Method for producing galactooligosaccharides

Abstract

A method for producing galactooligosaccharides (GOS) includes the steps of: (a) contacting a lactose-containing product with a first β-galactosidase enzyme to form a first composition; (b) inactivating the first β-galactosidase enzyme in the first composition; (c) adjusting the temperature of the first composition to a temperature within the range of 5-55°C; (d) contacting the first composition with a second β-galactosidase enzyme to form a second composition; (e) inactivating the second β-galactosidase enzyme in the second composition; and (f) membrane filtering the second composition to form a concentrated (GOS) composition. The GOS composition can contain 0.5-6 wt.% lactose and 24-50 wt.% GOS, based on carbohydrate, and often has a GOS:lactose mass ratio in the range of 6:1 to 45:1.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application is filed as a PCT international patent application on November 20, 2023, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 384,619, filed November 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates generally to the preparation of galactooligosaccharides from lactose-containing dairy streams. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] IUPAC Compendium of Chemical Terminology, 2nd Edition (1997) [Non-patent document 2] Standard Methods for the Examination of Dairy Products, 17th Edition (2004), American Public Health Association, Washington, DC Summary of the Invention [Means for solving the problem]

[0004] This Summary is provided to introduce some concepts in a simplified form that are further described herein. This Summary is not intended to identify required or essential features of the claimed subject matter. Nor is this Summary intended to be used to limit the scope of the claimed subject matter.

[0005] Methods for producing galactooligosaccharides are disclosed and described herein. Exemplary methods include: (a) contacting a lactose-containing product with a first β-galactosidase enzyme to form a first composition; (b) inactivating the first β-galactosidase enzyme in the first composition; (c) adjusting the temperature of the first composition to a range of 5 to 55°C; (d) contacting the first composition with a second β-galactosidase enzyme to form a second composition; (e) inactivating the second β-galactosidase enzyme in the second composition; and (f) filtering the second composition through a membrane to form a concentrated galactooligosaccharide (GOS) composition.

[0006] Galactooligosaccharide (GOS) compositions are also disclosed and described herein. Exemplary compositions may contain, on a carbohydrate basis, 0.5-6 wt.% lactose and 24-50 wt.% GOS (DP3+). The mass ratio of GOS:lactose in the composition is often in the range of 6:1 to 45:1.

[0007] Both the foregoing summary and the following detailed description are exemplary and explanatory only. Therefore, the foregoing summary and the following detailed description should not be considered limiting. Furthermore, features or variations may be provided in addition to those described herein. For example, certain aspects may be directed to combinations and subcombinations of various features described in the detailed description.

[0008] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description and examples. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows a schematic flow chart of a method for producing galactooligosaccharides (GOS) consistent with an embodiment of the present invention.

[0010] definition The following definitions are provided to more clearly define the terms used herein. Unless otherwise indicated, the following definitions apply to this disclosure. When a term is used in this disclosure but is not expressly defined herein, the definition from the IUPAC Compendium of Chemical Terminology, Second Edition (1997) may apply, unless it conflicts with any other disclosure or applicable definition herein and renders unclear or impossible any claim to which that definition may apply. To the extent that any definition or usage provided by any document incorporated by reference herein conflicts with the definition or usage provided herein, the definition or usage provided herein shall control.

[0011] Features of the subject matter are described herein such that various combinations of features may be envisioned within particular embodiments. For any and all embodiments and / or features disclosed herein, all combinations that do not adversely affect the designs, compositions, processes, and / or methods described herein are contemplated, regardless of whether a particular combination is explicitly described. Additionally, unless expressly stated otherwise, any of the embodiments and / or features disclosed herein may be combined to describe inventive designs, compositions, processes, and / or methods consistent with the present invention.

[0012] In this disclosure, compositions and methods are often described as "comprising" various components or steps, but unless otherwise specified, the compositions and methods can also "consist essentially of" or "consist of" those various components or steps. The terms "a," "an," and "the" are intended to include plural alternatives, e.g., at least one, unless otherwise specified.

[0013] In the disclosed methods, the term "contacting" includes combining components in any order, in any manner, and for any length of time, unless otherwise specified. For example, the components can be blended or mixed.

[0014] Several types of ranges are disclosed herein. When any type of range is disclosed or claimed, the intent is to individually disclose or claim each possible number that such range can reasonably encompass, including the endpoints of the range and any subranges, and combinations of subranges subsumed therein. For example, a lactose-containing product may contain 6-50 wt.% lactose in an embodiment of the invention. By disclosing that the amount of lactose in a lactose-containing product is 6-50 wt.%, the intent is to state that the amount of lactose can be any amount within that range, such as any range or combination of the 6-50 wt.% range, e.g., 8-40 wt.%, 10-30 wt.%, or 10-20 wt.%, etc. Similarly, all other ranges disclosed herein should be interpreted in a similar manner.

[0015] In general, an amount, size, formulation, parameter, range, or other quantity or characteristic is "about" or "approximately," whether or not expressly stated to be so. Whether or not modified by the term "about" or "approximately," the claims include the equivalent of that quantity or characteristic.

[0016] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods, devices, and materials are described herein.

[0017] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in the publications and patents that might be used in connection with the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] Disclosed herein is a method for producing galactooligosaccharides (GOS) from lactose-containing dairy streams. Lactose, also known as milk sugar, is a disaccharide composed of two sugar monomers, D-glucose and D-galactose, interconnected by a β1-4 bond, and has the chemical structure (β-D-galactopyranosyl-(1→4)-α-D-glucopyranose). Lactose is a component of all mammalian milk, ranging in concentration between 2 and 10 wt.%. Cow's milk is the most widely consumed milk in the United States, with an average lactose level of 4.8 wt.%. Lactose is considered a by-product of many dairy processing operations, such as cheese, yogurt, and membrane filtration. Lactose's relatively low sweetness index and poor solubility limit its use as a sweetener. While a valuable nutrient, especially for infants, lactose intolerance poses an additional challenge to its use as a sweetener. On average, approximately 65% ​​of the population is reported to be lactose intolerant to some degree. Lactose intolerance refers to an individual's impaired ability to digest lactose due to a lack of the lactase enzyme. Furthermore, lactose's high oxygen demand poses additional challenges for its disposal. All these factors point to the need for alternatives to maximize lactose utilization. Bioconversion of lactose into glucose-galactose syrup as a sweetener is one alternative. However, the relatively high cost and bitter taste of the syrup limit its application.

[0019] Another alternative is to create value-added ingredients from underutilized lactose streams, such as galactooligosaccharides (GOS), lactulose, polylactose, and lactobionic acid. GOS are a new class of natural dietary fiber with a wide range of variability in composition. GOS compositions can have 2-20 molecules of galactose and 1 molecule of glucose. Galactooligosaccharides occur naturally in breast milk, garlic, onion, soybean, and chicory root.

[0020] The prebiotic activity of GOS has been the subject of much research into its health benefits. Prebiotics are generally defined as indigestible food ingredients that beneficially affect the host by selectively stimulating the growth and / or activity of one or more limited bacterial species in the colon, thus improving host health. GOS are acid-resistant, allowing them to withstand the acidic environment of the stomach and remain undigested, with approximately 90% of ingested contents reaching the intestine. The human intestinal microbiota is inherently complex, consisting of over 500 bacterial species. These species possess various genes that enable them to ferment indigestible carbohydrates, producing a wide range of bioactive compounds, including short-chain fatty acids (SCFAs). SCFAs directly affect the function of the intestinal mucosa, serving as an energy source and regulating water and mineral absorption. Studies have shown that GOS specifically stimulates the growth of bifidobacteria and lactobacilli, the predominant bacteria in the human intestine. Other health benefits associated with GOS consumption include enhanced intestinal function, reduced incidence of intestinal inflammation, and modulation of the immune response to allergies.

