. In some embodiments, a fermented milk product can be yogurt. In some embodiments, lactic acid bacteria are used to ferment one or more milk products, including non-mammalian milk products, such as fresh, unprocessed milk. In some embodiments, lactic acid bacteria are used to ferment non-mammalian milk products, such as processed milk, including skimmed, semi-skimmed, and / or pasteurized milk. Fermentation may be used for the generation of one or more fermented milk products, such as yogurt. In some embodiments, a fermented substrate comprises a plurality of microbial cells, such as one or more microbial cells, or cell constituent(s), and / or compounds produced or derived therefrom during fermentation, such as, but not limited to, at least one microbial cell wall fragment, cell membrane fragment, exopolysaccharide, cell-wall anchored protein, pili, extracellular membrane vesicle(s), genetic material, or any other component derived from a microbial cell. The cell constituent and / or compound can also include a secreted microbial protein, metabolite, bioactive compound, short chain fatty acids, butyrate, anti-inflammatory metabolites, antibiotics, various B vitamins, such as Vitamin B 12 and riboflavin, alcohols, acetic monosodium glutamate, amino acids, and / or organic acids.

[0066] As used herein, the term “microorganism” refers to bacteria or yeast or other microscopic organism (i.e., microorganism). A microorganism will be understood to be a single-celled or multi-cellular organism. Microorganisms are very diverse group of organisms and include bacteria, archaea, protozoa, fungi, and algae, especially cells of plant pathogens and / or plant symbionts. Microorganisms include prokaryotic, such as, bacteria, and eukaryotic organisms, such as, yeast. A “microbe” will be understood to be a microorganism, i.e. a microscopic organism, which can be single celled or multicellular. Microorganisms are very diverse and include all the bacteria, archaea, protozoa, fungi, and algae, especially cells of plant pathogens and / or plant symbionts. Certain animals are also considered microbes, e.g. rotifers. The terms “microorganism” and “microbe” are used interchangeably herein.

[0067] The term “inoculation” as used herein refers to the introduction of an additive, such as a microorganism (or plurality of microbial cells), into a substrate or composition, including for example, introduction of a starter culture composition into a milk product. This may include placing the substrate in the presence of one or more microorganisms. Inoculation may include placing the substrate within a liquid containing one or more microorganisms or placing the microorganisms within a composition containing a substrate. For example, and without limitation, inoculation may include placing a microbial cell, or plurality of microbial cells, into a liquid solution containing a substrate, such as, for example, a milk product which includes a liquid milk product. In some embodiments, inoculation comprises placing a microbial starter culture composition, comprising a plurality of microbial cells, into a milk product, wherein said milk product comprises a liquid milk product, such as unprocessed or processed milk. The inoculation of a starter culture composition into said milk product can serve to generate a fermented milk product after fermentation of the milk product.

[0068] The term “milk” and “milk product” refers to any non-human, mammal milk and / or product derived from non-human, mammal milk comprising lactose. Some examples include cow milk or goat milk, or processed milk products derived therefrom. In some instances, milk can include fresh, unprocessed cow or goat milk that has or has not been maintained at a proper temperature (e.g., around 4°C) to prevent, for example, microbial outgrowth. Additional examples include milk that has been processed and / or pasteurized, such as ultra-pasteurized milk products, and / or skimmed, or semi-skimmed milk. Milk can include fat-free milk, low fat milk, full fat milk, concentrated milk, dry milk, evaporated milk, powdered milk. Fat-free milk is a nonfat or skim milk product. Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat. Full fat milk often contains about 3.25% fat. Milk can include any animal milk comprising lactose. Animal sources of milk include, but are not limited to, human, cow, sheep, goat, buffalo, camel, llama, mare, and deer.

[0069] A “substrate” for the purposes of this disclosure is a substance in which an organism obtains its nourishment. For example, a substrate may include a particular food wherein one or more microorganisms may metabolize the food during a fermentation process. Fermentation may include the supervision and controlling of one or more factors, such as pH levels, moisture levels, temperature levels, and / or oxygen levels that are associated with the substrate and the microorganisms.

[0070] Various embodiments of this disclosure may be presented in a range format. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.

[0071] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” or “is between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. The recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ^0 and =2 if the variable is inherently continuous.

[0072] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

[0073] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “increased”, “increase”, and the like encompass both a partial reduction and a significant increase compared to a control.

[0074] As used herein, the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0075] A “nutritional supplement”, for the purposes of this disclosure, is an edible substance intended to provide nutritional value to a subject when consumed.

[0076] As used herein, “w / v” refers to the weight present within a total volume (i.e., “weight / volume”, or “weight per volume”). For example, “w / v” can refer to, and be presented as, grams per 100 milliliters (i.e., grams per 0.1 L), such as the amount, in grams, of a substance, including, but not limited to, lactose, glucose, galactose, or GOS, present within a volume of a composition, such as a milk-based composition or a GOS composition. In some instances, “w / v” is presented as a numerical value, such as a numerical percentage. As used herein, 1% (w / v) refers to 1 g / 100 mb. The amount of a substance present within a volume can be determined by any suitable means available in the art, such as liquid chromatography or any method adapted therefrom, or utilizing liquid chromatography.

[0077] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0078] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0079] II. Overview of the Invention

[0080] Embodiments of the present disclosure include methods of generating compositions comprising GOS (“GOS compositions”) from a milk product using - hexosyltransferase (BHT) proteins to convert lactose to GOS, as described herein. BHT exhibits the ability to catalyze the hydrolysis of P~( 1 -4) glycosidic linkages to generate GOS composition(s) from lactose, including, but not limited to, GOS compositions with or without GlcNAc, as well as compositions comprising GOS with and without LacNAc, such as LacNAc -enriched GOS compositions that are generated in the presence of N- acetylglucosamine (GlcNAc). Certain p-hexosyltransferases, such as that of the basidiomycete yeast Sporobolomyces singularis (formerly Bullera singularis) are known to have transgalactosylation activity even at low lactose concentrations and very limited lactose hydrolysis. This variant of BHT in particular does not appear to be inhibited by lactose concentrations above 20%. Unlike, P-galactosidases, the BHT from .S', singularis simultaneously carries out glycosyl-hydrolase and P- hexosyltransferase activities, converting lactose to GOS without extracellular accumulation of galactose. Two molecules of lactose are required during the transgalactosylation event: one molecule is hydrolyzed and the second acts as galactose acceptor, generating the trisaccharide galactosyl-lactose (P-D-Gal(l-4)-p-D- Gal(l-4)-p-D-Glc) and residual glucose. Galactosyl-lactose can also act as acceptor of a new galactose to generate the tetrasaccharide galactosylgalactosyl -lactose (P-D-Gal(l-4)-p-D- Gal(l-4)-p-D-Gal(l-4)-p-D-Glc), and similarly for the tetrasaccharide and subsequent products. The tri, tetra, and penta saccharides accumulating in .S', singularis have been collectively designated GOS. As would be recognized by one of ordinary skill in the art based on the present disclosure, GOS generally refers to a galactose-containing polysaccharide with two or more sugar units such as Gal -Gal (Gal; galactose) or [Gal]n-Glc (l<n<8)(Glc; glucose), including -D-Gal( H4)- -D-Gal( H4)- -D-Glc, -D-Gal( H4)- -D-Gal(l - 4)- -D-Gal( H4)- 0 -D-Glc, and 0 -D-Gal( H4)- 0 -D-Gal( H4)- 0 -D-Gal( H4)- 0 - D-Gal( H4)- 13 -D-Glc. The use of any BHT, or active fragment thereof, capable of converting lactose into GOS is envisaged within the scope of the present disclosure.

[0081] Accordingly, “BHT activity”, as used herein, refers to the ability of a protein, or activity fragment thereof, to convert lactose to GOS. The BHT protein, or active fragment thereof, can be a .S', singularis protein, such as a BHT protein isolated from .S', singularis. Any variant of a .S', singularis BHT protein is envisaged for use in the methods and compositions disclosed herein, such as a BHT with at least 99%, at least 90%, at least 85%, or at least 80% sequence identity to a .S', singularis BHT. In some embodiments, the BHT having at least 99%, at least 90%, at least 85%, or at least 80% sequence identity to a .S', singularis BHT, can be derived from a .S', singularis BHT. The GOS produced using the BHT polypeptides can include one or more N- acetyllactosamine (LacNAc) units, such as by the addition ofN-acetylglucosamine (GlcNAc) during the production of GOS from a milk product in the presence of BHT. The production of LacNAc-containing, or LacNAc-enriched, GOS by the addition of GlcNAc during GOS production is envisaged for any of the methods of GOS production disclosed herein. For example, GlcNAc can be added to a composition comprising a milk product, a starter culture composition comprising a plurality of cells and / or cell constituents, BHT, such as cell-free BHT, and optionally further comprising a lactose hydrolyzing enzyme, such as a P-galactosidase. For example, the GlcNAc can be added to a milk product mixture (e.g., a milk product mixture comprising a milk product, a starter culture composition comprising a plurality of cells and / or cell constituents, BHT, such as cell-free BHT, and, optionally, P-galactosidase) prior to heating of the milk product, or milk product mixture. GOS can be produced by incubating a BHT polypeptide in a milk product, or milk product composition (e.g., a mixture comprising a milk product and a plurality of microbial cells and / or cell constituents), that comprises a disaccharide substrate such as for example lactose. The BHT, or active fragment thereof, of the present disclosure are useful for producing LacNAc-containing GOS (i.e., LacNAc-enriched GOS), and related GOS compositions. For purposes of the present disclosure, the term “LacNAc-enriched GOS” is used to describe GOS with LacNAc produced by the addition of BHT and GlcNAc to a milk product, according to the methods described herein. In some embodiments, the BHT of the present disclosure can be used for producing GOS compositions that do not include the addition of N-acetylglucosamine (GlcNAc). For example, in the absence of GlcNAc, BHT is useful for the production of GOS without, and not enriched with LacNAc. The addition of BHT and GlcNAc to the milk product is required to generate the LacNAc-enriched GOS of the present disclosure. Specifically, the addition of BHT and GlcNAc to a milk product generates a LacNAc-enriched GOS composition and results in higher levels of LacNAc than those generated, or present in GOS compositions generated, from a similar method performed using a milk product in the absence of BHT and GlcNAc as described herein.

[0082] Embodiments of the present disclosure further provide compositions related to the production of galactooligosaccharide (GOS) compositions from a milk product using a hexosyltransferase enzyme, such as a P-hexosyltransferase (BHT), or active fragment thereof. The GOS compositions disclosed herein comprise reduced lactose content and are generated from a milk product, such as a liquid milk product in a milk-based composition. Also disclosed are GOS compositions comprising the human milk oligosaccharide (HMO) N-acetyllactosamine (LacNAc). In some embodiments, the GOS compositions comprise N-acetyllactosamine (LacNAc)-enriched GOS. The compositions can include N-acetylglucosamine (GlcNAc) and a BHT added to a milk product to promote the reduction of lactose and production of GOS and, optionally, GOS comprising LacNAc. The compositions can also include a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof. The milk-based compositions and GOS compositions generated therefrom can have a pH ranging between 7.0-3.4, or below, due to the presence of a starter culture composition comprising a plurality of microbial cells that aids in fermentation of the milk product.

[0083] As would be recognized by one of ordinary skill in the art based on the present disclosure, P-hexosyltransferase proteins and polypeptides, or BHT proteins, includes full length BHT proteins and any fragments and / or variants thereof, which includes proteins encoded by naturally-occurring allelic variants of the BHT gene, as well as recombinantly- produced BHT proteins, which may contain some sequence changes relative to naturally- occurring BHT proteins. A recombinant protein can be a protein that results from the process of genetic engineering, which generally involves use of a corresponding recombinant nucleic acid molecule encoding the peptide that is inserted into an engineered host cell in order to express the nucleic acid molecule and the corresponding peptide. That is, the host cell has been transfected, transformed or transduced with a recombinant polynucleotide molecule, and thereby altered so as to cause the cell to express the desired polypeptide (e.g., rBHT). Examples of recombinantly produced BHT enzymes that are functional and can be used as a cell-free P-hexosyltransferase in the methods and compositions disclosed herein are thoroughly described in WO 2021 / 236664 and U.S. Patent No. 9,783,789, the contents of which are incorporated herein by reference in their entirety. For the purposes of this disclosure, “P-hexosyltransferase”, “BHT”, and “a BHT” are used interchangeably.

