Process for preparation of galactooligosaccharides

Abstract

A process for the preparation of galactooligosaccharide (GOS), the process comprising (a) contacting a lactose-containing product with a first beta-galactosidase to form a first composition, (b) inactivating the first beta-galactosidase in the first composition, (c) adjusting the temperature of the first composition to in the range of 5 to 55 DEG C, and (d) removing the first composition from the first composition. (d) contacting the first composition with a second beta-galactosidase to form a second composition, (e) inactivating the second beta-galactosidase in the second composition, and (f) subjecting the second composition to membrane filtration to form a concentrated (GOS) composition. The GOS compositions may contain from 0.5 to 6 wt% lactose and from 24 to 50 wt% GOS, based on carbohydrate, and typically have a GOS: lactose weight ratio ranging from 6: 1 to 45: 1.

Description

[0001] Citation of Related Applications

[0002] This application was filed as a PCT international patent application on November 20, 2023, and claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 384,619, filed on November 22, 2022. The disclosure of the U.S. Provisional Patent Application is hereby incorporated by reference in its entirety. Background of the Invention

[0003] The present invention generally relates to the preparation of galactooligosaccharides from lactose-containing dairy streams. Summary of the Invention

[0004] The Summary of the Invention is provided to introduce in a simplified form a selection of some concepts that are further described herein. The Summary of the Invention is not intended to identify essential or necessary features of the claimed subject matter. The Summary of the Invention is also not intended to be used to limit the scope of the claimed subject matter.

[0005] Disclosed and described herein are methods for preparing galactooligosaccharides. Representative processes can include (a) contacting a lactose-containing product with a first β-galactosidase to form a first composition, (b) inactivating the first β-galactosidase 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 to form a second composition, (e) inactivating the second β-galactosidase in the second composition, and (f) subjecting the second composition to membrane filtration to form a concentrated galactooligosaccharide (GOS) composition.

[0006] Also disclosed and described herein are galactooligosaccharide (GOS) compositions. Representative compositions can contain: 0.5 to 6 wt% lactose and 24 to 50 wt% GOS (DP3+) based on carbohydrates. Typically, the weight ratio of GOS:lactose in the composition ranges from 6:1 to 45:1.

[0007] The foregoing Summary of the Invention and the following Detailed Description both provide examples and are merely illustrative. Therefore, the foregoing Summary of the Invention and the following Detailed Description should not be considered restrictive. Further, features or variations can be provided in addition to those set forth herein. For example, certain aspects can relate to various combinations and sub-combinations of features described in the Detailed Description. Brief Description of the Drawings

[0008] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to the drawings in conjunction with the Detailed Description and Examples.

[0009] Figure 1Shows a schematic flow chart of a process for preparing galactooligosaccharides (GOS) according to an aspect of the present invention.

[0010] Definitions

[0011] To more clearly define the terms used herein, the following definitions are provided. Unless otherwise stated, the following definitions apply to the present disclosure. If a term is used in the present disclosure but not specifically defined herein, the definition from IUPAC Compendium of Chemical Terminology, 2 nd Ed (1997) may be applied, provided that the definition does not conflict with any other disclosure or definition applied herein, or render any claim to which the definition is applicable indefinite or unenforceable. If any definition or use provided by any document incorporated herein by reference conflicts with the definition or use provided herein, the definition or use provided herein shall prevail.

[0012] The features of the subject matter are described herein such that within a particular aspect, combinations of different features can be envisioned. For each and every aspect and / or feature disclosed herein, all combinations are contemplated, with or without an express description of a particular combination, provided that they do not have an adverse effect on the designs, compositions, processes, and / or methods described herein. Additionally, unless otherwise expressly stated, any aspect and / or feature disclosed herein can be combined to describe an inventive design, composition, process, and / or method according to the present invention.

[0013] In the present disclosure, although compositions and methods are generally described in terms of "comprising" various components or steps, unless otherwise stated, the compositions and methods may also "consist essentially of" or "consist of" the various components or steps. Unless otherwise stated, the terms "a", "an", and "the" are intended to include plural alternatives, such as at least one.

[0014] In the disclosed methods, unless otherwise stated, the term "contact" encompasses the combination of components in any order, in any manner, and for any length of time. For example, the components may be blended or mixed.