[0021] Traditionally, GOS are produced by acid hydrolysis of lactose, resulting in the formation of a complex mixture of di- and trisaccharides along with anhydrosugars. The harsh processing parameters and the complex nature of the products limit the feasibility of scaling up this method. Currently, GOS are often produced through the catalytic activity of glycoside hydrolases, also known as β-galactosidases. These enzymes function through two steps: hydrolysis and transgalactosylation. In the hydrolysis step, lactose is cleaved into its component monosaccharides, glucose and galactose. Transgalactosylation results in chain elongation, forming GOS chains of different lengths in which β(1→6)-linked galactopyranosyl units are attached to terminal glycopyranosyl residues via α(1→4) glycosidic bonds.

[0022] These two steps occur simultaneously. Nevertheless, the relatively low efficiency of the transgalactosylation reaction and the low yield of GOS are challenges of this method. This method results in a complex mixture containing glucose, galactose, a large amount of residual lactose, and GOS with different structures.

[0023] The objective of the present invention is to overcome the shortcomings of conventional methods for producing GOS, particularly to maximize the yield of GOS and reduce the residual lactose level. However, the majority of research has been done on lactose-rich streams that are by-products of the cheese industry. These streams will typically contain salts, cultures, or enzymes that can negatively impact the flavor profile, purity, and further processability of the stream.

[0024] The disclosed method can start with a relatively concentrated lactose-containing product or stream, which is then subjected to two sequential enzyme treatments, with a heat treatment after each enzyme treatment step and a membrane filtration step. In the first enzyme treatment step, the lactose product is treated with a beta-galactosidase enzyme to convert lactose to GOS. After a high yield of GOS is obtained, the enzyme is then heat-inactivated. The resulting composition contains a mixture of GOS of various degrees of polymerization (DP), residual lactose, DP2, glucose, and galactose. In the second step, the composition is treated with a different source of beta-galactosidase enzyme, which has a higher hydrolytic activity than the enzyme from the first step. Additionally, this enzyme selectively hydrolyzes lactose with minimal impact on GOS. This step reduces residual lactose while maximizing the retention of the GOS formed. This step is also followed by heat inactivation. Finally, membrane filtration is used for separation / concentration to retain the purer GOS stream, while glucose, galactose, DP2 sugars, and lactose permeate. The amount of GOS in the compositions herein is based on galactooligosaccharides with a degree of polymerization of 3 or greater (DP3+).

[0025] Method for producing galactooligosaccharides According to one aspect of the present invention, there is provided a method for producing galactooligosaccharides (GOS), which method can comprise (or consist essentially of, or consist of) the steps of: (a) contacting a lactose-containing product with a first β-galactosidase enzyme to form a first composition; (b) inactivating the first β-galactosidase enzyme in the first composition; (c) adjusting the temperature of the first composition to be within the range of 5 to 55°C; (d) contacting the first composition with a second β-galactosidase enzyme to form a second composition; (e) inactivating the second β-galactosidase enzyme in the second composition; and (f) membrane filtration of the second composition to form a concentrated galactooligosaccharide (GOS) composition.

[0026] Generally, the features of the methods (e.g., the lactose-containing product, the characteristics of the first β-galactosidase enzyme, the first composition, the characteristics of the second β-galactosidase enzyme, the characteristics of the second composition, the characteristics of the GOS composition, and the conditions under which any of the steps are carried out, among others) are described individually herein and can be combined in any combination to further describe the disclosed methods. Furthermore, unless otherwise specified, other steps can be performed before, during, and / or after any of the steps recited in the disclosed methods. Additionally, any GOS composition (e.g., a concentrated GOS composition) produced according to the disclosed methods is within the scope of the present disclosure and is encompassed herein.

[0027] Filtration techniques (e.g., ultrafiltration, nanofiltration, diafiltration, etc.) can separate or concentrate components in a mixture such as milk by passing the mixture through a membrane system (or selective barrier) under appropriate conditions (e.g., pressure). Concentration / separation can therefore be based on molecular size. The stream retained by the membrane is called the retentate (or concentrate). The stream that passes through the pores of the membrane is called the permeate.

[0028] Referring now to step (a), a lactose-containing product is contacted with a first β-galactosidase enzyme to form a first composition. Often, but not exclusively, the lactose-containing product can contain 6-50 wt.% lactose, such as 8-40 wt.%, 10-30 wt.%, or 10-20 wt.% lactose. In one embodiment, for example, the lactose-containing product can comprise a nanofiltration (NF) retentate fraction (typically from the ultrafiltration permeate fraction of whole milk or skim milk). Optionally, the lactose-containing product can comprise a concentrated NF retentate fraction (e.g., in which the NF retentate fraction has been concentrated by forward osmosis or reverse osmosis, or other suitable technique).

[0029] Whole milk before UF can be cow's milk, which contains approximately 87 wt.% water, 3-4 wt.% protein, 4-5 wt.% carbohydrates / lactose, 3-4 wt.% fat, and 0.3-0.8 wt.% minerals. Skim milk can be prepared by separating whole milk (e.g., by centrifugation or microfiltration) into skim milk and a fat-rich fraction (also called cream or butterfat). The fat-rich fraction typically contains high levels of fat (e.g., 20-50 wt.% fat, 30-50 wt.%, 35-45 wt.% fat, or 38-42 wt.%) and solids (e.g., 30-60 wt.%, 40-55 wt.%, 40-50 wt.%, or 42-47 wt.%), and often contains approximately 1-4 wt.% protein (or 1-3 wt.%, or 2-3 wt.%), 2-5 wt.% lactose (or 2.5-4 wt.%, or 2.5-3.5 wt.%), and 0.2-0.9 wt.% minerals (or 0.2-0.6 wt.%, or 0.2-0.4 wt.%), but is not limited thereto. In contrast, skim milk prior to UF typically contains very low levels of fat (e.g., 0.5 wt.% or less, 0.35 wt.% or less, or 0.2 wt.% or less) and significantly fewer solids than the fat-rich fraction (e.g., 7-13 wt.%, 8-12 wt.%, 8.5-10 wt.%, or 9-9.5 wt.%); skim milk often contains, but is not limited to, approximately 2-5 wt.% protein (or 3-4 wt.%, or 3.2-3.7 wt.%), 3-6 wt.% lactose (or 4-5.5 wt.%, or 4.5-5 wt.%), and 0.4-1.2 wt.% minerals (or 0.4-0.9 wt.%, or 0.5-0.9 wt.%).

[0030] In addition to the lactose content described above, the lactose-containing product of step (a) may contain, but is not necessarily limited to, 0.01-1 wt.%, 0.01-0.5 wt.%, or 0.01-0.2 wt.% fat. Additionally or alternatively, the lactose-containing product may contain 0.1-2 wt.%, 0.1-1.2 wt.%, or 0.2-1 wt.% protein. Additionally or alternatively, the lactose-containing product may contain 0.5-3 wt.%, 0.7-2.5 wt.%, or 1-2 wt.% minerals.

[0031] Any suitable β-galactosidase enzyme can be used as the first β-galactosidase enzyme in step (a). Generally, the first β-galactosidase enzyme is of a type that produces galactooligosaccharides (GOS) from lactose. In a particular embodiment of step (a), the first β-galactosidase enzyme can be derived from Bifidobacterium bifidum.