[0084] In particular, the present disclosure provides compositions and methods for converting lactose and N-acetylglucosamine (GlcNAc) from a milk product, such as raw, unprocessed liquid milk, into galactooligosaccharide (GOS) compositions, such as N- acetyllactosamine (LacNAc) -enriched GOS compositions, using BHT. III. Methods of Producing GOS Compositions with and without N-acetyllactosamine (LacNAc)

[0085] Disclosed herein are methods of producing galactooligosaccharide (GOS) compositions from a milk product using a hexosyltransferase enzyme, such as a P- hexosyltransferase (BHT). BHT functions in the presence of both low and high concentrations of lactose, and surprisingly can be added directly into a raw, unprocessed milk product to convert lactose (i.e., reduce lactose content) and generate a GOS composition. Adding, inter alia, BHT, directly into a raw and / or unprocessed milk product to contact said milk product, and convert lactose within the milk product, allows for rapid production of GOS, and food and beverage products, such as fermented or unfermented dairy products comprising the same. Elimination of the requirement for using a processed product results in a faster, more efficient method of generating GOS compositions. The milk product, such as a liquid milk product comprising fresh, unprocessed, or processed, milk, comprising lactose, can be included in a milk-based composition, further comprising a starter culture composition comprising a plurality of microbial cells and / or cell constituents.

[0086] The methods of making a GOS composition disclosed herein comprise contacting a milk product with a starter culture composition and a cell-free BHT, or active fragment thereof, (e.g., a milk-based composition) and heating the milk product, or milk-based composition comprising the milk product that has been contacted and mixed with a starter culture composition and a cell-free BHT, to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition. The starter culture compositions that are contacted with the milk product comprise a plurality of microbial cells, and / or cell constituents, that aid in fermentation of the milk product during GOS composition production, when the milk product, or milk-based composition is heated. During fermentation, the pH of the milk-based composition, and GOS composition resulting from the disclosed methods, will decrease, resulting in a GOS composition with a lower pH compared to the initial milk product and milk-based composition. In some instances, the pH of the milkbased composition, comprising a milk product, is between pH 6.5-7.0. In some instances, the GOS composition has a pH between 3.4-7.0. In some instances, the GOS composition has a pH at or below pH 4.8, such as around pH 4.6. Once produced, the GOS compositions can be cooled and stored at a temperature of around 4°C for up to 60 days while maintaining stable GOS levels within the composition. In some embodiments of the methods of generating a GOS composition from a milk product, N-acetylglucosamine (GlcNAc) is added to a milk product or milk-based composition comprising a liquid milk product to aid in production of a GOS composition comprising the human milk oligosaccharide (HMO) N-acetyllactosamine (LacNAc)- containing, or LacNAc-enriched, GOS. The amount of GlcNAc added to generate LacNAc can vary depending on not only the desired LacNAc output, but also factors such as the amount of lactose present. In preferred embodiments, the amount of GlcNAc added to the composition is dependent on the initial amount of lactose in the composition. Specifically, the amount of GlcNAc added to a milk product is determined based on the amount of lactose in said milk product. For example, to ensure the most effective production of LacNAc, the amount of GlcNAc added is calculated based on the amount of lactose present (i.e., lactose content) in the milk product or milk-based composition. In preferred embodiments of the methods for producing LacNAc-containing GOS, GlcNAc is added at a ratio of 1:8 with lactose (1:8 GlcNAc -to-lactose ratio).

[0087] In some embodiments, the BHT, or active fragment thereof, of the present disclosure are useful for producing LacNAc, LacNAc-containing GOS (i.e., LacNAc-enriched GOS), and related GOS compositions. The terms “P-hexosyltransferase”, “BHT”, p- hexosyltransferase enzyme”, “P-hexosyltransferase polypeptide”, “P-hexosyl-transferase” are used interchangeably herein. It will be readily appreciated by those of skilled in the art that an active fragment of a P-hexosyltransferase would function (i.e., retain p-hexosyltransferase activity) as a full-length, functional P-hexosyltransferase polypeptide or enzyme, and thus would be able to be readily interchangeable, unless specifically and / or otherwise noted. “BHT activity”, as used herein, refers to the ability of a protein, or activity fragment thereof, to convert lactose to GOS. The use of any BHT, or active fragment thereof, capable of converting lactose into GOS is envisaged within the scope of the present disclosure. Therefore, a skilled artisan, unless specifically instructed for the purposes of this disclosure, would not distinguish between a P-hexosyltransferase and an active fragment thereof. For the purposes of this disclosure, any reference to “P-hexosyltransferase” also includes any and all active fragments thereof, which can be readily interchanged with a full-length enzyme.

[0088] In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a total GOS concentration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 75% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a total GOS concentration of between 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 65-70%, 70-80%, or 75% or more of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 10% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 20% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 30% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 40% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 50% of initial lactose concentration. The method can also produce a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 60% of initial lactose concentration. In some embodiments, the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 70% of initial lactose concentration. In some embodiments, the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 75% of initial lactose concentration. For example, using an initial lactose-to-GlcNAc ratio of 1:8, the methods provided herein using BHT with about 50-100 g lactose and about 6.25- 12.5 g GlcNAc generate about 6.25g-12.5 g of LacNAc and about 12.5-25 g GOS. Initial lactose-to-GlcNAc ratios can range from about 1:20 to about 20: 1. For example, lactose -to-GlcN Ac ratios can be between 1:20-1: 10 lactose-to-GlcNAc, 1: 15-1:5 lactose -to-GlcN Ac, 1: 10-1: 1 lactose-to-GlcNAc, 1:5-5: 1 lactose-to-GlcNAc, 1: 1-10: 1 lactose -to-GlcN Ac, 5: 1-15: 1 lactose-to-GlcNAc, or 10: 1-20: 1 lactose-to-GlcNAc. In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 20% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 30% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 40% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 10% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 50% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 60% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N- acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 70% of initial lactose concentration. In some embodiments of the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc -enriched GOS yield of at least 80% of initial lactose concentration. For example, the method of generating a GOS composition comprising N-acetyllactosamine (LacNAc), the method produces a LacNAc-enriched GOS yield of between 5-15%, 10-20%, 15-25%, 20- 30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 75-85%, or great than 85% of initial lactose concentration.

[0089] In some embodiments, the BHT, or active fragment thereof, of the present disclosure are useful for producing LacNAc, and LacNAc-containing GOS (i.e., LacNAc-enriched GOS), and related GOS compositions.

[0090] In some embodiments, the BHT of the present disclosure can be used for producing GOS compositions that do not include N-acetylglucosamine (GlcNAc). Embodiments of the present disclosure includes materials and methods for producing GOS compositions lacking GlcNAc, which include reacting lactose, such as from a milk product, such as unprocessed or processed milk, with BHT polypeptides having the amino acid sequences provided herein under suitable conditions so as to produce GOS. Similar compositions and methods are described in related U.S. Pat. Nos. 10,513,695, and 9,783,789, both of which are herein incorporated by reference. As would be recognized by one of ordinary skill in the art based on the present disclosure, p-hexosyltransferase proteins, or BHT proteins, includes full length BHT proteins and any fragments and / or variants thereof, which includes proteins encoded by naturally-occurring allelic variants of the BHT gene, as well as recombinantly-produced BHT proteins, which may contain some sequence changes relative to naturally-occurring BHT proteins. A recombinant protein can be a protein that results from the process of genetic engineering, which generally involves use of a corresponding recombinant nucleic acid molecule encoding the peptide that is inserted into an engineered host cell in order to express the nucleic acid molecule and the corresponding peptide. That is, the host cell has been transfected, transformed or transduced with a recombinant polynucleotide molecule, and thereby altered so as to cause the cell to express the desired polypeptide (e.g., rBHT).

[0091] Examples of recombinantly produced BHT enzymes that are functional and can be used as a cell-free P-hexosyltransferase in the methods and compositions disclosed in the present application are thoroughly described in US 2023 / 0279368, the contents of which are incorporated herein by reference in their entirety.

[0092] Disclosed herein are methods of generating a galactooligosaccharide (GOS) composition from a milk product, wherein the method comprises: contacting said milk product with a starter culture composition comprising a plurality of microbial cells, and / or cell constituents; contacting said milk product with a cell-free P- hexosyltransferase (BHT), or active fragment thereof; and heating said milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition. Milk Product

[0093] The milk product used in the methods disclosed herein can comprise a liquid milk product, such as non-human, mammal liquid milk. Any form of non-human, mammal milk is envisaged for use as a liquid milk product within the milk-based composition. Examples of non-human, mammal liquid milk that can be used include cow milk and goat milk. Any form of non-human, mammal milk is envisaged for use as milk product for the methods of generating a GOS composition disclosed herein. In some instances, the milk is unprocessed, raw milk. Raw milk may have been stored at a temperature necessary to prevent microbial outgrowth (e.g., a temperature of around 4°C) prior to use in the methods disclosed herein. Alternatively, the milk can be modified and / or processed milk. For example, the milk can be a pasteurized milk product, that has undergone a pasteurization. Additionally, the liquid milk product can also be processed such that the milk is skimmed or semi-skimmed milk. The methods disclosed herein utilize BHT to produce GOS and reduce lactose from an initial milk product. The milk product used for the methods described herein can comprise a wide-ranging concentration of lactose. Specifically, the milk product used for the methods of producing GOS compositions, as described herein, can comprise between 2% (w / v) and 50% (w / v) lactose. BHT is unique to other commercially available enzymes in that it optimally performs in both minimal (around 2%) and higher concentration ranges (approximately 50%) without pre-processing the milk product. The milk products used in the methods disclosed herein to generate GOS compositions comprise between 2% and 50% (w / v) lactose content. For example, the milk products can comprise at least 2% (w / v), at least 3% (w / v), at least 4% (w / v), at least 5% (w / v), at least 6% (w / v), at least 7% (w / v), at least 8% (w / v), at least 9% (w / v), at least 10% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), at least 50% (w / v), or more than 50% (w / v) lactose content. In some embodiments, the milk products comprise between 2%-4% (w / v), 3%-5% (w / v), 4%-6% (w / v), 5%-7% (w / v), 6%-8% (w / v), 7%-9% (w / v), 8%- 10% (w / v), 9-20% (w / v), 10%-25% (w / v), 20%-30% (w / v), 25%-35% (w / v), 30%-40% (w / v), 35%-45% (w / v), 40%-50% (w / v), or greater than 50% (w / v) lactose content. As the milk product used in the methods disclosed herein can comprise unprocessed, milk the initial pH of the milk product can be expected to that of natural, unprocessed milk. For example, the pH of the milk product that is contacted with a starter culture composition and a cell-free BHT, or active fragment thereof, can be between pH 6.5 and pH 7.0. The pH of the milk product can be at least pH 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the pH of the milk product can be between 6.5-6.7, 6.6-6.8, 6.7- 6.9, or 6.8-7.0. In some embodiments, the milk product used for the methods of producing GOS composition disclosed herein comprises a liquid milk product that is contacted with a plurality of microbial cells, and / or cell constituents and a cell-free P-hexosyltransferase enzyme, or active fragment thereof, wherein the pH of said composition is between pH 6.5 and pH 7.0. In some embodiments, the pH of the milk product contacted with a starter culture composition and a cell-free BHT, or active fragment thereof, can be between 6.5-6.7, 6.6-6.8, 6.7-6.9, or 6.8-7.0. In some embodiments, the pH of the milk product contacted with a starter culture composition and a cell-free BHT, or active fragment thereof, can be between 6.5-6.7, 6.6-6.8, 6.7-6.9, or 6.8-7.0, prior to the step of heating the milk product, such as prior to heating to a temperature of between 40°C-44°C. If necessary, a skilled artisan would understand how to measure the pH of the milk product prior to contacting the milk product with a starter culture composition and BHT, or active fragment thereof, and prior to heating the milk product. If necessary, the pH of the milk product can be adjusted prior to the step of heating the milk product, using an method understood in the art to measure and adjust the pH.