[0015] Several types of ranges are disclosed in the present invention. When any type of range is disclosed or claimed, each possible number that such a range could reasonably encompass is intended to be separately disclosed or claimed, including the endpoints of the range and any sub-ranges and combinations of sub-ranges subsumed therein. For example, in some aspects of the present invention, a lactose-containing product can contain from 6 to 50% by weight of lactose. By the disclosure, the amount of lactose in the lactose-containing product is from 6 to 50% by weight, and it is intended to enumerate that the amount of lactose can be any amount within this range, and for example, can include any range or combination within the range of 6 to 50% by weight, such as from 8 to 40% by weight, from 10 to 30% by weight, or from 10 to 20% by weight of lactose, etc. Similarly, all other ranges disclosed herein should be interpreted in a manner similar to this example.

[0016] Generally, a quantity, size, formulation, parameter, range, or other quantity or property is “about” or “approximately” whether or not so stated explicitly. The claims include equivalents of the quantity or property whether or not modified by the term “about” or “approximately”.

[0017] 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, typical methods, devices, and materials are described herein.

[0018] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications and patents, which can be used in conjunction with the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] This disclosure relates to a process for preparing 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 β 1-4 linkage and having the chemical structure (β-D-galactopyranosyl-(1→4)-α-D-glucopyranose). Lactose is a component of milk in all mammals, with concentrations ranging 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 byproduct of many dairy processing operations such as cheese, yogurt, and membrane filtration. The relatively low sweetness index and low solubility of lactose limit its use as a sweetener. Although a valuable nutrient, especially for infants, lactose intolerance poses an additional challenge for its use as a sweetener. On average, approximately 65% of the population is reported to be lactose intolerant to some degree. Lactose intolerance means that due to a lack of lactase, an individual's ability to digest lactose is impaired. Additionally, the high oxygen demand of lactose makes lactose digestion an additional challenge. All these factors indicate a need for alternatives to maximize lactose utilization. Bioconverting lactose to glucose-galactose syrup as a sweetener is one alternative. However, the relatively high cost and bitter taste of the syrup limit its application.

[0021] Another alternative is to produce value-added components from underutilized lactose streams, such as galactooligosaccharides (GOS), lactulose, polylactose, and lactobionic acid. GOS represents a class of emerging natural dietary fibers with a wide range of compositional variability. GOS compositions can have from 2 to 20 galactose molecules and 1 glucose molecule. Galactooligosaccharides occur naturally in breast milk, garlic, onions, soybeans, and chicory root.

[0022] The prebiotic activity of GOS has been the subject of many studies related to 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 of a limited number of bacteria in the colon, thereby improving host health. GOS is acid-tolerant and can thus withstand the acidic environment of the stomach without being digested, with approximately 90% of the ingested content reaching the intestine. The human gut microbiota is naturally complex, consisting of over 500 bacterial species. These species have a diverse range of genes that enable them to ferment indigestible carbohydrates and produce a variety of bioactive compounds, including short-chain fatty acids (SCFAs). SCFAs have a direct effect on the function of the intestinal mucosa, serve as an energy source, and regulate water and mineral absorption. Studies have shown that GOS specifically stimulates the growth of bifidobacterium bifidum and lactobacilli that are predominantly present in the human intestine. Other health benefits associated with GOS consumption include enhanced intestinal function, a reduced incidence of intestinal inflammation, and regulation of the immune response to allergies.

[0023] Traditionally, GOS has been produced by acid hydrolysis of lactose, which results in a complex mixture of disaccharides and trisaccharides together with anhydro sugars. The harsh process parameters and the complex nature of the product limit the feasibility of scaling up this process. GOS is now commonly produced through the catalytic activity of glycoside hydrolases, also known as β-galactosidases. These enzymes operate in two steps: hydrolysis and transgalactosylation. In the hydrolysis step, lactose is cleaved into its monosaccharide components: glucose and galactose. Transgalactosylation results in the elongation and formation of GOS chains of different lengths, with β(1→6)-linked galactopyranosyl units attached to the terminal glucopyranosyl residue via an α(1→4) glycosidic bond.

[0024] These two steps occur simultaneously. However, the relatively low transgalactosylation reaction efficiency and low GOS yield mean that this method is challenging. This method produces a complex mixture containing glucose, galactose, a large amount of residual lactose, and GOS of different structures.