[0032] Step (a) can be carried out under any suitable temperature and time conditions. Without limitation, step (a) can be carried out at a first temperature ranging from 20 to 70°C, such as 25 to 65°C, 30 to 70°C, 30 to 60°C, or 35 to 55°C. In one aspect, step (a) can be carried out at a temperature higher than room temperature (e.g., 20 to 25°C), and thus the lactose-containing product and the first enzyme can be heated to a desired high temperature and contacted for a suitable time to form the first composition.

[0033] With respect to the duration of step (a), step (a) is often carried out for a first time period ranging from 15 minutes to 10 hours. Other illustrative ranges for the first time period include 30 minutes to 8 hours, 45 minutes to 6 hours, 1 hour to 4 hours, or 90 minutes to 150 minutes.

[0034] As one skilled in the art would readily understand, the amount of the first enzyme used in step (a) can vary widely based on, among other considerations, the first temperature, the first time period, and the amount of lactose in the lactose-containing product. Without limitation, the amount of the first β-galactosidase enzyme can range from 0.05 to 1 wt.%, 0.1 to 0.8 wt.%, or 0.15 to 0.65 wt.%, based on the total weight of the lactose-containing product. Based on the starting amount of lactose, the amount of the first β-galactosidase enzyme can range from 1 to 15 wt.%, 1.5 to 6 wt.%, 1.5 to 4 wt.%, or 2 to 3 wt.%, based on the amount of lactose in the lactose-containing product, although, as noted above, this amount can vary based on the temperature and time conditions in step (a).

[0035] Referring now to step (b), the first β-galactosidase enzyme in the first composition is inactivated. In step (b), any suitable method for inactivating the enzyme can be utilized, but inactivation is conveniently achieved by exposure to a relatively high temperature for a suitable period of time. Thus, the inactivation step in step (b) can include subjecting the first composition to high temperature or heat-treating the first composition at high temperature.

[0036] Step (b) can be carried out under any temperature and time conditions that sufficiently inactivate the first β-galactosidase enzyme in the first composition. The inactivation step of step (b) can include, but is not limited to, subjecting the first composition to a temperature in the range of 75 to 150°C, such as 80 to 120°C, 80 to 115°C, or 85 to 105°C, or heat-treating the first composition at a temperature in the range of 75 to 150°C, such as 80 to 120°C, 80 to 115°C, or 85 to 105°C.

[0037] Regarding the duration of step (b), the inactivation step of step (b) often involves subjecting the first composition to the above-mentioned temperature for a time period ranging from 1 minute to 6 hours, or heat-treating the first composition at the above-mentioned temperature for a time period ranging from 1 minute to 6 hours. Other illustrative ranges of the heat treatment time for inactivating the enzyme in step (b) include 1 minute to 30 minutes, 2 minutes to 20 minutes, or 4 minutes to 15 minutes.

[0038] In step (c), the temperature of the first composition is adjusted to within the range of 5 to 55° C. Thus, for example, when high-temperature enzyme inactivation is utilized in step (b), the first composition is cooled to a temperature within the range of 5 to 55° C. In one embodiment, the temperature of the first composition in step (c) is 10 to 55° C., while in another embodiment, the temperature is 20 to 50° C., in yet another embodiment, the temperature is 25 to 45° C., and in yet another embodiment, the temperature is 30 to 45° C. Other suitable temperatures and ranges can be used if desired.

[0039] At this stage of the process (before step (d) and addition of the second enzyme), the first composition can often contain, but is not limited to, 1-5 wt.%, 1.5-4.5 wt.%, or 2-3.5 wt.% lactose. Additionally or alternatively, the first composition can contain 4-10 wt.%, 5-9 wt.%, or 6-8.5 wt.% GOS. However, any combination of the above amounts of lactose and GOS can be present in the first composition, and beneficially, the GOS:lactose mass ratio can be in the range of 1.6:1-3.5:1, 2:1-3:1, or 2.2:1-2.8:1, but is not limited to these ranges.

[0040] The first composition may contain 10-20 wt.%, 12-18 wt.%, or 13-17 wt.% lactose based on the carbohydrates in the first composition. Additionally or alternatively, the first composition may contain 30-44 wt.%, 32-42 wt.%, or 34-40 wt.% GOS based on the carbohydrates.

[0041] After the first enzyme treatment, the first composition (before step (d)) contains significantly less lactose than is present in the lactose-containing product of step (a). Advantageously, the first composition contains 60-95 wt.% less lactose than is present in the lactose-containing product of step (a). More often, the first composition contains 70-90 wt.% or 75-85 wt.% less lactose than is present in the lactose-containing product of step (a).

[0042] In step (d), the first composition is contacted with a second β-galactosidase enzyme to form a second composition. Any suitable β-galactosidase enzyme can be used as the second β-galactosidase enzyme in step (d). Generally, the second β-galactosidase enzyme is of a type that preferentially hydrolyzes lactose to form glucose and galactose. In certain embodiments of step (d), the second β-galactosidase enzyme can be derived from Kluyveromyces lactis.

[0043] Step (d) can be carried out under any suitable temperature and time conditions. Without limitation, step (d) can be carried out at a second temperature ranging from 10 to 60°C, such as 20 to 50°C, 25 to 45°C, or 30 to 45°C. In one embodiment, step (d) can be carried out at a temperature higher than room temperature (e.g., 20 to 25°C), whereby the first composition and the second enzyme can be heated to a desired high temperature (or controlled at a desired high temperature) and contacted for an appropriate time to form the second composition.

[0044] With respect to the duration of step (d), step (d) is often carried out for a second time period ranging from 15 minutes to 10 hours. Other illustrative ranges for the second time period include 30 minutes to 8 hours, 45 minutes to 6 hours, 1 hour to 4 hours, or 90 minutes to 150 minutes.

[0045] As will be readily understood by those skilled in the art, the amount of the second enzyme used in step (d) can vary widely based on the second temperature, the second time period, and the amount of lactose in the first composition, among other considerations. Without limitation, the amount of the second β-galactosidase enzyme can range from 0.01 to 0.6 wt.%, 0.03 to 0.3 wt.%, or 0.04 to 0.1 wt.%, based on the total weight of the first composition. The amount of the second β-galactosidase enzyme can range from 0.35 to 20 wt.%, 1 to 10 wt.%, or 1.4 to 4 wt.%, based on the amount of lactose in the first composition, although, as noted above, this amount can vary based on the temperature and time conditions in step (d).

[0046] Referring now to step (e), the second β-galactosidase enzyme in the second composition is inactivated. In step (e), any suitable method for inactivating the enzyme can be utilized, but inactivation is conveniently achieved by exposure to a relatively high temperature for a suitable period of time. Thus, the inactivation step in step (e) can include subjecting the second composition to high temperature or heat-treating the second composition at high temperature.

[0047] Step (e) can be carried out under any temperature and time conditions that sufficiently inactivate the second β-galactosidase enzyme in the second composition. The inactivation step of step (e) can include, but is not limited to, subjecting the second composition to a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C, or heat-treating the second composition at a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C.

[0048] Regarding the duration of step (e), the inactivation step of step (e) often involves subjecting the second composition to the above-mentioned temperature for a time period ranging from 1 minute to 6 hours, or heat-treating the second composition at the above-mentioned temperature for a time period ranging from 1 minute to 6 hours. Other illustrative ranges of the heat treatment time for inactivating the enzyme in step (e) include 1 minute to 30 minutes, 2 minutes to 20 minutes, or 4 minutes to 15 minutes.