[0094] Any volume of milk product is envisaged within the scope of the present application. In some instances, the volume of milk product that is used to generate a GOS composition can be between about 1 mL and about 10,000 liters (L). For example, the volume of milk product can be between 1-100 mL, 50-500 mL, 100 mL-1 L, 500 mL-5 L, 1 L-10 L, 5 L- 50 L, 10 L-100 L, 50 L-500 L, 100 L-1,000 L, 500 L-5,000 L, or 1,000 L-10, 000 L. In some instances, the volume of milk product can be greater than 10,000 L.

[0095] Contacting said milk product with a starter culture composition and B-hexosyltransferase (BHT)

[0096] To generate a GOS composition, the milk product, such as a liquid milk product is contacted with a starter culture composition and a cell-free BHT, or active fragment thereof. The starter culture composition comprises a plurality of microbial cells, and / or cell constituents that may be helpful in promoting fermentation of the milk product into a fermented milk product under the appropriate conditions. The steps of contacting the milk product with a starter culture composition comprising a plurality of microbial cells and / or cell constituents and a cell-free BHT can occur simultaneously, or subsequently, and occur before heating of said milk product mixture. Contacting the milk product, starter culture composition and BHT can occur through any means known to one skilled in the art that would enable the milk product to contact the BHT and starter culture and enable production of a GOS composition. Contacting for example can include inoculation of the BHT and starter culture composition directly in a liquid milk product. Once inoculated, the milk product, starter culture composition and BHT enzyme can be mixed to ensure thorough contact of the milk product with the starter culture composition and BHT, or active fragment thereof. For example, contacting can also include any means necessary to ensure adequate contact of the milk product, such as a liquid milk product containing lactose, and the starter culture composition and BHT, or active fragment thereof, such as mixing, stirring, and / or agitation after the components are inoculated. For example, a milk product can be inoculated with a lactose transferase enzyme, P- hexosyltransferase (BHT), followed by subsequent addition of a starter culture composition. Alternatively, a milk product can be inoculated with a starter culture composition, followed by subsequence addition of a lactose transferase enzyme, P- hexosyltransferase (BHT). In some embodiments, the step of contacting comprises agitation. In some embodiments, the contacting occurs under constant temperature.

[0097] In some instances, the milk product is at an initial pH value between pH 6.5-7.0 when inoculated with the starter culture and BHT enzyme. In some instances, the milk product is at a pH value between pH 6.5-7.0 after being contacted with the starter culture composition and BHT enzyme, or active fragment thereof. For example, the starter culture composition and BHT, or active fragment thereof can be at a pH similar to the milk product, such that when they are contacted, such as by inoculation, the initial pH value of the milk product is not changed and remains between pH 6.5 -7.0. In some embodiments, the starter culture composition comprises a plurality of microbial cells, and / or cell constituents, wherein the plurality of microbial cells comprises at least one lactic acid bacterium (LAB). In some instances, the lactose transferase enzyme, P-hexosyltransferase (BHT) is added to the milk product, or milk product and starter culture composition mixture at concentrations between 0. 1-5.0 U / g of lactose present in the milk product.

[0098] In a preferred embodiment, a milk product at an initial pH value between 6.5-7.0 is inoculated with a starter culture composition, comprising a plurality of microbial cells, and / or cell constituents, wherein the plurality of microbial cells comprises at least one lactic acid bacterium (LAB), and a lactose transferase enzyme, P-hexosyltransferase (BHT), or active fragment thereof, at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product. Once the milk product, starter culture composition and BHT, or active fragment thereof, are contacted, such as to create a pre-heating mixture comprising a milk product, such as a liquid milk product, a starter culture composition, and a cell-free BHT, or active fragment thereof, at a concentration of between 0.01 U / g to 5.0 U / g lactose in the milk product, the composition (e.g., the pre-heating mixture, such as the milk product) is heated. Heating can comprise any means known in the art to effectively heat the milk product (e.g., pre-heating mixture) that allows for the production of the GOS composition. Heating can comprise heating the milk product to a temperature of between 40°C-44°C, for between 6-24 hours. In some embodiments, the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for between 6-24 hours. The milk product can be heated by any means available to a skilled artisan, which may depend on the volume of the heated milk product. Any volume of milk product is envisaged within the scope of the application and will be determined by the desired volume and amount of GOS composition being produced. In some instances, the GOS composition being produced comprises N- acetyllactosamine (LacNAc)-enriched GOS. In such instances, the method disclosed herein can further comprise the step of contacting said milk product with N-acetylglucosamine (GlcNAc) prior to heating said milk product. For example, GlcNAc can be added prior to or after contacting said milk product with either the starter culture and cell- free BHT. In some instances, a mixture comprising a starter culture, BHT, and, optionally, GlcNAc can be generated by mixing the starter culture, BHT, and, optionally, GlcNAc in a pre-mixture, and said pre-mixture can be added to the milk product in a single step. For the purposes of GOS composition production, the order in which the milk product is contacted with the starter culture, BHT and GlcNAc can be any order. Following addition of the GlcNAc to the milk product, the milk product composition is heated to a temperature of between 40°C-44°C, for between 6-24 hours. In some embodiments, the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for between 6-24 hours. LacNAc is generated by mixing lactose, such as lactose-containing milk product, such as fresh, unprocessed milk, or processed milk, and GlcNAc with BHT polypeptides. In some embodiments, the addition of BHT and GlcNAc to the milk product catalyzes the addition of galactose to GlcNAc when the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for between 6-24 hours.

[0099] BHT and N-acetylglucosamine (GlcNAc)

[0100] The concentration and amount of BHT and GlcNAc that are added to and present within the milk product can impact the amount of GOS, LacNAc and LacNAc -containing GOS produced by the methods disclosed herein.

[0101] In some embodiments of the methods of producing a GOS composition disclosed herein, wherein the milk product is contacted with a starter culture composition and a BHT and, optionally, GlcNAc, the BHT, or active fragment thereof, is added to the milk product at a concentration of between 0.1 U / g and 5.0 U / g. For example, BHT can be added in an amount of at least 0.1 U / g of lactose or greater than 0.1 U / g of lactose, such as at least 0.5 U / g of lactose, at least 1.0 U / g lactose, at least 1.5 U / g lactose, at least 2.0 U / g lactose, at least 2.5 U / g lactose, at least 3.0 U / g lactose, at least 3.5 U / g lactose, at least 4.0 U / g lactose, at least 4.5 U / g lactose, or at least 5.0 U / g lactose. As the amount of BHT is added per gram of lactose present within the milk product, the concentration and amount of BHT added to the milk product can and will vary depending on the amount of lactose present within said composition. A skilled artisan will understand that the amount of BHT added to the milk product will be dependent upon the amount of lactose in the milk product, and will be capable of determining the amount of lactose in the milk product, and subsequently calculating the amount of BHT to added to the milk product. For example, the amount of BHT added can be between 0. 1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5-4.0, or 4.5- 5.0 U / g lactose present in the milk product. In preferred embodiments, the amount of BHT added to the milk product is between 0.5 U / g lactose and 5.0 U / g of lactose. The BHT can also be added to a starter culture composition that is then subsequently added to the milk product. When the BHT is added to a starter culture composition, the final concentration of BHT in the milk product is between 0.1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5- 4.0, or 4.5-5.0 U / g lactose present in the milk product.

[0102] The methods disclosed herein can further comprise the step of contacting said milk product with a lactose hydrolyzing enzyme, or active fragment thereof, such as by adding a lactose hydrolyzing enzyme, or active fragment thereof, to the milk product. In some instances, the lactose hydrolyzing enzyme, or active fragment thereof, comprises a P- galactosidase, or active fragment thereof. The step of contacting the milk product with a P- galactosidase, or active fragment thereof, can occur simultaneously, or subsequently, to contacting the milk product with a starter culture comprising a plurality of microbial cells and / or cell constituents, a cell-free BHT, and, optionally, GlcNAc, and occurs before heating of said milk product mixture. In some instances, a lactose hydrolyzing enzyme, such as a P- galactosidase, or active fragment thereof, is added to the milk product comprising a cell-free P-hexosyltransferase enzyme and a starter culture composition and, optionally, GlcNAc. A P- galactosidase enzyme can be added at any concentration that is suitable for the necessary lactose hydrolysis. For example, P-galactosidase can be added at a physiological concentration to reach similar enzymatic activity that is found in human break milk, which can vary. The terms “P-galactosidase” and “P-gal” are used interchangeably herein. It will be readily appreciated by those of skilled in the art that an active fragment of a P-galactosidase would be expected to function as a full-length, functional P-galactosidase polypeptide or enzyme, and thus would be able to be readily interchangeable, unless specifically and / or otherwise noted. Therefore, a skilled artisan, unless specifically instructed for the purposes of this disclosure, not distinguish between a P-galactosidase and an active fragment thereof. For the purposes of this disclosure, any reference to “P-galactosidase” includes any and all active fragments thereof, which can be readily interchanged with a full-length enzyme. GlcNAc can be added to the milk product comprising lactose to promote LacNAc production, such as to promote production of LacNAc -containing GOS production. Any concentration of GlcNAc that is effective for the production of LacNAc is envisaged as being able to be added to the milk product in the methods disclosed herein. In some instances, the amount of GlcNAc added to the milk product is dependent upon the amount of lactose within the composition. Specifically, a skilled artisan, will calculate and add an amount of GlcNAc to the milk product based on the amount of lactose in the milk product. In a preferred embodiment, GlcNAc will be added such that there is a 1 : 8 GlcNAc-lactose ratio in the milk product once GlcNAc is added. For example, GlcNAc can be added at a 1:8 ratio with lactose (e.g., 25 g / L GlcNAc to 200 g / L lactose) for the reaction to produce the desired GOS composition proportions. In some embodiments, the milk-based composition comprises between 1 g / L and 65 g / L GlcNAc. For example, the amount of GlcNAc added to the milk product can be between 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, or 60-65 g / L of GlcNAc. GlcNAc can be added such that it is present in the milk product in an amount between about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 55-60, or 60-65 g / L. In some embodiments, GlcNAc is added such that it is present in the milk product in an amount between 2.5 g / L and 62.5 g / L GlcNAc.

[0103] Starter culture composition

[0104] To produce a GOS composition, a milk-based composition comprising a liquid milk product is contacted with a starter culture composition comprising a plurality of microbial cells, and / or cell constituents. The starter culture composition added to the liquid milk product can comprise a plurality of microbial cells with any number of microbial cells necessary for fermentation of the milk product. The microbial cells aid in fermentation of the milk product and can comprise any microorganism capable of fermenting the milk product. For example, in some instances, the plurality of microbial cells comprises lactic acid bacteria and / or cell constituents, such as at least one lactic acid bacteria and / or cell constituents. The starter culture added to the milk product comprises a plurality of microbial cells, and / or cell constituents wherein the number of microbial cells within the composition can be at least 1 - 1010microbial cells, such as I- IO10lactic acid bacteria. In some instances, the number of microbial cells is greater than 1010microbial cells. In some instances, the number of microbial cells is less than 1010microbial cells (e.g., between 109and 108microbial cells, between 108and 107microbial cells, between 107and 106microbial cells, between 106and I05microbial cells, between 105and 104microbial cells, between 104and 103microbial cells, between 103and 102microbial cells, between 102and 10 microbial cells, between 10 and 1 microbial cell(s) or less than 1 microbial cell).