[0025] An object of the present invention is to overcome the disadvantages of traditional GOS preparation methods, in particular to maximize the GOS yield and reduce the residual lactose level. However, most of the research has been carried out on lactose-rich streams that are by-products of the cheese industry. These streams usually contain salts, cultures, or enzymes that can adversely affect the flavor characteristics, purity, and further processability of the stream.

[0026] In this article, the disclosed process can start from a relatively concentrated lactose-containing product or lactose stream, subject it to two consecutive enzymatic treatments, followed by heat treatment after each enzymatic step, and then a membrane filtration step. In the first enzymatic step, the lactose product is treated with a β-galactosidase that converts lactose to GOS, and then the enzyme is heat inactivated after obtaining a high GOS yield. The composition obtained from this step contains a mixture of GOS of different degrees of polymerization (DP), residual lactose, DP2, glucose, and galactose. In the second step, the composition is treated with a β-galactosidase from a different source that has higher hydrolytic activity compared to the enzyme in the first step. In addition, this enzyme selectively hydrolyzes lactose with a minimal effect on GOS. This step reduces the residual lactose while maximizing the retention of the formed GOS, and heat inactivation is also carried out after this step. Finally, membrane filtration is used for separation / concentration to retain a higher purity GOS stream while permeating glucose, galactose, DP2 sugars, and lactose. The amount of GOS in the compositions herein is based on galactooligosaccharides with a degree of polymerization of 3 or higher (DP3+).

[0027] Method for preparing galactooligosaccharides

[0028] According to one aspect of the present invention, there is provided a process for preparing galactooligosaccharides (GOS), and in this aspect, the process may comprise the following steps (or consist essentially of the following steps, or consist of the following steps): (a) contacting a lactose-containing product with a first β-galactosidase to form a first composition, (b) inactivating the first β-galactosidase 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 to form a second composition, (e) inactivating the second β-galactosidase in the second composition, and (f) subjecting the second composition to membrane filtration to form a concentrated galactooligosaccharides (GOS) composition.

[0029] Generally, the characteristics of the process (such as the lactose-containing product, the characteristics of the first β-galactosidase, the first composition, the characteristics of the second β-galactosidase, the characteristics of the second composition, the characteristics of the GOS composition, and the conditions when performing any step, etc.) are described independently herein, and these characteristics can be combined in any combination to further describe the disclosed process. In addition, unless otherwise stated, other steps can be carried out before, during, and / or after any step listed in the disclosed process. Additionally, any GOS composition (such as a concentrated GOS composition) prepared according to the disclosed process is within the scope of the present disclosure and is covered herein.

[0030] Filtration techniques (such as 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 suitable conditions (such as pressure). Thus, the concentration / separation can be based on molecular size. The stream retained by the membrane is called the retentate (or concentrate). The stream passing through the pores of the membrane is called the permeate.

[0031] Now referring to step (a), a lactose-containing product is contacted with a first β-galactosidase to form a first composition. Although not limited thereto, the lactose-containing product generally may contain 6 to 50 wt% lactose, such as 8 to 40 wt%, 10 to 30 wt%, or 10 to 20 wt% lactose, etc. In one aspect, for example, the lactose-containing product may comprise a nanofiltration (NF) retentate portion (usually the ultrafiltration permeate portion of whole milk or skim milk). Optionally, the lactose-containing product may comprise a concentrated NF retentate portion (such as where the NF retentate portion is concentrated by forward osmosis or reverse osmosis or other suitable techniques).

[0032] The whole milk before UF can be cow's milk, which contains about 87% by weight of water, 3 - 4% by weight of protein, 4 - 5% by weight of carbohydrates / lactose, 3 - 4% by weight of fat, and 0.3 - 0.8% by weight of minerals. Skim milk can be prepared by separating (e.g., by centrifugation or microfiltration) the whole milk into skim milk and a fat-rich portion (also known as cream or butterfat). The fat-rich portion typically contains a high fat level (e.g., 20 - 50% by weight of fat, 30 - 50%, 35 - 45% by weight of fat, or 38 - 42%) and solids (e.g., 30 - 60% by weight, 40 - 55%, 40 - 50% by weight, or 42 - 47%), and typically contains about 1 - 4% by weight of protein (or 1 - 3% or 2 - 3%), 2 - 5% by weight of lactose (or 2.5 - 4% or 2.5 - 3.5%), and 0.2 - 0.9% by weight of minerals (or 0.2 - 0.6% or 0.2 - 0.4%), but is not limited thereto. In contrast, the skim milk before UF typically contains a very low fat level (e.g., less than or equal to 0.5% by weight, less than or equal to 0.35% by weight, or less than or equal to 0.2% by weight) and much lower solids than the fat-rich portion (e.g., 7 - 13% by weight, 8 - 12%, 8.5 - 10% by weight, or 9 - 9.5%), and the skim milk typically contains about 2 - 5% by weight of protein (or 3 - 4% or 3.2 - 3.7%), 3 - 6% by weight of lactose (or 4 - 5.5% or 4.5 - 5%), and 0.4 - 1.2% by weight of minerals (or 0.4 - 0.9% or 0.5 - 0.9%), but is not limited thereto.