[0049] When heat treatment is used for the quenching in step (e), the method can further comprise the step of cooling the second composition to a temperature in the range of 5 to 50°C (before step (f)). In one embodiment, the temperature of the second composition can be cooled or adjusted to a temperature in the range of 10 to 45°C or 20 to 50°C before step (f), while in another embodiment, the temperature of the second composition can be cooled or adjusted to a temperature in the range of 5 to 25°C or 8 to 20°C before step (f). In one embodiment, the temperature of the second composition before step (f) can be below room temperature (e.g., 20 to 25°C), and thus the second composition can be cooled to a temperature of 25°C or below, 20°C or below, 18°C ​​or below, or 15°C or below, with typical ranges including 8 to 18°C ​​and 10 to 15°C.

[0050] At this stage of the process (prior to step (f) and concentration by membrane filtration), the second composition can often contain, but is not limited to, 0.05-5 wt.%, 0.05-1.5 wt.%, 0.1-4 wt.%, 0.1-1 wt.%, 0.2-2 wt.%, or 0.2-0.9 wt.% lactose. Additionally or alternatively, the second composition can contain 4-15 wt.%, 4-9 wt.%, 5-12 wt.%, 5-8 wt.%, 5.5-9.5 wt.%, or 5.5-7.5 wt.% GOS. Any combination of the above amounts of lactose and GOS can be present in the second composition. However, the mass ratio of GOS:lactose in the second composition can beneficially be in the range of 4:1 to 40:1, 7:1 to 35:1, or 8:1 to 30:1, although this ratio is not limited to these ranges.

[0051] The second composition may contain 0.5 to 5 wt.%, 1 to 5 wt.%, or 2 to 4 wt.% lactose based on the carbohydrates in the second composition. Additionally or alternatively, the second composition may contain 20 to 45 wt.%, 25 to 40 wt.%, or 32 to 39 wt.% GOS based on the carbohydrates.

[0052] After the second enzyme treatment, the second composition (before step (f)) contains significantly less lactose than is present in the lactose-containing product of step (a). Advantageously, the second composition contains 80-99 wt.% less lactose than is present in the lactose-containing product of step (a). More often, the second composition contains 85-98 wt.% less, 90-99 wt.% less, 92-99 wt.% less, or 93-98 wt.% less lactose than is present in the lactose-containing product of step (a).

[0053] However, it is beneficial for the amount of GOS present in the second composition to be substantially the same as in the first composition. Typically, prior to step (f), the second composition contains no more than 3 wt.% less GOS than the GOS present in the first composition. More often, prior to step (d), the second composition contains no more than 2 wt.%, no more than 1 wt.%, no more than 0.8 wt.%, no more than 0.5 wt.%, or no more than 0.3 wt.% less GOS than the GOS in the first composition.

[0054] In step (f), the second composition is subjected to membrane filtration to form a concentrated galactooligosaccharide (GOS) composition. In one embodiment, step (f) can include nanofiltration of the second composition through a polymeric membrane (or alternatively, through a ceramic membrane) to form a concentrated GOS composition (which can also be referred to as an NF retentate). In another embodiment, step (f) can include diafiltration of the second composition through a nanofiltration membrane (polymeric or ceramic). Without wishing to be bound by theory, it is believed that polymeric membrane filtration is generally more suitable than ceramic membrane filtration in step (f) due to the need for smaller pore sizes and lower molecular weight cutoffs. Furthermore, diafiltration of the second composition through a nanofiltration membrane can include diafiltration of a mixture of the second composition and water. Any suitable water source, such as RO permeate, can be used in the mixture.

[0055] Nanofiltration (or diafiltration) can be performed using nanofiltration membranes with pore sizes typically in the range of 0.001 to 0.01 microns, e.g., pore sizes in the range of 0.001 to 0.008 μm. In some embodiments, the nanofiltration process utilizes a membrane system with pore sizes in the range of 0.001 to 0.01 μm.

[0056] Nanofiltration in the dairy industry typically uses a membrane element that retains particles having a molecular weight above a certain molecular weight (measured in Daltons, Da). Nanofiltration is a pressure-driven process in which the starting composition is forced through a membrane under pressure, and materials having a molecular weight greater than a specified cutoff are largely retained, while smaller particles largely pass through the membrane pores. Nanofiltration can therefore perform both concentration and separation simultaneously.

[0057] Regarding the membrane filtration in step (f), membranes can often be specified based on their molecular weight cut-off (MWCO) rather than pore size. Membrane systems, such as polymer nanofiltration systems, generally retain materials with molecular weights greater than their MWCO numbers. The second composition can be subjected to membrane filtration using a membrane with a molecular weight cut-off (MWCO) of at least 150 Da and less than 1000 Da. Thus, suitable membranes may have a MWCO in the range of from 150 Da to 900 Da, alternatively from 150 Da to 500 Da, alternatively from 150 to 300 Da, alternatively from 200 to 900 Da, alternatively from 200 to 800 Da, alternatively from 300 to 900 Da, alternatively from 300 to 800 Da, alternatively from 300 to 700 Da, alternatively from 300 to 500 Da, alternatively from 500 to 1000 Da, alternatively from 500 to 800 Da, alternatively from 500 to 700 Da, alternatively from 600 to 900 Da, or alternatively from 600 to 800 Da. For example, the step of performing membrane filtration on the second composition using a membrane having a molecular weight cut-off (MWCO) in the range of 300 to 800 Da includes the use of a membrane having a MWCO of 300 to 500 Da, the use of a membrane having a MWCO of 500 to 700 Da, and the use of a membrane having a MWCO of 600 to 800 Da.

[0058] In one embodiment, the MWCO of the membrane utilized in step (f) ranges from 200 to 800 Da, while in another embodiment, the MWCO is from 300 to 800 Da, in another embodiment, the MWCO is from 300 to 700 Da, in yet another embodiment, the MWCO is from 500 to 800 Da, and in yet another embodiment, the MWCO is from 500 to 700 Da.

[0059] Step (f) can be performed at a filtration temperature in the range of, but not limited to, 5 to 50° C. In one embodiment, the filtration temperature in step (f) can be in the range of 10 to 45° C. or 20 to 50° C., and in another embodiment, the filtration temperature in step (f) can be in the range of 5 to 25° C. or 8 to 20° C. In one embodiment, the filtration temperature in step (f) can be set to room temperature or lower (e.g., 20 to 25° C.), and therefore the filtration temperature can be 25° C. or lower, 20° C. or lower, 18° C. or lower, or 15° C. or lower, with typical ranges including 8 to 18° C. and 10 to 15° C.

[0060] The operating pressure of the membrane filtration step (f) is not particularly limited, but is generally in the range of 100 to 1000 psig. In one embodiment, step (f) is carried out at a pressure in the range of 200 to 800 psig, while in another embodiment, the pressure is in the range of 300 to 650 psig, and in yet another embodiment, the pressure is in the range of 320 to 560 psig.

[0061] At this stage of the process (after step (f) and concentration / separation by membrane filtration), the concentrated GOS composition can often contain, but is not limited to, 0.1-3 wt.%, 0.5-2 wt.%, or 0.7-1.8 wt.% lactose. Additionally or alternatively, the concentrated GOS composition can contain 10-20 wt.%, 12-20 wt.%, or 14-18 wt.% GOS. However, any combination of the above amounts of lactose and GOS can be present in the concentrated GOS composition, and beneficially, the mass ratio of GOS:lactose in the concentrated GOS composition can range from 6:1 to 45:1, 7:1 to 35:1, or 8:1 to 30:1, but is not limited to these ranges.