[0105] In some embodiments, the number of microbial cells within the starter culture composition is about 1-10, about 10-102, about 102-l 03, about 103-l 04, about 104-l 05, about 105-106, about 106-107, about 107- 108, about 108-109, about 1O9-1O10, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells within the starter culture composition is about 108- 109, about 109- 1010, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells within the starter culture composition is between about 1 and about 1012microbial cells. In some embodiments, the number of microbial cells within the starter culture composition is about 1- 1012microbial cells. In some embodiments, the plurality of microbial cells within the starter culture composition comprises at least one lactic acid bacteria and / or cell constituents. Heating the milk product

[0106] The method of producing a GOS composition disclosed herein comprises the step of heating said milk product, after contacting the milk product with a starter culture composition and cell-free BHT, or active fragment thereof (e.g., creating a milk-based composition), or a milk-based composition to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition. Once the milk product, starter culture and BHT are contacted, and for example, mixed, the milk product is heated. For example, in some instances, the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for between 6-24 hours to generate a GOS composition. In some instances, the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for about 24 hours, or more than 24 hours. In some instances, the milk product is heated to 40°C-44°C and maintained at 40°C-44°C for at least 6, at least 7 at least 8 at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 hours. For example, the milk product is heated to 40°C-44°C and maintained at 40°C- 44°C for 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, 18-20, 19-21, 20-22, 21-23, 22-24, or more than 24 hours to generate a GOS composition. Any length of time necessary to generate a GOS composition, optionally, to generate a GOS composition comprising LacNAc -containing, or LacNAc -enriched, GOS is envisaged within the methods of the present disclosure. The milk product can be heated by any means available to a skilled artisan, which may depend on the volume of the heated milk product. Any volume of milk product is envisaged within the scope of the application and will be determined by the desired volume and amount of GOS composition being produced. In some embodiments, the step of heating said milk product to a temperature of between 40-44°C comprises maintaining the milk product at a temperature of between 40-44°C. In some embodiments, the step of heating the milk product comprising exposing the milk product to a constant temperature of between 40-44°C. In some embodiments, the step of heating said milk product comprising agitating the milk product at a temperature of between 40-44°C. The step of heating can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 or more than 24 hours. In some embodiments, the step of heating to a temperature of between 40-44°C is about 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-15 hours, 15-20 hours, 20- 24 hours, or more than 24 hours. In some embodiments, the heating step is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours.

[0107] In a preferred embodiment, a milk product at an initial pH value between 6.5-7.0 is inoculated with a starter culture composition, comprising a plurality of microbial cells, and / or cell constituents, wherein the plurality of microbial cells comprises at least one lactic acid bacterium (LAB), and a lactose transferase enzyme, P-hexosyl transferase (BHT), or active fragment thereof, at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product and is heated to a temperature of between 40°C-44°C and maintained at 40°C- 44°C for between 6-24 hours. In another preferred embodiment, a milk product at an initial pH value between 6.5-7.0 is inoculated with a starter culture composition, comprising a plurality of microbial cells, and / or cell constituents, wherein the plurality of microbial cells comprises at least one lactic acid bacterium (LAB), and a BHT, or active fragment thereof, at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product is heated to a temperature of between 40°C-44°C and maintained at 40°C-44°C for between 6-24 hours.

[0108] Fermentation of the milk product

[0109] In some instances, the heating step results in fermentation of the milk product to generate a fermented milk product. For example, when the milk product is at maintained at 40°C-44°C, the milk product is fermented to generate a fermented milk product, and the pH of the milk product, or GOS composition produced during the GOS production will decrease. During fermentation, the pH of the milk product and associated composition will decrease over time. While the milk product is maintained at 40°C-44°C, the pH of the milk product, and GOS composition that is generated therefrom, is expected to decrease and as a result will be lower than the initial milk product, and / or milk-based composition. Specifically, the pH of the milk-based composition and GOS composition generated therefrom will begin to decrease below the milk product initial pH of 6.5-7.0.

[0110] Lactic acid may be produced during fermentation of an organic substrate, such as a milk product, by lactic acid bacteria under anaerobic conditions. In some embodiments, during lactic acid fermentation, glucose or another sugar molecule is broken into pyruvic acid. The pyruvic acid may then be reduced to lactic acid in the absence of alcohol. In some embodiments, during lactic acid fermentation, one or more microbial cells may die or lyse, degrading microbial cells and releasing cellular constituents. This release of microbial cell constituents may include, but is not limited to release of bacteriocins, organic acids, carbonic substances, and enzymes. In some embodiments, fermentation of a milk product may include regulating a temperature of the milk product, milk-based composition or GOS composition comprising a starter culture composition. In some embodiments, fermentation may further include regulating various factors, such as pH levels, temperature, moisture, oxygen concentration (or lack thereof), and / or duration of time for fermentation. In some embodiments, fermentation may include the introduction of a culture media or growth media. In some embodiments, the culture media may aid in the growth and metabolic activity of the microorganisms during the fermentation process. In some embodiments, fermentation may include the introduction of one or more culture media as described above to aid in the fermentation process.

[0111] By heating the milk product, starter culture composition and BHT, or active fragment thereof, at a temperature of between 40°C-44°C for between 6-24 hours, the amount of lactose in the milk product will decrease overtime, while the GOS levels will increase, resulting in GOS composition that may comprise a fermented milk product. The pH of the resulting GOS composition will decrease from the initial pH of the milk product due to fermentation that is aided by the plurality of microbial cells in contact with the milk product under conditions conducive for fermentation. The milk product, milk-based composition, and resulting GOS composition generated therefrom, can be maintained at a temperature between 40°C-44°C until the pH of the resulting GOS composition is at or below any desired pH. For example, the milk product can be maintained at a temperature between 40°C-44°C until the pH is below pH 7.0, below pH 6.5, below pH 6.0, below pH 5.5, below pH 5.0, below pH 4.5, below pH 4.0, or below pH 3.5. The milk product can be maintained at a temperature between 40°C-44°C until the pH is between 7.0-6.0, 6.5-5.5, 6.0-5.0, 5.5-4.5, 5.0-4.0, 4.5-3.5, 4.0-3.5, or below pH 3.5. In a preferred embodiment of the method disclosed herein, the milk product (i.e., the milk-based composition) is maintained at a temperature between 40°C-44°C until the pH of the resulting GOS composition is at or below pH 4.8, such as at or below pH 4.6. Specifically, the milk product (i.e., the milk-based composition) is maintained at a temperature between 40°C-44°C until the pH of the GOS composition generated therefrom is at or below pH 4.8, such as between pH 4.8-4.6, or at or below pH 4.6, such as between pH 4.6-3.4.

[0112] The GOS compositions generated from the methods disclosed herein comprise lactose content reduced from the initial milk product, such as the lactose content present in the initial raw, unprocessed or processed liquid milk product. Lactose content is reduced due to the addition of BHT, or active fragment thereof, and, in some instances, the corresponding addition of a P-galactosidase, or active fragment thereof. Lactose content will decrease when the milk product is heated, specifically, when the milk product is heated to, and maintained at, a temperature between 40°C-44°C. In some embodiments of the methods disclosed herein, the milk product, milk-based composition, and GOS composition generated therefrom, is heated to and maintained at a temperature between 40°C-44°C until lactose content in said GOS composition is reduced between 20% and 99% compared to lactose content in said milk product. For example, the lactose content can be reduced at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the initial lactose content in the milk product. In some embodiments, the lactose content can be reduced between 20%-30%, 25%-35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%-65%, 60%-70%, 65%-75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-99%, or 95%-99% compared to the initial lactose content in the milk product. In some instances, the milk product is heated to and maintained at a temperature between 40°C-44°C until lactose content in said GOS composition is reduced to a specifically desired amount. For example, until the lactose content in the GOS composition is at most 500 g / L, 400 g / L, 300 g / L, 200 g / L, 100 g / L, 50 g / L, 25 g / L, 15 g / L, 10 g / L, 5 g / L, 1 g / L or 0.5 g / L. In some embodiments, the milk product is heated to and maintained at a temperature between 40°C-44°C until the lactose content in the GOS composition is between 500-400 g / L, 450-350 g / L, 400-300, 350-250, 300-200, 250-150, 200-100, 150-50, 100-25, 50-10, 25-15, 10-5, or 15-1, or 5-0.5 g / L.

[0113] The addition of a p-hexosyltransferase, or active fragment thereof, and a P- galactosidase, or active fragment thereof, to said milk product can result in greater efficacy of lactose reduction compared to said method when performed without addition of a P- hexosyltransferase, or active fragment thereof. For example, the addition of a P- hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product can result in a larger amount of lactose reduction in a shorter amount of time compared to said method when performed without addition of a P- hexosyltransferase, or active fragment thereof. In some embodiments, lactose content can be reduced in an at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% shorter amount of time in a method comprising the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, compared to said method when performed without addition of a P- hexosyltransferase, or active fragment thereof. In some embodiments, the lactose content can be reduced in an amount of time between 5%-l 5%, I0%-20%, 15-25%, 20%-30%, 25%- 35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%-65%, 60%-70%, 65%- 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-99%, or 95%-99% shorter amount of time in a method comprising the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, compared to said method when performed without addition of a P-hexosyltransferase, or active fragment thereof. In some embodiments, the lactose content can be reduced at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% greater in the same amount of time in a method comprising the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, compared to said method when performed without addition of a P-hexosyltransferase, or active fragment thereof. In some embodiments, the lactose content can be reduced between 5%-l 5%, I0%-20%, 15- 25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%- 65%, 60%-70%, 65%-75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-99%, or 95%- 99% greater in the same amount of time in a method comprising the addition of a P- hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, compared to said method when performed without addition of a P-hexosyltransferase, or active fragment thereof.

[0114] Disclosed herein are methods of generating a GOS composition from a milk product, such as a fresh, unprocessed, or processed milk product, comprising the step of heating said milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition. The methods disclosed herein are particularly effective at generating GOS compositions, such as GOS compositions with and without LacNAc, for example, LacNAc- enriched GOS compositions (e.g., by the addition of GlcNAc to the milk product) because the methods minimize the amount of GOS levels that are decreased throughout GOS production, specifically, for example, the heating step of the milk product when GOS is being generated. In some embodiments of the methods disclosed herein, the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, the GOS levels are decreased no more than 1% during said heating step. For example, the GOS levels are decreased no more than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% during said heating step. In some embodiments of the methods disclosed herein, GOS levels are decreased no more than 0.5%-2%, l%-3%, 2-4%, 3%-5%, 4%-6%, 5%-7%, 6%-8%, 7%-9%, or 8-10% during said heating step.

[0115] Once the GOS composition is generated, the methods disclosed herein further comprise the step of cooling the GOS composition. Cooling the GOS composition allows for long-term storage without a reduction in GOS levels. Following the step of heating the milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition, the GOS composition can be cooled to a temperature of about 4°C. The GOS composition can be cooled to and maintained at a temperature of no more than 8°C, no more than 7°C, no more than 6°C, no more than 5 °C, no more than 4°C, no more than 3 °C, no more than 2°C, or no more than 1°C for 1-60 days, or more than 60 days. In some embodiments, the method comprises cooling and maintaining the GOS composition at a temperature of between 1°C-4°C, 2°C-5°C, 3°C-6°C, or 4°C-8°C for 1-60 days, or more than 60 days.

[0116] The cooling to about 4°C can occur over a period of time between at most 0. 1, at most 0.5, at most 1, at most 5, at most 10, at most 15, at most 20, at most 30, at most 40, at most 50, at most 100, at most 200, at most 300, at most 400, or at most 500 mins. In some embodiments, the cooling to about 4°C can occur over a period of time between 0.1-1, between 0.5-5, 1-10, 5-15, 10-20, 15-30, 20-40, 30-50, 40-100, 50-200, 100-300, 200-400, 300-500, or more than 500 mins. Once the GOS composition is cooled to a temperature of about 4°C, the GOS composition can be maintained at a temperature of no higher than about 4°C for between 1-60 days. In some instances, the GOS composition can be maintained at a temperature of about 4°C for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or more than 60 days. Once the GOS composition is cooled and stored, the pH of the composition should be maintained at around a constant value throughout storage. Also disclosed herein are methods of producing a GOS composition, wherein once the GOS composition is cooled to a temperature of about 4°C, wherein the pH of the GOS composition is no higher than pH 4.6 during storage at 4°C. In some instances, the pH of the GOS composition is no higher than pH 4.6 during storage at 4°C for 1-60 days, or more than 60 days. In some embodiments, the pH is no higher than pH 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, or 3.4 during storage at 4°C. In some embodiments, the pH is maintained at between pH 3.5 and 4.6 during storage of the GOS composition at about 4°C for 1-60 days, or more than 60 days. For example, the pH is maintained at between pH 3.5-3.7, 3.6-3.8, 3.7-3.9, 3.8-4.0, 3.9-4. 1, 4.0- 4.2, 4.1-4.3, 4.2-4.4, 4.3-4.5, or 4.4-4.6 during storage at 4°C.