[0033] In addition to the above lactose content, the lactose-containing product in step (a) can contain 0.01 to 1% by weight, 0.01 to 0.5% by weight, or 0.01 to 0.2% by weight of fat, but is not necessarily limited thereto. Additionally or alternatively, the lactose-containing product can contain 0.1 to 2% by weight, 0.1 to 1.2% by weight, or 0.2 to 1% by weight of protein. Additionally or alternatively, the lactose-containing product can contain 0.5 to 3% by weight, 0.7 to 2.5% by weight, or 1 to 2% by weight of minerals.

[0034] Any suitable β-galactosidase can be used as the first β-galactosidase in step (a). Generally, the first β-galactosidase is of the type that produces galactooligosaccharides (GOS) from lactose. In a particular aspect of step (a), the first β-galactosidase can be derived from bifidobacterium bifidum.

[0035] Step (a) can be carried out under any suitable temperature and time conditions. Although not limited thereto, the first temperature range at which step (a) can be carried out is from 20 to 70 °C, such as from 25 to 65 °C, from 30 to 70 °C, from 30 to 60 °C, or from 35 to 55 °C, etc. In one aspect, step (a) can be carried out at a temperature higher than the ambient temperature (for example, 20 - 25 °C), so that the lactose-containing product and the first enzyme can be heated to the desired high temperature and contacted for a suitable period of time to form the first composition.

[0036] Now referring to the time period of step (a), generally the first time period range for step (a) to be carried out is from 15 min to 10 h. Other illustrative ranges of the first time period include from 30 min to 8 h, from 45 min to 6 h, from 1 h to 4 h, or from 90 min to 150 min, etc.

[0037] As will be readily appreciated by those skilled in the art, the amount of the first enzyme used in step (a) can vary significantly based on factors such as the first temperature, the first time period, and the amount of lactose in the lactose-containing product, etc. Although not limited thereto, based on the total weight of the lactose-containing product, the amount range of the first β-galactosidase can be from 0.05 to 1 wt%, from 0.1 to 0.8 wt%, or from 0.15 to 0.65 wt%. The amount range of the first β-galactosidase based on the starting amount of lactose can be from 1 to 15 wt%, from 1.5 to 6 wt%, from 1.5 to 4 wt%, or from 2 to 3 wt%, but as described above, this amount based on the amount of lactose in the lactose-containing product can vary based on the temperature and time conditions in step (a).

[0038] Now referring to step (b), inactivate the first β-galactosidase in the first composition. Although any suitable method for inactivating the enzyme in step (b) can be employed, inactivation by exposure to a relatively high temperature for a suitable time is conveniently achieved. Therefore, the inactivation in step (b) can include subjecting the first composition to a high temperature, or heat-treating the first composition at a high temperature.

[0039] Step (b) can be carried out under any temperature and time conditions that can sufficiently inactivate the first β-galactosidase in the first composition. Although not limited thereto, the inactivation in step (b) can include 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).

[0040] Now referring to the time period of step (b), the inactivation in step (b) generally includes subjecting the first composition to the above temperature, or heat-treating the first composition at the above temperature for a time range of 1 min to 6 hr. Other illustrative ranges for the heat-treatment time used to inactivate the enzyme in step (b) can include 1 min to 30 min, 2 min to 20 min, or 4 min to 15 min, etc.

[0041] 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 employed in step (b), the first composition is cooled to a temperature within the range of 5 to 55 °C. In one aspect, the temperature of the first composition in step (c) is 10 to 55 °C, while in another aspect, the temperature is 20 to 50 °C, and in yet another aspect, the temperature is 25 to 45 °C, and in yet another aspect, the temperature is 30 to 45 °C. Other suitable temperatures and ranges can be used if desired.