[0062] The concentrated GOS composition can contain 0.5-6 wt.%, 1-5.5 wt.%, 2-6 wt.%, or 2.5-5 wt.% lactose based on the carbohydrates in the concentrated GOS composition. Additionally or alternatively, the concentrated GOS composition can contain 24-50 wt.%, 30-48 wt.%, 33-50 wt.%, or 38-46 wt.% GOS based on the carbohydrates in the concentrated GOS composition.

[0063] Optionally, the concentrated GOS composition can be heat-treated after step (f). In one embodiment, the heat-treating step can include pasteurization at a temperature ranging from 80°C to 95°C for a time ranging from less than 1 minute to 15 minutes, e.g., 2 to 15 minutes. In another embodiment, the heat-treating step can include UHT sterilization at a temperature ranging from 135°C to 145°C for a time ranging from 1 to 10 seconds. In yet another embodiment, the heat-treating step can include UHT sterilization at a temperature ranging from 148°C to 165°C for a time ranging from 0.05 to 1 second, e.g., 150°C to 155°C for a time ranging from 0.08 to 0.2 seconds. Other suitable pasteurization or sterilization temperature and time conditions will be readily apparent from the present disclosure. Furthermore, the present invention is not limited by the method or equipment used to carry out the pasteurization / sterilization process; any suitable techniques and equipment can be used, whether the operation is batch or continuous.

[0064] Typical UHT sterilization techniques include indirect heating, direct steam injection, and direct steam infusion. In the case of indirect heating, the GOS composition is not directly contacted with a heat source or heat medium, such as a heat exchanger. Indirect heating requires a longer time for sterilization due to heat transfer limitations. In embodiments of the present invention, the GOS composition is advantageously heat-treated using direct UHT sterilization. In direct steam injection, high-temperature steam is injected into a pipe or other container containing the GOS composition, thus rapidly sterilizing the GOS composition. Direct steam injection is generally performed continuously, where a continuous flow of the GOS composition is combined with continuous injection of steam. In direct steam injection, the GOS composition is sprayed into a chamber containing the steam, thus rapidly and uniformly sterilizing the GOS composition. Like direct steam injection, direct steam injection is generally performed continuously. After the heat treatment step, the heat-treated GOS composition can be cooled to any suitable temperature, for example, in the range of 5°C to 40°C or 10°C to 30°C.

[0065] The methods for producing galactooligosaccharides described herein can be carried out in either a batch or continuous mode. While the following examples are batch experiments, any step, or any combination of steps, in the methods can alternatively be carried out continuously. Any suitable container (e.g., tank, silo, etc.) can be used to carry out any step, any combination of steps, or all steps in the methods. The methods can further include packaging (aseptically or otherwise) the GOS composition in any suitable container under any suitable conditions; illustrative, non-limiting examples of typical containers include cups, bottles, bags, or pouches. The container can be made of any suitable material, such as glass, metal, plastic, and the like, as well as combinations thereof.

[0066] In some embodiments, the method for producing galactooligosaccharides can further include (i) measuring the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof, in the first composition prior to step (d), and (ii) adjusting the first temperature, the first time period, the amount of the first β-galactosidase enzyme, or any combination thereof, in step (a) based on the measured compositional characteristics of the first composition. For example, if the amount of lactose in the first composition is too high, the first temperature can be adjusted, the first time period can be adjusted, the amount of the first β-galactosidase enzyme can be adjusted, or any combination thereof can be adjusted, so that the amount of lactose in the first composition is reduced (e.g., to a desired target amount).

[0067] Similarly, the method for producing galactooligosaccharides can further include (i) measuring the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof, in the second composition prior to step (f), and (ii) adjusting the second temperature, the second time, the amount of the second β-galactosidase enzyme, or any combination thereof, in step (d) based on the measured compositional characteristics of the second composition. For example, if the amount of lactose in the second composition is too high, the second temperature can be adjusted, the second time can be adjusted, the amount of the second β-galactosidase enzyme can be adjusted, or any combination thereof can be adjusted, so that the amount of lactose in the second composition is reduced (e.g., to a desired target amount).

[0068] An illustrative, non-limiting example of a representative method 100 for producing galactooligosaccharides (GOS) consistent with embodiments of the present invention is shown in Figure 1. Initially, a lactose-containing product 105, which in Figure 1 contains 10-20 wt.% lactose, is heated to a temperature in the range of 35-55°C 110, then contacted with a first β-galactosidase enzyme, which may be derived from Bifidobacterium bifidum 115, and then incubated at an appropriate temperature and time combination, such as 35-55°C for 90-150 minutes 120. The resulting first composition is then heat-treated at 85-105°C for 5-10 minutes 125 to inactivate the first β-galactosidase enzyme. After cooling to a temperature of 30-45°C 130, a second β-galactosidase enzyme, which can be derived from Kluyveromyces lactis, is added 135 and then incubated at a suitable temperature and time combination, such as 30-45°C for 90-150 minutes 140. The resulting second composition is then heat treated at 85-105°C for 5-10 minutes 145 to inactivate the second β-galactosidase enzyme, followed by membrane filtration 150 to form a retentate fraction, which is the enriched galactooligosaccharide (GOS) composition 155.

[0069] Galactooligosaccharide (GOS) composition An illustrative, non-limiting example of a galactooligosaccharide (GOS) composition consistent with the present invention can contain 0.5-6 wt.% lactose and 24-50 wt.% GOS (DP3+) based on carbohydrate. Another illustrative, non-limiting example of a GOS composition consistent with the present invention contains lactose and GOS in a GOS:lactose mass ratio ranging from 6:1 to 45:1. Another illustrative, non-limiting example of a GOS composition consistent with the present invention can contain 0.5-6 wt.% lactose and 24-50 wt.% GOS based on carbohydrate, with the GOS:lactose mass ratio in the composition ranging from 6:1 to 45:1. These illustrative, non-limiting examples of GOS compositions consistent with the present invention can also have any of the characteristics listed below in any combination, unless otherwise specified.

[0070] In one aspect, any GOS composition described herein can contain 1-5.5 wt.% lactose, or 30-48 wt.% GOS, or both 1-5.5 wt.% lactose and 30-48 wt.% GOS. In another aspect, the GOS composition can contain 2-6 wt.% lactose, or 33-50 wt.% GOS, or both 2-6 wt.% lactose and 33-50 wt.% GOS. Additionally or alternatively, the GOS composition can contain 2.5-5 wt.% lactose, or 38-46 wt.% GOS, or both 2.5-5 wt.% lactose and 38-46 wt.% GOS. The respective amounts of lactose and GOS in the composition are based on total carbohydrate. Additionally or alternatively, the mass ratio of GOS:lactose in the GOS composition may fall within the ranges of 7:1 to 35:1, 7:1 to 15:1, 8:1 to 30:1, or 8:1 to 12:1.

[0071] As will be readily understood by those skilled in the art, a GOS composition is not limited to only lactose and GOS components. For example, a GOS composition can further comprise glucose, galactose, and a DP2 carbohydrate; alternatively, glucose and galactose; alternatively, glucose; alternatively, galactose; or alternatively, a DP2 carbohydrate. In one embodiment, the amount of galactose (or glucose) present in the GOS composition is greater than the amount of lactose. For example, the mass ratio of galactose to lactose in the GOS composition can fall within the ranges of 1.5:1 to 35:1, 1.7:1 to 10:1, 1.7:1 to 3:1, 2:1 to 30:1, 2:1 to 10:1, or 2:1 to 3:1. The mass ratio of glucose:lactose in GOS compositions can be higher, with typical ranges including 3:1 to 40:1, 3:1 to 20:1, 3:1 to 7:1, 4:1 to 40:1, 4:1 to 10:1, or 4:1 to 6:1. Without limitation, any GOS composition described herein can contain 5 to 20 wt.% galactose, e.g., 6 to 15 wt.% or 7 to 10 wt.% galactose, and / or 15 to 30 wt.% glucose, e.g., 18 to 26 wt.% or 20 to 24 wt.% glucose. These amounts are based on total carbohydrate.