[0117] Also disclosed herein are methods comprising adding to said milk product and / or said GOS composition at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder. For example, the at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder can be added to said milk product, milk-based composition, or GOS composition generated therefrom by the methods disclosed herein at any time due production of the GOS composition. For example, at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder can be added to the milk product, or GOS composition generated therefrom, prior to, during or after heating said milk product to a temperature of between 40°C-44°C. When at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder is added to the milk product or GOS composition after heating said milk product to a temperature of between 40°C-44°C, the at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder can be added prior to storage at 4°C.

[0118] Food or beverage product

[0119] Also disclosed herein are methods of generating a GOS composition, wherein the method comprises: contacting said milk product with a starter culture composition comprising a plurality of microbial cells, and / or cell constituents; contacting said milk product with a cell-free P-hexosyltransferase, or active fragment thereof; and heating said milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition, wherein said GOS composition comprises a food or beverage product, such as a dairy-based food or a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula. In some embodiments, the methods comprise adding to said milk product and / or said GOS composition at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder. The GOS compositions generated by the methods disclosed herein can be further formulated into a food or beverage product, such as a nutritional supplement or a prebiotic.

[0120] As would be recognized by one of ordinary skill in the art based on the present disclosure, GOS compositions, including GOS with or without GlcNAc and LacNAc - enriched GOS compositions, are widely used as prebiotic supplements in foods and beverages around the world. These highly prized non-digestible sugars are able to mimic human milk oligosaccharides (HMOs) by having a positive influence on the growth and metabolism of gastrointestinal (GI) bacteria (probiotics). Addition of prebiotics to the diet has shown a substantiated improvement in overall health of the host by reducing GI discomfort, managing the immune system, and reducing pathogenic and opportunistic bacteria and viruses. Embodiments of the present disclosure demonstrate novel methods for generating food or beverage products, including dairy-based food or beverage products or prebiotics, comprising the milk-based compositions and / or GOS compositions of the present disclosure to generate LacNAc and / or GOS from lactose in raw, unprocessed or processed milk and GlcNAc and to significantly increase the amount of LacNAc -containing GOS in a food and / or beverage product.

[0121] The food or beverage product disclosed herein can comprise between about 0.01-5 grams of GOS or LacNAc-containing GOS. For example, the food or beverage product can comprise at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 grams of GOS or LacNAc-containing GOS. In some embodiments, the food or beverage product can comprise between 0.01-0.1, 0.05-0.5, 0.1-1.0, 0.5-2, 1-3, 2-4, 3-5, 4-10, or 5-20 grams of GOS or LacNAc-containing GOS.

[0122] The food or beverage product can be any food or beverage product intended for human or animal consumption, non-limiting examples of which a food or beverage product, such as a dairy-based food or a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula. In some embodiments, the food or beverage product comprises a semisolid, such as yogurt, or a solid, such as gelatin. In some embodiments, the food or beverage product comprises a powder intended to be mixed with a fluid. When the GOS composition comprises a powder, the powder can be added to a liquid food or beverage product. In some instances, the food or beverage product is a liquid, such as a smoothie or a protein shake. In some embodiments, the GOS composition comprises a powder, such as a lyophilized powder. The food or beverage product can also be a solid, or semi-solid. For example, the food or beverage product can be a yogurt, a yogurt-containing food or beverage product, such as a granola bar, a smoothie, or a protein shake.

[0123] IV. Compositions Comprising GOS with and without N-acetyllactosamine (LacNAc)

[0124] Disclosed herein are galactooligosaccharide (GOS) compositions (e.g., GOS with or without GlcNAc, as well as LacNAc -containing, or LacNAc-enriched, GOS) and compositions useful for generating the same. The GOS compositions or compositions useful for generating GOS compositions described herein can include, for example, but are not limited to: a milk based composition comprising a liquid milk product, a plurality of microbial cells, and / or cell constituents, and a BHT enzyme, or active fragment thereof, wherein the pH of the milk-based composition is between pH 6.5 and 7.0; a GOS composition comprising a plurality of microbial cells, and / or cell constituents, wherein the pH of the GOS composition is between pH 3.4 and 7.0, optionally wherein the GOS composition comprises LacNAc (i.e., LacNAc-containing GOS), and, optionally, wherein the GOS composition comprises a fermented milk product; and a GOS composition comprising a plurality of microbial cells, and / or cell constituents, wherein the pH of the GOS composition is pH 4.8 or less than pH 4.8, optionally, wherein the GOS composition comprises comprises LacNAc (i.e., LacNAc-enriched GOS), and optionally, wherein said GOS composition comprises a fermented milk product. In some embodiments, milked based compositions and / or GOS compositions disclosed herein further comprises a cell-free p-hexosyltransferase enzyme, or active fragment thereof and, optionally, a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof. The compositions disclosed herein can comprise N-acetylglucosamine (GlcNAc). Also disclosed herein are food or beverage products comprising the milk-based compositions or GOS compositions described herein. In some embodiments, the food or beverage product comprises a dairy-based food, such as a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula.

[0125] Milk-based compositions

[0126] Disclosed herein are methods of producing GOS compositions from a milk product, such as fresh, unprocessed milk or processed milk. The method comprises contacting a milk product, such as contacting fresh milk with a starter culture, comprising a plurality of microbial cells. Included in the present disclosure are milk-based compositions comprising a milk product, including, but not limited to a liquid milk product, such as fresh, unprocessed milk. In some embodiments, the liquid milk product is a processed liquid milk product. The milk compositions comprising a liquid milk product can further comprise a starter culture composition comprising a plurality of microbial cells, and / or cells constituents. The plurality of microbial cells comprises at least one microorganism, such as one lactic acid bacteria. The milk-based compositions of the present disclosure can be at any pH suitable for generating a GOS composition. As the milk-based compositions can comprise fresh, unprocessed milk, it is expected, without wishing to be bound by theory, that the milk-based compositions have a pH that is similar to fresh, unprocessed milk. For instance, the milk-based compositions can have a pH between about 6.0 and about 8.0. The pH of the composition can be adjusted by any means known to a skilled artisan, such as addition of buffers. The pH can be about 6.0- 8.0, about 6.5-8.0, about 7.0-8.0, or about 7.6-8.0. In some embodiments, the pH can be 6.0- 6.5, 6.5-7.0, 7.0-7.5, or 7.6-8.0. The milk products used in the compositions disclosed herein to generate GOS compositions comprise between 2% and 50% (w / v) lactose content. For example, the milk products can comprise at least 2% (w / v), at least 3% (w / v), at least 4% (w / v), at least 5% (w / v), at least 6% (w / v), at least 7% (w / v), at least 8% (w / v), at least 9% (w / v), at least 10% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), at least 50% (w / v), or more than 50% (w / v) lactose content. In some embodiments, the milk products comprise between 2%-4% (w / v), 3%-5% (w / v), 4%-6% (w / v), 5%-7% (w / v), 6%-8% (w / v), 7%-9% (w / v), 8%-l 0% (w / v), 9-20% (w / v), 10%-25% (w / v), 20%-30% (w / v), 25%-35% (w / v), 30%-40% (w / v), 35%-45% (w / v), 40%-50% (w / v), or greater than 50% (w / v) lactose content. To produce a GOS composition, a milk-based composition comprising a liquid milk product is contacted with a plurality of microbial cells, and / or cell constituents. The plurality of microbial cells can be added to the liquid milk product within a starter culture composition comprising any number of microbial cells necessary for generating a GOS composition described herein. The plurality of microbial cells aids in fermentation of the liquid milk product and can comprise any microorganism that aids in fermentation of said milk product. In some instances, the plurality of microbial cells comprises lactic acid bacteria. The milkbased compositions disclosed herein comprise a plurality of microbial cells, and / or cell constituents wherein the number of microbial cells within the composition can be at least 1- 1010microbial cells. In some instances, the number of microbial cells is greater than IO10microbial cells. In some instances, the number of microbial cells is less than 1010microbial cells (e.g., between 109and 108microbial cells, between 108and 107microbial cells, between IO7and 106microbial cells, between 106and 105microbial cells, between 105and IO4microbial cells, between 104and 103microbial cells, between 103and 102microbial cells, between 102and 10 microbial cells, between 10 and 1 microbial cell(s) or less than 1 microbial cell). In some embodiments, the number of microbial cells within the plurality of microbial cells is about 1-10, about I0-102, about 102-103, about 103-104, about 104-105, about 105-106, about 106-107, about 107-108, about 108-109, about 1O9-1O10, about 1010-10n, or about 1011- 1012microbial cells. In some embodiments, the number of microbial cells within the plurality of microbial cells is about 108- 109, about 109- 1010, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells is between about 1 and about 1012microbial cells. In some embodiments, the number of microbial cells is about 1- 1012microbial cells. In some embodiments, the plurality of microbial cells comprises at least one lactic acid bacteria.

[0127] In some embodiments, the milk-based composition disclosed herein comprises a liquid milk product, a plurality of microbial cells, and / or cell constituents; and a cell-free - hexosyltransferase enzyme, or active fragment thereof, wherein the pH of said composition is between pH 6.5 and pH 7.0. For example, the pH of the milk-based composition can be between pH 6.5-6.6, 6.6-6.7, 6.7-6.8, 6.8-6.9, or 6.9-7.0.

[0128] The milk-based composition comprising a liquid milk product can further comprise N-acetylglucosamine (GlcNAc). GlcNAc can be added to the composition to promote LacNAc production, such as to promote production of LacNAc-containing GOS production. Any concentration of GlcNAc that is effective for the production of LacNAc is envisaged as being added to the composition. In some instances, the amount of GlcNAc added to the milk- based composition is added depending on the amount of lactose within the composition. For example, GlcNAc can be added at a 1:8 ratio with lactose (e.g., 25 g / L GlcNAc to 200 g / L lactose) for the reaction to produce the desired GOS composition proportions. In some embodiments, the milk-based composition comprises between 1 g / L and 65 g / L GlcNAc. The amount of GlcNAc in the composition can be between about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 55-60, or 60-65 g / L. In some embodiments, the milkbased composition comprises between 2.5 g / L and 62.5 g / L GlcNAc.

[0129] The liquid milk product used in the milk-based composition can be non-human, mammal milk, such as cow milk or goat milk. Any form of non-human, mammal milk is envisaged for use as a liquid milk product within the milk-based composition. In some instances, the milk is unprocessed, raw milk. Alternatively, the milk can be modified and / or processed milk. For example, the milk can be a pasteurized milk product, that has undergone a pasteurization. Additionally, the liquid milk product can also be processed such that the milk is skimmed or semi-skimmed milk.

[0130] In some instances, a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof, can be added to the milk-based composition, such that the composition comprises a cell-free p-hexosyltransferase enzyme, or active fragment thereof, and a P- galactosidase enzyme, or active fragment thereof. A p-galactosidase enzyme can be added at any concentration that is suitable for lactose hydrolysis necessary to generate a GOS composition according to the methods described herein. For example, P-galactosidase, or active fragment thereof, can be added at a physiological concentration to reach similar enzymatic activity that is found in human break milk, which is known to vary.