[0042] At this stage of the process (before step (d) and the addition of the second enzyme), the first composition can generally contain 1 to 5 wt%, 1.5 to 4.5 wt%, or 2 to 3.5 wt% of lactose, but is not limited thereto. Additionally or alternatively, the first composition can contain 4 to 10 wt%, 5 to 9 wt%, or 6 to 8.5 wt% of GOS. Any combination of the above lactose and GOS amounts can be present in the first composition. However, the weight ratio of GOS:lactose can advantageously be in the range of 1.6:1 to 3.5:1, 2:1 to 3:1, or 2.2:1 to 2.8:1, but the ratio is not limited to these ranges.

[0043] Based on the carbohydrates in the first composition, the first composition can contain 10 to 20 wt%, 12 to 18 wt%, or 13 to 17 wt% of lactose. Additionally or alternatively, based on carbohydrates, the first composition can contain 30 to 44 wt%, 32 to 42 wt%, or 34 to 40 wt% of GOS.

[0044] After the first enzyme treatment, the first composition (before step (d)) contains significantly less lactose than the lactose present in the lactose-containing product in step (a). Advantageously, the first composition contains 60 to 95 wt% less lactose than the lactose-containing product in step (a). More generally, the first composition contains 70 to 90 wt% less lactose than the lactose-containing product in step (a), or 75 to 85 wt% less.

[0045] In step (d), the first composition is contacted with a second β-galactosidase to form a second composition, and any suitable β-galactosidase can be used as the second β-galactosidase in step (d). Generally, the second β-galactosidase is of the type that preferentially hydrolyzes lactose to form glucose and galactose. In a particular aspect of step (d), the second β-galactosidase can be derived from Kluyveromyces lactis.

[0046] Step (d) can be carried out under any suitable temperature and time conditions. Although not limited thereto, a second temperature range in which step (d) can be carried out is from 10 to 60 °C, such as from 20 to 50 °C, from 25 to 45 °C, or from 30 to 45 °C, etc. In one aspect, step (d) can be carried out at a temperature higher than the ambient temperature (e.g., 20 - 25 °C), so that the first composition and the second enzyme can be heated to (or controlled at) the desired high temperature and contacted for a suitable period of time to form the second composition.

[0047] Now referring to the period of time of step (d), generally, the second period of time in which step (d) is carried out ranges from 15 min to 10 h. Other illustrative ranges of the second period of time include from 30 min to 8 h, from 45 min to 6 h, from 1 h to 4 h, or from 90 min to 150 min, etc.

[0048] As will be readily appreciated by those skilled in the art, the amount of the second enzyme used in step (d) can vary significantly based on factors such as the second temperature, the second period of time, and the amount of lactose in the first composition, etc. Although not limited thereto, based on the total weight of the first composition, the amount of the second β-galactosidase can range from 0.01 to 0.6 wt%, from 0.03 to 0.3 wt%, or from 0.04 to 0.1 wt%. Based on the amount of lactose in the first composition, the amount of the second β-galactosidase can range from 0.35 to 20 wt%, from 1 to 10 wt%, or from 1.4 to 4 wt%, although as described above, this amount based on the amount of lactose in the first composition can vary based on the temperature and time conditions in step (d).

[0049] Now referring to step (e), the second β-galactosidase in the second composition is inactivated. Although any suitable method for inactivating the enzyme in step (e) can be employed, inactivation by exposure to a relatively high temperature for a suitable time is conveniently achieved. Thus, the inactivation in step (e) can include subjecting the second composition to a high temperature, or heat-treating the second composition at a high temperature.

[0050] Step (e) can be carried out under any temperature and time conditions that sufficiently inactivate the second β-galactosidase in the second composition. Although not limited thereto, the inactivation in step (e) can include subjecting the second composition to a temperature within the following ranges, or heat-treating the second composition at a temperature within the following ranges: 75 to 150 °C, 80 to 120 °C, 80 to 115 °C, or 85 to 105 °C.

[0051] Now referring to the time period of step (e), the inactivation in step (e) generally includes subjecting the second composition to the above temperature for a certain period of time, or heat-treating the second composition at the above temperature for a certain period of time, and the range of said period of time is 1 min to 6 h. Other illustrative ranges of the heat-treatment time period for inactivating the enzyme in step (e) can include 1 min to 30 min, 2 min to 20 min, or 4 min to 15 min, etc.