[0072] Similarly, GOS compositions are not limited to carbohydrates and can also contain fat, protein, and minerals. For example, without limitation, GOS compositions can contain 0.01-2 wt.% fat, more often 0.03-1 wt.% or 0.03-0.2 wt.% fat. Additionally or alternatively, GOS compositions can contain 0.3-3 wt.% protein, more often 0.4-1.5 wt.% or 0.6-1.2 wt.% protein. Additionally or alternatively, GOS compositions can contain 0.5-5 wt.% minerals, more often 1-3 wt.% or 1.5-2.5 wt.% minerals. [Example]

[0073] The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the present invention in any way. Various other embodiments, modifications thereof, and equivalents may occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention or the scope of the appended claims.

[0074] Total solids (wt.%) was measured using a CEM Turbo Solids and Moisture Analyzer (CEM Corporation, Matthews, North Carolina) according to procedure SMEDP 15.10C. Ash is the residue remaining at a certain mass after ignition in a suitable apparatus at 550°C; such treatment at 550°C typically eliminates all organic matter, and the remaining material is primarily mineral (Standard Methods for the examination of dairy products, 17th ed. (2004), American Public Health Association, Washington, DC). Ash testing was performed using a Phoenix (CEM Corporation microwave oven), heating samples at 550°C for 30 minutes. Mineral content (in wt.%) is generally equivalent to ash content (in wt.%); therefore, ash test results are used to quantify total mineral content in this disclosure. Protein and fat contents were determined by AOAC (Association of Official Analytical Chemists) methods.

[0075] The carbohydrate profile (glucose, galactose, lactose, DP2, DP3, DP4, and DP4+) was measured by high-performance liquid chromatography (HPLC) using various detectors. HPLC-RI (refractive index) was used to quantify galactose, glucose, DP2, DP3, DP4, and DP4+. DP2 GOS was separated from lactose using the HPAEC-PAD (pulsed amperometric detection) method.

[0076] Example 1 Figure 1 summarizes the method used in Example 1, and Table I summarizes the carbohydrate degradation of certain starting, intermediate, and final compositions. The lactose-containing starting products used in the examples had 17-22 wt.% solids and a pH of 5.8-7.0, and contained 0.01-0.10 wt.% fat, 0.2-0.8 wt.% protein, 1-2 wt.% minerals, and 15-20 wt.% carbohydrates (the remainder was water).

[0077] In the first enzyme treatment step, each liter of lactose-containing product was combined with 3.6 g of beta-galactosidase enzyme (derived from Bifidobacterium bifidum) in a stirred tank at 55°C for 120 minutes to form a first composition, maximizing GOS yield. This first composition was gently mixed throughout to prevent lactose precipitation in the tank and enhance the interaction between the enzyme and GOS components. Enzyme deactivation was then performed at 95°C for 7.5 minutes to minimize or stop the hydrolytic action of the enzyme. If deactivation is not performed for an appropriate time, the reaction equilibrium may shift toward GOS hydrolysis, resulting in a decrease in GOS yield.

[0078] After the first enzyme treatment step, the temperature was lowered to 37°C. The first composition contained a mixture of GOS with various degrees of polymerization (DP), residual lactose, DP2, glucose, and galactose (see Table I). In the second enzyme treatment step, the first composition was contacted with approximately 0.06 wt.% of a different beta-galactosidase enzyme (derived from Kluyveromyces lactis) at a temperature of 37°C for 120 minutes to form a second composition. This step reduced the amount of residual lactose by hydrolyzing it to glucose and galactose. The enzyme in this step had higher hydrolytic activity, was highly selective for the degradation of lactose, and had a lower affinity for GOS. Gentle mixing was maintained throughout this step to enhance the interaction between the second enzyme and the residual lactose. The temperature and time utilized in the second enzyme treatment step are generally selected to achieve significant lactose reduction (e.g., 65-85%) while maintaining nearly all GOS (e.g., 95% or more).

[0079] A second enzyme inactivation was performed at 95°C for 7.5 minutes to minimize or stop enzymatic cleavage of GOS. If the inactivation is not performed for the appropriate time, the yield of GOS will be reduced. After the second enzyme treatment step, the temperature was reduced to 5-10°C. The second composition contained a mixture of GOS of various degrees of polymerization (DP), residual lactose, DP2, glucose, and galactose (see Table I).

[0080] The second composition was subjected to membrane filtration at temperatures ranging from 10 to 20°C and pressures ranging from 360 to 560 psig to concentrate the GOS approximately two-fold (high molecular weight) and permeate other (lower molecular weight) sugars, including DP2, lactose, glucose, and galactose. A nanofiltration unit was used to form the GOS composition (retentate) and the NF permeate. The nanofiltration unit used a polymeric membrane filter with a MWCO of 500 to 700 daltons.

[0081] Advantageously, in the method of Example 1 shown in Table 1, the first composition contained 6.8 wt.% GOS and only 2.8 wt.% lactose (a mass ratio of GOS:lactose equal to 2.4:1). On a carbohydrate basis, the first composition contained 36 wt.% GOS and only 15 wt.% lactose, meaning the first composition contained 80 wt.% less lactose than was present in the lactose-containing starting product.

[0082] Table I shows the surprisingly high amount of GOS relative to lactose in the second composition. The second composition contained 6.5 wt.% GOS (only 0.2-0.3 wt.% less than in the first composition) and only 0.7 wt.% lactose (a GOS:lactose mass ratio equal to 9.3:1). On a carbohydrate basis, the second composition contained 35 wt.% GOS and only 3.8 wt.% lactose, meaning the second composition contained 95 wt.% less lactose than the lactose present in the lactose-containing starting product.

[0083] After the membrane filtration process, the concentrated GOS composition unexpectedly had a high relative amount of GOS to lactose, and the total amount of carbohydrates was increased (higher % solids). The GOS composition contained 15.6 wt.% GOS (DP3+) and only 1.5 wt.% lactose (GOS:lactose mass ratio equal to 10.3:1). On a carbohydrate basis, the GOS composition contained 43 wt.% GOS and only 4 wt.% lactose. In Table I, the GOS composition had 35-36 wt.% solids and contained approximately 0.09 wt.% fat, 0.95 wt.% protein, and 2.1 wt.% minerals.

[0084] [Table 1]

[0085] Examples 2 to 4 In Examples 2-4, representative first compositions having 18-21 wt.% total solids with the ingredients and amounts shown in Table II were treated with different amounts of the second enzyme (0.06 wt.%, 0.1 wt.%, 0.4 wt.%) and incubated at a fixed temperature of 37°C for different times (90, 120, 180 minutes) to determine the effect on lactose, GOS(DP3+), and the GOS:lactose mass ratio in the second compositions (total solids ranged from 18-21 wt.%). The results are summarized in Table II.

[0086] Higher enzyme loadings generally decreased the amount of both lactose and GOS in the second composition, and generally increased the GOS:lactose ratio. However, surprisingly high GOS:lactose ratios were often observed when the amount of GOS in the second composition was significantly lower (e.g., lower GOS yield). Longer incubation times also generally decreased the amount of both lactose and GOS in the second composition, and generally increased the GOS:lactose ratio.