[0131] The milk-based composition can comprise a P-galactosidase enzyme, or active fragment thereof, and / or P-hexosyltransferase enzyme, or active fragment thereof, at a concentration of at least 0.1 U / g of lactose, or greater than 0.1 U / g of lactose. As the amount of BHT is added per gram of lactose present within the milk-based composition, the concentration and amount of BHT within the composition can vary depending on the amount of lactose present within said composition. For example, the amount of BHT added to the milk-based composition can be between 0.1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5-4.0, or 4.5-5.0 U / g of lactose. In some instances, the amount of BHT within the composition can be between about 0.5 U / g of lactose and about 5.0 U / g of lactose.

[0132] The presence of the P-galactosidase and / or p-hexosyltransferase enzymes, or active fragments thereof, within the composition aids in the reduction of lactose and production of GOS. Prior to the reduction of lactose and increase in GOS, the milk-based composition comprising the liquid milk product and a plurality of microbial cells, and / or cell constituents and BHT may comprise relatively low amounts of GOS. In some instances, the composition may further comprise GlcNAc and relative low amounts of GOS, or LacNAc -containing GOS. In some instances, the milk-based composition comprises a galactooligosaccharide (GOS) content of 0.5% (w / v) or lower. In some instances, the GOS content is no more than 0.5% (w / v) of the milk-based composition. For example, the GOS content may be between 0.5% (w / v) and 0.01% (w / v). In some instances, the GOS content is between 0.5% (w / v) and 0.4% (w / v), 0.4% (w / v) and 0.3% (w / v), 0.3% (w / v) and 0.2% (w / v), 0.2% (w / v) and 0.1% (w / v), 0.1% (w / v) and 0.05% (w / v), 0.05% (w / v) and 0.01% (w / v), or less than 0.01% (w / v) of the milk-based composition.

[0133] GOS Compositions at a pH between 3,4 and 7,0,

[0134] In some instances of the methods of generating a GOS composition disclosed herein, the milk product or milk-based composition (i.e., milk-based composition comprising a liquid milk product) comprising a plurality of microbial cells and a P-hexosyltransferase, or active fragment thereof, is heated to a temperature of between 40°C-44°C for between 6-24 hours to generate a composition comprising GOS (i.e., a GOS composition). The milk-based composition can also comprise GlcNAc and / or a P-galactosidase. As the step of heating the milk product can promote the production of GOS, the amount of GOS present in the composition may increase over time as the milk-based composition is heated to, and maintained at, 40°C-44°C, resulting in a GOS composition comprising GOS. In some instances, the milk product has undergone fermentation and the composition comprises a fermented milk product. As a result, the pH of the milk-based composition may decrease over time as fermentation occurs, resulting in a GOS composition with a higher amount of GOS and a lower pH compared to the milk-based compositions disclosed herein. In some instances, the GOS can be LacNAc-containing GOS, or LacNAc -enriched GOS.

[0135] Disclosed herein are GOS compositions comprising a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is between pH 3.4 and pH 7.0. In some instances, when GlcNAc has been added to the milk-based composition used to produce a GOS composition, the resulting GOS composition comprises LacNAc (i.e., LacNAc- containing GOS), and a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is between pH 3.4 and pH 7.0. The GOS composition comprising LacNAc (e.g., GOS with LacNAc) can have a pH between pH 3.4 and pH 7.0. The pH can be about 3.4-7.0, about 3.6-7.0, about 3.8-7.0, about 4.0-7.0, about 4.2-7.0, about 4.4-7.0, about 4.6-7.0, about 4.8-7,0, about 5.0-7.0, about 5.2-7.0, about 5.4-7.0, about 5.6-7.0, about 5.8- 7.0, about 6.0-7.0, about 6.2-7.0, about 6.4-7.0, about 6.6-7.0, or about 6.8-7.0. In some embodiments, the pH can be 3.4-5.0, 4.0-6.0, or 5.0-7.0.As the milk-based composition comprises a liquid milk product comprising between 2% to 50% lactose, without wishing to be bound by theory, it is thought that the GOS compositions will have less lactose than the milk-based composition, as the lactose in the milk product is in contact with the BHT, and optionally P-galactosidase, resulting in GOS production, and lactose hydrolysis. LacNAc is generated by mixing lactose and GlcNAc with BHT. While GlcNAc can be added to the milk-based composition, addition of GlcNAc is envisaged to any GOS composition disclosed herein. The amount of GlcNAc in the GOS composition is typically expected to be lower than the amount of GlcNAc in the milk-based composition used to generate said GOS composition. For example, GlcNAc may be added at a 1:8 ratio with lactose (e.g., 25 g / L GlcNAc to 200 g / L lactose) in milk -based composition comprising a liquid milk product for the reaction to produce the desired GOS composition proportions. In some instances, the final concentration of GlcNAc does not exceed more than 50% of the initial GlcNAc added to a milk-based composition (i.e., between 1 g / L and 65 g / L). In some embodiments, the milkbased composition comprises between 1 g / L and 65 g / L GlcNAc. In some embodiments, the amount of GlcNAc in the GOS composition, wherein the pH of said composition is between pH 3.4 and 7.0 is no more than 65 g / L GlcNAc. In some instances, the amount of GlcNAc is less than 65 g / L. The amount of GlcNAc in the composition can be between about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 55-60, or 60-65 g / L. In some embodiments, the GOS composition comprises between 2.5 g / L and 62.5 g / L GlcNAc. In some instances, the amount of GlcNAc in the GOS composition is between 2.5 g / L and 62.5 g / L.

[0136] To generate a GOS composition comprising a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is between pH 3.4 and pH 7.0, a milk -based composition comprising a liquid milk product is contacted with a plurality of microbial cells that aids in fermentation of the milk product. The plurality of microbial cells can be within a starter culture composition comprising at least one microbial cell. For example, the at least one microbial cell can be a lactic acid bacteria, or any microbial cell that can aid in fermentation of the milk product. As the milk-based composition comprising at least one microbial cell, such as at least one lactic acid bacteria, is heated to a temperature of about 40°C-44°C, it is possible that the number of microbial cells within the composition increases over time. In some instances, the number of microbial cells within a GOS composition will be increased compared to the number of microbial cells in a milk-based composition used to generate a GOS composition. The number of microbial cells within the GOS composition can be at least 1-1O10microbial cells. In some instances, the number of microbial cells is greater than IO10microbial cells. It is possible that the number of microbial cells increased during fermentation. In some instances, the number of microbial cells is less than 1010microbial cells (e.g., between 109and 108microbial cells, between 108and 107microbial cells, between 107and 106microbial cells, between 106and 105microbial cells, between 105and 104microbial cells, between 104and 103microbial cells, between 103and 102microbial cells, between 102and 10 microbial cells, between 10 and 1 microbial cell(s) or less than 1 microbial cell). During fermentation, or during incubation at 40°C-44°C, the number of microbial cells within the composition may increase over time. For example, the number of microbial cells within the composition after fermentation has started can be greater than the number of microbial cells within the plurality of microbial cells contacted with a milk product. For example, the number of microbial cells within the composition can be at least 105microbial cells, at least 106microbial cells, at least 107microbial cells, at least 108microbial cells, at least 109microbial cells, at least 1010microbial cells, or greater than at least 1010microbial cells. In some embodiments, the number of microbial cells within the GOS composition is about 105-106, about 106-107, about 107-108, about 108-109, about 1O9-1O10, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells within the GOS composition is about 108-109, about 1O9-1O10, about 1010-10n, or about 1011- 1012microbial cells. In some embodiments, the number of microbial cells within the GOS composition is between about 1 and about 1012microbial cells. In some embodiments, the number of microbial cells within the GOS composition is about 1-1012microbial cells.

[0137] The milk-based composition can comprise a P-galactosidase enzyme, or active fragment thereof, and / or P-hexosyltransferase enzyme, or active fragment thereof, at a concentration of at least 0.1 U / g of lactose, or greater than 0.1 U / g of lactose. As the amount of BHT is added per gram of lactose present within the milk-based composition, the concentration and amount of BHT within the composition can vary depending on the amount of lactose present within said composition. For example, the amount of BHT added to the milk-based composition can be between 0. 1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5-4.0, or 4.5-5.0 U / g of lactose. In some instances, the amount of BHT within the composition can be between about 0.5 U / g of lactose and about 5.0 U / g of lactose.

[0138] The presence of the P-galactosidase and / or p-hexosyltransferase enzymes, or active fragments thereof, within the GOS composition aids in the reduction of lactose and enhances production of GOS. Prior to the reduction of lactose, the milk-based composition comprises a liquid milk product comprising between 2% (w / v) and 50% (w / v) lactose content. In some instances, the milk-based composition comprises a galactooligosaccharide (GOS) content of 0.5% (w / v) or lower. In some instances, the lactose content of the GOS composition comprising a plurality of microbial cells, and / or cell constituents is no more than 10% (w / v) of the GOS composition at a pH between 3.4 and 7.0. For example, the lactose content may be between 0.5% (w / v) and 10% (w / v) of the GOS composition at a pH between 3.4 and 7.0. In some instances, the GOS content is between 0.5% (w / v) and 2% (w / v), 1% (w / v) and 3% (w / v), 2% (w / v) and 4% (w / v), 3% (w / v) and 5% (w / v), 4% (w / v) and 6% (w / v), 5% (w / v) and 7% (w / v), 6% (w / v) and 8% (w / v), 7% (w / v) and 9% (w / v), or 8% (w / v) and 10% (w / v) of the GOS composition at a pH between 3.4 and 7.0.

[0139] Free glucose may result when lactose is contacted with BHT or BHT and P- galactosidase in a milk product comprising a plurality of microbial cells, resulting in a GOS composition comprising excess free glucose. Without wishing to be bound by theory, the amount of excess glucose may be linked to the amount of BHT present within the composition, such that a higher concentration of BHT may be associated with increased glucose accumulation over time. When the GOS composition comprises BHT or BHT and - galactosidase and a plurality of microbial cells, glucose can be present in the range of between 0.001% (w / v) and 1% (w / v). For instance, there can be between .01 g / L and 10 g / L glucose in the GOS composition.

[0140] When BHT is present in the composition comprising a milk product, the amount of free galactose is expected to be relatively low. For instance, the GOS composition may have galactose in the range of between 0.001% (w / v) and 1.5% (w / v). That is, free galactose may be present between .01 g / L and 15 g / L in the GOS composition.

[0141] GOS Compositions at a pH at a pH of 4,8, or below pH 4,8, such as pH 4,6

[0142] Also disclosed herein are GOS compositions comprising LacNAc -enriched GOS, and a plurality of microbial cells, and / or cell constituents. The pH of the GOS compositions comprising LacNAc-enriched GOS and a plurality of microbial cells can be between pH 4.8 or less than pH 4.8. In some instances, the pH of said composition is between pH 3.4 and pH 4.8.

[0143] The pH of the GOS composition can be an acidic pH because the composition has undergone fermentation due to the presence of a plurality of microbial cells. The plurality of microbial cells within the GOS composition comprises at least one lactic acid bacteria. To produce a GOS composition, a milk-based composition comprising a liquid milk product is contacted with a plurality of microbial cells. The plurality of microbial cells aid in fermentation of the milk product. Therefore, the milk-based compositions disclosed herein comprise a plurality of microbial cells, and / or cell constituents. The number of microbial cells within the composition can be at least 1-1O10microbial cells. In some instances, the number of microbial cells is greater than 1010microbial cells. In some instances, the number of microbial cells is less than 1010microbial cells (e.g., between 109and 108microbial cells, between 108and 107microbial cells, between 107and 106microbial cells, between 106and IO5microbial cells, between 105and 104microbial cells, between 104and 103microbial cells, between 103and 102microbial cells, between 102and 10 microbial cells, between 10 and 1 microbial cell(s) or less than 1 microbial cell). During fermentation, it is possible that the number of microbial cells within the composition increases over time. For example, the number of microbial cells within the composition after fermentation has started can be greater than the number of microbial cells within the plurality of microbial cells mixed with the organic mixture. For example, the number of microbial cells within the composition during fermentation, or after fermentation, can be at least IO5microbial cells, at least 106microbial cells, at least 107microbial cells, at least 108microbial cells, at least 109microbial cells, at least IO10microbial cells, or greater than at least 1010microbial cells.