[0087] [Table 2]

[0088] Examples 5 to 9 Examples 5-9 were conducted to determine the effect of molecular weight cut-off (MWCO) of polymeric nanofiltration membranes on the distribution of carbohydrates (sugars and GOS) in NF retentates and NF permeates. In Example 5 (200 Da MWCO) and Example 8 (600-800 Da MWCO), a representative first pre-filtration composition (18.7 wt.% solids, 0.05 wt.% fat, 0.66 wt.% protein, and 1.15 wt.% minerals) having the components and amounts shown in Table III was subjected to membrane filtration at temperatures ranging from 5 to 50°C and pressures ranging from 200 to 500 psig. In Example 6 (MWCO of 150-300 Da) and Example 7 (MWCO of 300-500 Da), a representative first composition before filtration (21.2 wt.% solids, 0.05 wt.% fat, 0.66 wt.% protein, and 1.22 wt.% minerals) having the components and amounts shown in Table III was subjected to membrane filtration at temperatures ranging from 5 to 45°C and pressures ranging from 350 to 550 psig.

[0089] The NF permeates of Examples 5-7 contained very little GOS, and therefore the NF retentates contained almost all of the GOS present in the pre-filtration compositions. This means that the GOS yields were very good. However, these NF permeates also did not contain significant amounts of sugars (DP2, glucose, galactose, lactose). This indicates that much of the sugar was also present in the NF retentate, i.e., both sugars and GOS were concentrated. Therefore, these MWCOs are useful when GOS yield is critical but a high GOS-to-sugar concentration is not required (note the GOS / sugar ratio in the NF retentate).

[0090] The NF permeate of Example 8 contained a large amount of GOS, and the GOS / sugar ratio in the NF retentate was extremely high. The MWCO of Example 8 allowed a significant amount of GOS to permeate, resulting in a low GOS yield in the NF retentate, but a high concentration of GOS relative to sugars was observed in the NF retentate.

[0091] Example 9 (MWCO 500-700 Da) was performed using different starting materials and membrane filtration of a representative second pre-filtration composition (19.3 wt.% solids, 0.06 wt.% fat, 0.4 wt.% protein, and 1.07 wt.% minerals) having the components and amounts shown in Table III at temperatures ranging between 10 and 20° C. and pressures ranging between 320 and 560 psig. This example demonstrated a good balance between the yield of GOS in the NF retentate (relatively low GOS in the NF permeate) and the concentration of GOS relative to sugars in the NF retentate (ratio of GOS / NF retentate to pre-filtration composition). The NF retentate (GOS composition) of Example 9 in Table III had 35-36 wt.% solids and contained approximately 0.05 wt.% fat, 1 wt.% protein, and 2 wt.% minerals.

[0092] [Table 3] [Explanation of symbols]

[0093] 100 Typical methods for producing galactooligosaccharides (GOS) 105 Lactose-containing products 110 Heating 115 contact with the first β-galactosidase enzyme 120 Incubation 125 Heat Treatment 130 Cooling 135 Addition of a second β-galactosidase enzyme 140 Incubation 145 Heat Treatment 150 Membrane filtration 155 Concentrated Galactooligosaccharide (GOS) Composition

Claims

1. (a) contacting a lactose-containing product with a first β-galactosidase enzyme to form a first composition; (b) inactivating the first β-galactosidase enzyme in the first composition; (c) adjusting the temperature of the first composition to within the range of 5 to 55°C; (d) contacting the first composition with a second β-galactosidase enzyme to form a second composition; (e) inactivating the second β-galactosidase enzyme in the second composition; and (f) subjecting the second composition to membrane filtration to form a concentrated galactooligosaccharide (GOS) composition. A method comprising:

2. 2. The method of claim 1, wherein the lactose-containing product comprises 6 to 50 wt.%, 8 to 40 wt.%, 10 to 30 wt.%, or 10 to 20 wt.% lactose.

3. The lactose-containing product 0.01-1 wt.%, 0.01-0.5 wt.%, or 0.01-0.2 wt.% fat, and / or 0.1-2 wt.%, 0.1-1.2 wt.%, or 0.2-1 wt.% protein, and / or 0.5-3wt.%, 0.7-2.5wt.%, or 1-2wt.% minerals 3. The method of claim 1 or 2, comprising:

4. 4. The method of claim 1, wherein the lactose-containing product comprises a nanofiltration (NF) retentate fraction of an ultrafiltration (UF) permeate fraction of whole milk or skim milk.

5. 5. The method of claim 4, wherein the lactose-containing product comprises an enriched NF retentate fraction.

6. the amount of the first β-galactosidase enzyme is 1 to 15 wt.%, 1.5 to 6 wt.%, 1.5 to 4 wt.%, or 2 to 3 wt.%, based on the amount of lactose in the lactose-containing product; and / or the amount of the first β-galactosidase enzyme is 0.05 to 1 wt.%, 0.1 to 0.8 wt.%, or 0.15 to 0.65 wt.%, based on the total weight of the lactose-containing product; and / or The first β-galactosidase enzyme is derived from Bifidobacterium bifidum.

6. The method according to any one of claims 1 to 5.

7. step (a) is carried out at a first temperature in the range of 20 to 70°C, 25 to 65°C, 30 to 70°C, 30 to 60°C, or 35 to 55°C; and / or step (a) is carried out for a first time period ranging from 15 minutes to 10 hours, from 30 minutes to 8 hours, from 45 minutes to 6 hours, from 1 hour to 4 hours, or from 90 minutes to 150 minutes; 7. The method according to any one of claims 1 to 6.

8. the quenching step of step (b) comprises subjecting the first composition to a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C, or heat treating the first composition at a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C; and / or the quenching step of step (b) comprises subjecting the first composition to the temperature for a time ranging from 1 minute to 6 hours, from 1 minute to 30 minutes, from 2 minutes to 20 minutes, or from 4 minutes to 15 minutes, or heat treating the first composition at the temperature for a time ranging from 1 minute to 6 hours, from 1 minute to 30 minutes, from 2 minutes to 20 minutes, or from 4 minutes to 15 minutes; 8. The method according to any one of claims 1 to 7.

9. 9. The method of any one of claims 1 to 8, wherein the temperature of the first composition in step (c) is 10 to 55°C, 20 to 50°C, 25 to 45°C, or 30 to 45°C.

10. Prior to step (d), the first composition comprises: 1-5 wt.%, 1.5-4.5 wt.%, or 2-3.5 wt.% lactose, and / or 4-10 wt.%, 5-9 wt.%, or 6-8.5 wt.% GOS, and / or a GOS:lactose mass ratio of 1.6:1 to 3.5:1, 2:1 to 3:1, or 2.2:1 to 2.8:1, and / or 10-20 wt.%, 12-18 wt.%, or 13-17 wt.% lactose based on carbohydrates, and / or 30-44 wt.%, 32-42 wt.%, or 34-40 wt.% GOS based on carbohydrates, and / or 60 to 95 wt.% less, 70 to 90 wt.% less, or 75 to 85 wt.% less lactose than in the lactose-containing product of step (a).

10. The method according to any one of claims 1 to 9, comprising:

11. the amount of the second β-galactosidase enzyme is 0.01 to 0.6 wt.%, 0.03 to 0.3 wt.%, or 0.04 to 0.1 wt.%, based on the total weight of the first composition; and / or the amount of the second β-galactosidase enzyme is 0.35 to 20 wt.%, 1 to 10 wt.%, or 1.4 to 4 wt.%, based on the amount of lactose in the first composition; and / or the second β-galactosidase enzyme is derived from Kluyveromyces lactis; 11. The method according to any one of claims 1 to 10.