[0144] In some embodiments, the number of microbial cells within the composition during fermentation, or after fermentation, is about 105-106, about 106-107, about 107-108, about 108- I09, about 1O9-1O10, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells within the composition during fermentation, or after fermentation, is about 108- 109, about 109- 1010, about 1010-10n, or about 10n-1012microbial cells. In some embodiments, the number of microbial cells within the composition during fermentation, or after fermentation, is between about I05and about 1012microbial cells. In some embodiments, the number of microbial cells within the composition during fermentation, or after fermentation, is about 105-1012microbial cells.

[0145] The milk-based composition can comprise a P-galactosidase enzyme, or active fragment thereof, and / or P-hexosyltransferase enzyme, or active fragment thereof, at a concentration of at least 0. 1 U / g of lactose, or greater than 0. 1 U / g of lactose. As the amount of BHT is added per gram of lactose present within the milk-based composition, the concentration and amount of BHT within the composition can vary depending on the amount of lactose present within said composition. For example, the amount of BHT added to the milk-based composition can be between 0.1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5-4.0, or 4.5-5.0 U / g of lactose. In some instances, the amount of BHT within the composition can be between about 0.5 U / g of lactose and about 5.0 U / g of lactose. The presence of the P-galactosidase and / or p-hexosyltransferase enzymes, or active fragments thereof, within the GOS composition aids in the reduction of lactose and enahnces production of GOS. Prior to the reduction of lactose, the milk-based composition comprises a liquid milk product comprising between 2% (w / v) and 50% (w / v) lactose content. In some instances, the milk-based composition comprises a galactooligosaccharide (GOS) content of 0.5% (w / v) or lower. In some instances, the lactose content of the GOS composition comprising a plurality of microbial cells, and / or cell constituents is no more than 10% (w / v) of the GOS composition at a pH between 3.4 and 7.0.

[0146] When the GOS composition is at a pH between pH 4.8, or less than pH 4.8, the lactose content, glucose content and galactose content are expected to be relatively low. For instance, the lactose content is expected to be lower than the initial lactose content in the liquid milk product used to produce the GOS composition. The GOS composition may comprise lactose in the range of between 0.5% (w / v) and 3% (w / v). For example, lactose may be present between 5 g / L and 30 g / L in the GOS composition. For example, the lactose content may be between 0.5% (w / v) and 3% (w / v) of the GOS composition at a pH of 4.8, or less than pH 4.8. In some instances, the GOS content is between 0.5% (w / v) and 2% (w / v) or 1% (w / v) and 3% (w / v) of the GOS composition at a pH 4.8 or less than pH 4.8.

[0147] When the GOS composition is at a pH between pH 4.8, or less than pH 4.8, glucose may be present in the range of between 0.6% (w / v) and 1% (w / v). For example, glucose is between 6 g / L and 10 g / L in the GOS composition. In some instances, the glucose content may be between 0.6% (w / v) and 0.8% (w / v), 0.7% (w / v) and 0.9% (w / v), or 0.8% (w / v) and 1% (w / v) of the GOS composition at a pH 4.8 or less than pH 4.8.

[0148] When the GOS composition is at a pH between pH 4.8, or less than pH 4.8, galactose may be present in the range of between 0.6% (w / v) and 1.5% (w / v). For example, galactose is between 6 g / L and 15 g / L in the GOS composition. In some instances, the galactose content is between 0.6% (w / v) and 1% (w / v), 0.8% (w / v) and 1.2% (w / v), and 1% (w / v) and 1.5% (w / v) of the GOS composition at a pH 4.8 or less than pH 4.8.

[0149] Also disclosed herein are the milk-based compositions and GOS compositions of the present disclosure further comprising at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder. For example, the at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder can be added to said milk product, milk-based composition, or GOS composition generated therefrom by the methods disclosed herein at any time due production of the GOS composition. In some embodiments, the milk-based compositions and GOS compositions are in the form of a liquid, solid, semi-solid, or powder. In some instances, the composition comprises a semisolid, such as yogurt, or a solid, such as gelatin. In some embodiments, the composition comprises a powder intended to be mixed with a fluid. In some embodiments, the composition comprises a powder, such as a lyophilized powder. When the milk-based composition or GOS composition comprises a powder, the powder is a dried or lyophilized power that can be added to a liquid food or beverage product. In some instances, the food or beverage product is a liquid, such as a smoothie or a protein shake. The food or beverage product can also be a solid, or semi-solid. For example, the food or beverage product can be a yogurt, a yogurt-containing food or beverage product, such as a granola bar, a smoothie, or a protein shake.

[0150] Food or beverage product

[0151] Also disclosed herein are food or beverage products comprising any of the milk-based compositions or GOS compositions described in the present application. In some embodiments, the food or beverage products comprises a dairy-based food or a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula. In some embodiments, the methods comprise adding to said milk product and / or said GOS composition at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder. The GOS compositions generated by the methods disclosed herein can be further formulated into a food or beverage product, such as a nutritional supplement or a prebiotic.

[0152] As would be recognized by one of ordinary skill in the art based on the present disclosure, GOS compositions, including GOS with or without GlcNAc and LacNAc- enriched GOS compositions, are widely used as prebiotic supplements in foods and beverages around the world. These highly prized non-digestible sugars are able to mimic human milk oligosaccharides (HMOs) by having a positive influence on the growth and metabolism of gastrointestinal (GI) bacteria (probiotics). Addition of prebiotics to the diet has shown a substantiated improvement in overall health of the host by reducing GI discomfort, managing the immune system, and reducing pathogenic and opportunistic bacteria and viruses. Embodiments of the present disclosure demonstrate novel methods for generating food or beverage products, including dairy-based food or beverage products or prebiotics, comprising the milk-based compositions and / or GOS compositions of the present disclosure to generate LacNAc and / or GOS from lactose in raw, unprocessed or processed milk and GlcNAc and to significantly increase the amount of LacNAc -containing GOS in a food and / or beverage product.

[0153] The food or beverage product disclosed herein can comprise between about 0.01-5 grams of GOS or LacNAc-containing GOS. For example, the food or beverage product can comprise at least 0.01, at least 0.05, at least 0. 1, at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 grams of GOS or LacNAc-containing GOS. In some embodiments, the food or beverage product can comprise between 0.01-0.1, 0.05-0.5, 0.1-1.0, 0.5-2, 1-3, 2-4, 3-5, 4-10, or 5-20 grams of GOS or LacNAc-containing GOS.

[0154] The food or beverage product can be any food or beverage product intended for human or animal consumption, non-limiting examples of which a food or beverage product, such as a dairy-based food or a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula. In some embodiments, the food or beverage product comprises a semisolid, such as yogurt, or a solid, such as gelatin. In some embodiments, the food or beverage product comprises a powder intended to be mixed with a fluid. When the GOS composition comprises a powder, the powder can be added to a liquid food or beverage product. In some instances, the food or beverage product is a liquid, such as a smoothie or a protein shake. In some embodiments, the GOS composition comprises a powder, such as a lyophilized powder. The food or beverage product can also be a solid, or semi-solid. For example, the food or beverage product can be a yogurt, a yogurt-containing food or beverage product, such as a granola bar, a smoothie, or a protein shake.

[0155] EXAMPLES

[0156] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0157] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0158] EXAMPLE 1: Materials and Methods for Composition Analysis

[0159] Reaction Product Analyses

[0160] Products and substrates of the reactions are analyzed by high-performance liquid chromatography (HPLC) (Shimadzu Corp., Japan) under isocratic conditions at 65°C and at 0.4 mL min"1flow rate. The mobile phase is 5 mM sulfuric acid (H2SO4) using an Altech IOA-IOOO organic acids column (300 mm by 7.8 mm) (Altech, IL) coupled to a refractive- index detector.

[0161] EXAMPLE 2: Production of milk products utilizing only BHT for GOS compositions and reduced lactose content

[0162] A milk product (i.e., whole milk) at an initial pH value between 6.5 -7.0 was inoculated with a starter culture composition comprising at least one lactic acid bacterium (LAB) and a lactose transferase enzyme, P-hexosyltransferase (BHT), at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product. The milk product inoculated with the starter culture and BHT were incubated together at 42°C for a duration of 6-24 h, or until the milk product reached a pH value at or below 4.8, or at or below pH 4.6. The fermented milk product was removed from the incubation temperature of 42°C and placed at 4°C. The fermented milk product enriched with fibers was be stored at 4°C for up to 60 days.

[0163] FIG. 1 shows fiber accumulation, lactose reduction, pH reduction and glucose accumulation in the milk-based composition (i.e., whole milk inoculated with a starter culture composition and BHT) overtime using multiple concentrations of BHT (0.5 U / g; 1.0 U / g; 1.5 U / g; and 2.0 U / g). The composition was incubated at 42°C for 24 hours, followed by a shift to 4°C for the time period as indicated. As depicted in FIG. 1, increasing concentration of BHT between 0.5 U / g lactose and 2.0 U / g lactose was generally associated with increased fiber accumulation, increased lactose reduction, increased pH reduction, and increased glucose accumulation in the composition. Surprisingly, increasing concentration of BHT was also generally associated with increased rates of fiber accumulation, lactose reduction, pH reduction, and glucose accumulation in the liquid milk composition. These results demonstrate that BHT can effectively convert lactose to GOS, thereby reducing lactose content, when inoculated into fresh, unprocessed milk in the presence of a starter culture composition, which aids in fermentation of the milk product, as demonstrated by the reduction in pH of the composition over time. This demonstrates that BHT can be used to effectively produce GOS and reduce lactose content, in an unprocessed milk product, and when combined with a starter culture composition can be utilized to produce dairy-food product, such as a fermented dairy-food product including yogurt, enriched in GOS.

[0164] EXAMPLE 3: Production of milk products utilizing only BHT for GOS compositions enriched with the human milk oligosaccharide (HMO) N-acetyllactosamine (LacNAc) and reduced lactose content

[0165] A milk product (e.g., a liquid milk product) at an initial pH value between 6.5-7.0 is inoculated with a starter culture containing at least one lactic acid bacterium (LAB) and a lactose transferase enzyme, P-hexosyl transferase (BHT), at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product. N-acetylglucosamine (GlcNAc) is added at a ratio of 8: 1 lactose to N-acetylglucosamine. The milk product inoculated with the starter culture and BHT with N-acetylglucosamine are incubated together at 42°C for a duration of 6-24 h, or until the milk product reaches a pH value at or below 4.8, or at or below pH 4.6. The fermented milk product is removed from the incubation temperature of 42°C and placed at 4°C. The fermented milk product enriched with fibers can be stored at 4°C for up to 60 days.

[0166] EXAMPLE 4: Production of milk products utilizing both BHT & -galactosidase for GOS compositions and reduced lactose content

[0167] A milk product (i.e., whole milk) at an initial pH value between 6.5-7.0 was inoculated with a starter culture composition comprising at least one lactic acid bacterium (LAB) and a lactose transferase enzyme, -hexosyltransferase (BHT), and a lactose hydrolase enzyme, active -galactosidase, at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product. The milk product inoculated with the starter culture and first and second lactose hydrolyzing enzymes were incubated together at 42°C for a duration of 6-24 h, or until the milk product reached a pH value at or below 4.8, or at or below pH 4.6. The fermented milk product was removed from the incubation temperature of 42°C and placed at 4°C. The fermented milk product enriched with fibers was be stored at 4°C for up to 60 days. FIG. 2 shows fiber accumulation, lactose reduction, galactose accumulation and glucose accumulation in the milk-based composition (i.e., whole milk inoculated with a starter culture composition, BHT, and P-galactosidase) over time using multiple concentrations of BHT (1.5 U / g; 2.0 U / g; 2.5 U / g; and 3.0 U / g). The composition was incubated at 42°C for 8 hours, followed by a shift to 4°C for the time period as indicated. As depicted in FIG. 2, increasing concentrations of BHT between 1.5 U / g lactose and 3.0 U / g lactose were generally associated with increased fiber accumulation, increased lactose reduction, and increased glucose accumulation in the composition. Similarly, the highest galactose accumulation was observed using BHT at 3.0 U / g lactose after 8 hours at 42°C. Surprisingly, increasing concentration of BHT was generally associated with increased rates of fiber accumulation, lactose reduction and glucose accumulation in the liquid milk composition in the presence of P-galactosidase. Notably, within 8 hours lactose content was reduced between 45%-55% (from 50 g / U to between 22.5-27.5 g / U) in the milk composition, while fiber accumulation ranged between 10-20 g / U within the same period of time. These results demonstrate that BHT can effectively reduce lactose and generate GOS compositions when inoculated into fresh, unprocessed milk in the presence of a starter culture composition and P-galactosidase within a period of 8 hours.

[0168] FIG. 3 shows fiber accumulation, lactose reduction, galactose accumulation and glucose accumulation in a milk-based composition (i.e., whole milk inoculated with a starter culture composition, BHT, and P-galactosidase) over time BHT compared to a similar milkbased composition treatment where BHT was not added. These data allow a direct comparison between a method using p-galactosidase alone compared to P-galactosidase and BHT in combination. The compositions were incubated at 42°C for either 6 hours or 24 hours, followed by a shift to 4°C for a period of around 60 days to assess stability of these metrics within the composition during storage at 4°C. As depicted in FIG. 3, the presence of BHT was generally associated with increased fiber accumulation, increased lactose reduction, lower galactose accumulation, and increased glucose accumulation in the composition. Increasing the length of time - from 6 hours to 24 hours - incubated at 42°C was associated with increased fiber accumulation, increased lactose reduction, lower galactose accumulation, and increased glucose accumulation in the composition. Notably, fiber accumulation and glucose accumulation were dependent upon the presence of BHT. Surprisingly, the presence of BHT resulted in significantly higher fiber accumulation and lactose reduction compared to P-galactosidase alone. These results demonstrate that BHT can effectively reduce lactose and generate GOS compositions when inoculated into fresh, unprocessed milk in the presence of a starter culture composition and P-galactosidase and that the levels of fiber, lactose, galactose, and glucose remain stable in compositions during the incubation at 4 °C for up to 60 days.

[0169] EXAMPLE 5: Production of milk products utilizing both BHT & P-galactosidase for GOS compositions enriched with the human milk oligosaccharide (HMO) N- acetyllactosamine (LacNAc) and reduced lactose content

[0170] A milk product (e.g., a liquid milk product) at an initial pH value between 6.5-7.0 is inoculated with a starter culture containing at least one lactic acid bacterium (LAB) and a lactose transferase enzyme, P-hexosyltransferase (BHT), and a lactose hydrolase enzyme, active P-galactosidase, at concentrations between 0.01 U / g and 5.0 U / g of lactose present in the milk product. N-acetylglucosamine is added at a ratio of 8: 1 lactose to N- acetylglucosamine. The milk product inoculated with the starter culture and first and second lactose hydrolyzing enzymes with N-acetylglucosamine are incubated together at 42°C for a duration of 6-24 h, or until the milk product reaches a pH value at or below 4.8, or at or below pH 4.6. The fermented milk product is removed from the incubation temperature of 42°C and placed at 4°C. The fermented milk product enriched with fibers can be stored at 4°C for up to 60 days.

[0171] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0172] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0173] While various aspects of the invention are described herein, it is not intended that the invention be limited by any particular aspect. On the contrary, the invention encompasses various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Furthermore, where feasible, any of the aspects disclosed herein may be combined with each other (e.g., the feature according to one aspect may be added to the features of another aspect or replace an equivalent feature of another aspect) or with features that are well known in the art, unless indicated otherwise by context.

Claims

What is claimed is:

1. A method of generating a galactooligosaccharide (GOS) composition from a milk product, said method comprising a. contacting said milk product with a starter culture composition comprising a plurality of microbial cells, and / or cell constituents, b. contacting said milk product with a cell-free P-hexosyltransferase, or active fragment thereof, and c. heating said milk product to a temperature of between 40°C-44°C for between 6-24 hours to generate a GOS composition.

2. The method of claim 1, wherein said method further comprises the step of contacting said milk product with N-acetylglucosamine (GlcNAc) prior to heating said milk product.

3. The method of claim 1 or 2, wherein said GOS composition comprises N- acetyllactosamine (LacNAc).

4. The method of any one of claims 1-3, wherein the milk product is non-human, mammal milk, such as cow milk or goat milk.

5. The method of claim 4, wherein said milk is unprocessed, raw milk.

6. The method of claim 4, wherein said milk is modified and / or processed milk.

7. The method of claim 6, wherein said milk is pasteurized milk.

8. The method of claim 6 or 7, wherein said milk is skimmed or semi-skimmed milk.

9. The method of any one of claims 1-8, wherein said milk product comprises between 2% and 50% (w / v) lactose.

10. The method of any one of claims 1-9, wherein said plurality of microbial cells comprises at least one lactic acid bacteria.

11. The method of any one of claims 1-10, wherein said milk product is maintained at a temperature of about 42°C for between 6 hours to 24 hours.

12. The method of any one of claims 1-11, wherein said P-hexosyltransferase, or active fragment thereof, is added to said milk product at a concentration of between 0.5 U / g and 5.0 U / g of lactose.

13. The method of any one of claims 1-12, wherein said heating step results in fermentation of said milk product.

14. The method of any one of claims 1-13, wherein the pH of the milk product prior to heating in step (c) is between pH 6.5 and pH 7.0.

15. The method of any one of claims 1-14, wherein said milk product is maintained at a temperature between 40°C-44°C until the pH of the GOS composition is at or below pH 4.8, or at or below pH 4.6.

16. The method of any one of claims 1-15, wherein said milk product is maintained at a temperature between 40°C-44°C until lactose content in said GOS composition is reduced between 20% and 99% compared to lactose content in said milk product.

17. The method of any one of claims 1-16, further comprising step (d) cooling the GOS composition to a temperature of about 4°C.

18. The method of any one of claims 1-17, further comprising the step of contacting said milk product with a lactose hydrolyzing enzyme, or active fragment thereof.

19. The method of claim 18, wherein said lactose hydrolyzing enzyme, or active fragment thereof, is contacted with said milk product prior to heating in step (c).

20. The method of claim 18 or 19, wherein said lactose hydrolyzing enzyme, or active fragment thereof, comprises a P-galactosidase, or active fragment thereof.

21. The method of any one of claims 18-20, wherein said method comprises the addition of a P-hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, resulting in greater efficacy of lactose reduction compared to said method when performed without addition of a P-hexosyltransferase, or active fragment thereof.

22. The method of any one of claims 18-21, wherein said method comprises the addition of a -hexosyltransferase, or active fragment thereof, and a P-galactosidase, or active fragment thereof, to said milk product, wherein GOS levels are decreased no more than 1% during said heating step.

23. The method of any one of claims 1-22, wherein said method further comprises maintaining the GOS composition at a temperature of no higher than about 4°C for 1-60 days.

24. The method of claim 23, wherein the pH of said GOS composition is no higher than pH 4.6 during storage at 4°C; optionally, wherein the pH is maintained at between pH 3.5 and 4.6.

25. The method of any one of claims 1-24, wherein said method comprises adding to said milk product and / or said GOS composition at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable product, such as salt or cocoa powder.

26. The method of any one of claims 1-25, wherein said GOS composition comprises a food or beverage product, such as a dairy-based food or a fermented or unfermented dairybased food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairybased beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula.

27. A milk-based composition, wherein said composition comprises a. a liquid milk product; b. a plurality of microbial cells, and / or cell constituents; and c. a cell-free P-hexosyltransferase enzyme, or active fragment thereof, wherein the pH of said composition is between pH 6.5 and pH 7.0.

28. The milk-based composition of claim 27, wherein said composition further comprises N-acetylglucosamine (GlcNAc).

29. The milk-based composition of claim 27 or 28, wherein said milk product is nonhuman, mammal milk, such as cow milk or goat milk.

30. The milk-based composition of any one of claims 27-29, wherein said milk is unprocessed, raw milk.

31. The milk-based composition of any one of claims 27-29, wherein said milk is modified and / or processed milk.

32. The milk-based composition of claim 31, wherein said milk is a pasteurized milk.

33. The milk-based composition of claim 31 or 32, wherein said milk is skimmed or semiskimmed milk.

34. The milk-based composition of any one of claims 27-33, wherein said plurality of microbial cells comprises at least one lactic acid bacteria.

35. The milk-based composition of any one of claims 27-34, further comprising a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof.

36. The milk-based composition of any one of claims 27-35, wherein said P- hexosyltransferase enzyme and / or said lactose hydrolyzing enzyme, or active fragment(s) thereof, is present at a concentration of 2.5 U / g or below.

37. The milk-based composition of any one of claims 27-36, wherein said composition comprises a galactooligosaccharide (GOS) content of 0.5% (w / v) or below.

38. A galactooligosaccharide (GOS) composition, wherein said GOS composition comprisesN-acetyllactosamine (LacN Ac) -containing GOS, and / or GOS without LacNAc; and a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is between pH 3.4 and pH 7.0.

39. The GOS composition of claim 38, wherein said composition further comprises N- acetylglucosamine (GlcNAc).

40. The GOS composition of claim 38 or 39, wherein said plurality of microbial cells comprises at least one lactic acid bacteria.

41. The GOS composition of any one of claims 38-40, wherein said composition comprises a p-hexosyltransferase, or active fragment thereof.

42. The GOS composition of any one of claims 38-41, wherein said composition comprises a lactose hydrolyzing enzyme, such as a P-galactosidase, or active fragment thereof.

43. The GOS composition of any one of claims 38-42, wherein said composition comprises lactose in the range of between 0.5% and 10% (w / v).

44. The GOS composition of any one of claims 38-43, wherein said composition comprises glucose in the range of between 0.001% and 1% (w / v).

45. The GOS composition of any one of claims 38-44, wherein said composition comprises galactose in the range of between 0.001% and 1.5% (w / v).

46. A galactooligosaccharide (GOS) composition, wherein said GOS composition comprisesN-acetyllactosamine (LacNAc)-enriched GOS and / or GOS without LacNAc; and a plurality of microbial cells, and / or cell constituents, wherein the pH of said composition is pH 4.8 or less than pH 4.8.

47. The GOS composition of claim 46, wherein the pH of said composition is between pH 3.4 and pH 4.8.

48. The GOS composition of claim 46 or 47, wherein said plurality of microbial cells comprises at least one lactic acid bacteria.

49. The GOS composition of any one of claims 46-48, wherein said composition comprises a P-hexosyltransferase, or active fragment thereof.

50. The GOS composition of any one of claims 46-49, wherein said composition comprises a lactose hydrolyzing enzyme, such as a -galactosidase, or active fragment thereof.

51. The GOS composition of any one of claims 46-50, wherein said composition comprises lactose in the range of between 0.5% and 3% (w / v).

52. The GOS composition of any one of claims 46-51, wherein said composition comprises glucose in the range of between 0.6% and 1% (w / v).

53. The GOS composition of any one of claims 46-52, wherein said composition comprises galactose in the range of between 0.6% and 1.5% (w / v).

54. The milk-based composition of any one of claims l- 1 or the GOS composition of any one of claims 38-53, wherein said composition further comprises at least one vitamin, mineral, protein, fat, fruit, nutritional supplement, coloring agent, and / or food-based consumable.

55. The milk-based composition of any one of claims 27-37 or 54 or the GOS composition of any one of claims 38-54, wherein said composition is in the form of a liquid, solid, semi-solid, or powder.

56. A food or beverage product comprising the milk-based composition of any one of claims 27-37 or 54-55 or the GOS composition of any one of claims 38-55.

57. The food or beverage product of claim 56, wherein said product comprises a dairybased food, such as a fermented or unfermented dairy-based food, a yogurt-based product, a yogurt containing product, a yogurt powder, a dairy-based beverage, a pasteurized food or beverage, a protein shake, a smoothie, a cheese-based product, an ice-cream-based product, a fruit beverage, a fruit preparation, a milk-based powder, an energy beverage, a meal replacement beverage, or baby or infant formula.

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