12. step (d) is carried out at a second temperature in the range of 10 to 60°C, 20 to 50°C, 25 to 45°C, or 30 to 45°C; and / or step (d) is carried out for a second time period ranging from 15 minutes to 10 hours, from 30 minutes to 8 hours, from 45 minutes to 6 hours, from 1 hour to 4 hours, or from 90 minutes to 150 minutes; 12. The method according to any one of claims 1 to 11.

13. the quenching step of step (e) comprises subjecting the second composition to a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C, or heat treating the second composition at a temperature in the range of 75 to 150°C, 80 to 120°C, 80 to 115°C, or 85 to 105°C; and / or the quenching step of step (e) comprises subjecting the second composition to the temperature for a time ranging from 1 minute to 6 hours, from 1 minute to 30 minutes, from 2 minutes to 20 minutes, or from 4 minutes to 15 minutes, or heat treating the second composition at the temperature for a time ranging from 1 minute to 6 hours, from 1 minute to 30 minutes, from 2 minutes to 20 minutes, or from 4 minutes to 15 minutes; 13. The method according to any one of claims 1 to 12.

14. 14. The method of any one of claims 1 to 13, further comprising, prior to step (f), cooling the second composition to a temperature within a range of 5 to 50°C, 10 to 45°C, 20 to 50°C, 5 to 25°C, or 8 to 20°C.

15. Prior to step (f), the second composition comprises: 0.05 to 5 wt.%, 0.05 to 1.5 wt.%, 0.1 to 4 wt.%, 0.1 to 1 wt.%, 0.2 to 2 wt.%, or 0.2 to 0.9 wt.% lactose, and / or 4-15 wt.%, 4-9 wt.%, 5-12 wt.%, 5-8 wt.%, 5.5-9.5 wt.%, or 5.5-7.5 wt.% GOS, and / or a GOS:lactose mass ratio of 4:1 to 40:1, 7:1 to 35:1, or 8:1 to 30:1, and / or 0.5-5 wt.%, 1-5 wt.%, or 2-4 wt.% lactose based on carbohydrates, and / or 20-45 wt.%, 25-40 wt.%, or 32-39 wt.% GOS based on carbohydrates, and / or 80 to 99 wt.% less, 85 to 98 wt.% less, 90 to 99 wt.% less, 92 to 99 wt.% less, or 93 to 98 wt.% less lactose than the lactose in the lactose-containing product of step (a), and / or 3 wt.% or less, 2 wt.% or less, 1 wt.% or less, 0.8 wt.% or less, 0.5 wt.% or less, or 0.3 wt.% or less GOS than in the first composition.

15. The method according to any one of claims 1 to 14, comprising:

16. step (f) comprises nanofiltration of the second composition through a polymeric membrane; and / or step (f) comprising diafiltering the second composition with a nanofiltration membrane; 16. The method according to any one of claims 1 to 15.

17. 17. The method of any one of claims 1 to 16, wherein the second composition is subjected to membrane filtration using a membrane having a molecular weight cut-off (MWCO) in the range of 150 Da to 1000 Da, 150 Da to 900 Da, 150 Da to 500 Da, 150 to 300 Da, 200 to 900 Da, 200 to 800 Da, 300 to 900 Da, 300 to 800 Da, 300 to 700 Da, 300 to 500 Da, 500 to 1000 Da, 500 to 800 Da, 500 to 700 Da, 600 to 900 Da, or 600 to 800 Da.

18. Step (f) is at a filtration temperature in the range of 5-50°C, 10-45°C, 20-50°C, 5-25°C, or 8-20°C; and / or Pressure ranges of 100 to 1000 psig, 200 to 800 psig, 300 to 650 psig, or 320 to 560 psig The method according to any one of claims 1 to 17, wherein

19. the concentrated GOS composition 0.1 to 3 wt.%, 0.5 to 2 wt.%, or 0.7 to 1.8 wt.% lactose, and / or 10-20 wt.%, 12-20 wt.%, or 14-18 wt.% GOS, and / or a GOS:lactose mass ratio of 6:1 to 45:1, 7:1 to 35:1, or 8:1 to 30:1, and / or 0.5-6 wt.%, 1-5.5 wt.%, 2-6 wt.%, or 2.5-5 wt.% lactose based on carbohydrates, and / or 24-50wt.%, 30-48wt.%, 33-50wt.%, or 38-46wt.% GOS based on carbohydrates 19. The method of any one of claims 1 to 18, comprising:

20. 20. The method of any one of claims 1 to 19, further comprising, after step (f), heat treating the GOS composition.

21. (i) measuring the amount of lactose, the amount of GOS, the ratio of GOS:lactose, or any combination thereof, in the first composition prior to step (d); and (ii) adjusting the first temperature, the first time period, the amount of the first β-galactosidase enzyme, or any combination thereof, in step (a) based on the determined compositional characteristics of the first composition.

21. The method of any one of claims 1 to 20, further comprising:

22. (i) measuring the amount of lactose, the amount of GOS, the ratio of GOS:lactose, or any combination thereof, in the second composition prior to step (f); and (ii) adjusting the second temperature, the second time period, the amount of the second β-galactosidase enzyme, or any combination thereof, in step (d) based on the determined compositional characteristics of the second composition.

22. The method of any one of claims 1 to 21, further comprising:

23. 23. A concentrated GOS composition produced by the method of any one of claims 1 to 22.

24. 0.5-6 wt.% lactose and 24-50 wt.% GOS based on carbohydrates, and / or GOS:lactose mass ratio of 6:1 to 45:1 A composition comprising:

25. 1-5.5 wt.% lactose and / or 30-48 wt.% GOS, or 2-6 wt.% lactose and / or 33-50 wt.% GOS, or 2.5-5 wt.% lactose and / or 38-46 wt.% GOS 25. The composition of claim 23 or 24, comprising:

26. 26. The composition of any one of claims 23 to 25, wherein the mass ratio of GOS:lactose is from 7:1 to 35:1, from 7:1 to 15:1, from 8:1 to 30:1, or from 8:1 to 12:

1.

27. and / or further comprising galactose in a galactose:lactose weight ratio ranging from 1.5:1 to 35:1, 1.7:1 to 10:1, 1.7:1 to 3:1, 2:1 to 30:1, 2:1 to 10:1, or 2:1 to 3:

1. and / or further comprising glucose in a weight ratio of glucose:lactose ranging from 3:1 to 40:1, 3:1 to 20:1, 3:1 to 7:1, 4:1 to 40:1, 4:1 to 10:1, or 4:1 to 6:

1. and / or further comprising 5-20 wt.%, 6-15 wt.%, or 7-10 wt.% galactose based on total carbohydrates; Further containing 15 to 30 wt.%, 18 to 26 wt.%, or 20 to 24 wt.% glucose based on the total carbohydrates; 27. The composition of any one of claims 23 to 26.

28. 0.01-2 wt.%, 0.03-1 wt.%, or 0.03-0.2 wt.% fat, and / or 0.3-3 wt.%, 0.4-1.5 wt.%, or 0.6-1.2 wt.% protein, and / or 0.5-5wt.%, 1-3wt.%, or 1.5-2.5wt.% minerals 28. The composition of any one of claims 23 to 27, further comprising:

29. 29. A composition according to any one of claims 23 to 28, produced by a method according to any one of claims 1 to 22.

30. 29. The method of any one of claims 1 to 22, wherein the composition produced is as defined in any one of claims 23 to 28.

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