[0052] If heat treatment is used for inactivation in step (e), the process can further include the step of cooling the second composition to a temperature within the range of 5 to 50 °C (before step (f)). In one aspect, before step (f), the temperature of the second composition can be cooled or adjusted to a temperature within the range of 10 to 45 °C or 20 to 50 °C, while in another aspect, the temperature of the second composition can be cooled or adjusted to a temperature within the range of 5 to 25 °C or 8 to 20 °C. In one aspect, the temperature of the second composition before step (f) can be equal to or lower than the ambient conditions (for example, 20 - 25 °C), so the second composition can be cooled to a temperature of less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 18 °C, or less than or equal to 15 °C; typical ranges include 8 - 18 °C and 10 - 15 °C.

[0053] At this stage of the process (before step (f) and before concentration by membrane filtration), the second composition generally can contain 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% of lactose, but not limited thereto. Additionally or alternatively, the second composition can contain 4 to 15 wt%, 4 to 9 wt%, 5 to 12 wt%, 5 to 8 wt%, 5.5 to 9.5 wt%, or 5.5 to 7.5 wt% of GOS. Any combination of the above amounts of lactose and GOS can be present in the second composition. However, the advantageous range of the weight ratio of GOS:lactose in the second composition can be 4:1 to 40:1, 7:1 to 35:1, or 8:1 to 30:1, but the ratio is not limited to these ranges.

[0054] Based on the carbohydrates in the second composition, the second composition may contain 0.5 to 5 wt%, 1 to 5 wt% or 2 to 4 wt% of lactose. Additionally or alternatively, based on the carbohydrates, the second composition may contain 20 to 45 wt%, 25 to 40 wt% or 32 to 39 wt% of GOS.

[0055] After the second enzymatic treatment, the second composition (before step (f)) contains significantly less lactose than the lactose present in the lactose-containing product in step (a). Advantageously, the second composition contains 80 to 99 wt% less lactose than the lactose-containing product in step (a). More typically, the second composition contains 85 to 98 wt%, 90 to 99 wt%, 92 to 99 wt% or 93 to 98 wt% less lactose than the lactose-containing product in step (a).

[0056] However, advantageously, the amount of GOS present in the second composition is substantially the same as in the first composition. Typically, before step (f), the second composition contains less than 3 wt% less GOS than the GOS present in the first composition. More typically, the second composition contains less than 2 wt%, less than 1 wt%, less than 0.8 wt%, less than 0.5 wt% or less than 0.3 wt% less GOS than the GOS present in the first composition before step (d).

[0057] In step (f), the second composition is subjected to membrane filtration to form a concentrated galactooligosaccharide (GOS) composition. In one aspect, step (f) may include nanofiltration of the second composition with a polymeric membrane (or alternatively, with a ceramic membrane) to form a concentrated GOS composition (also referred to as the NF retentate). In another aspect, step (f) may include diafiltration of the second composition with a nanofiltration membrane (polymeric or ceramic). Without wishing to be bound by the following theory, it is believed that in step (f), polymeric membrane filtration is generally more suitable than ceramic membrane filtration because smaller pore sizes and lower molecular weight cut-offs are required. Further, diafiltration of the second composition with a nanofiltration membrane may include diafiltration of a mixture of the second composition and water. Any suitable source of water, such as RO permeate, may be used in the mixture.

[0058] Nanofiltration (or diafiltration) may be carried out using a nanofiltration membrane with a pore size generally in the range of 0.001 to 0.01 microns, for example, the pore size range is 0.001 to 0.008 μm. In some aspects, the membrane system used in the nanofiltration step has a pore size range of 0.001 to 0.01 μm.

[0059] Nanofiltration in the dairy industry typically uses membrane elements that retain particles with 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 the membrane under pressure, and materials with a molecular weight greater than the specified retention value are typically retained, while smaller particles typically pass through the membrane pores. Thus, nanofiltration can perform concentration and separation simultaneously.

[0060] Regarding the membrane filtration in step (f), the membrane can typically be identified based on the molecular weight cut-off (MWCO) rather than the pore size. Membrane systems (such as polymer nanofiltration systems) typically retain materials with a molecular weight greater than the MWCO number. A membrane with a molecular weight cut-off (MWCO) of at least 150 Da and less than 1000 Da can be employed to subject the second composition to membrane filtration. Thus, a suitable membrane can have an MWCO range of 150 Da to 900 Da; alternatively, 150 Da to 500 Da; alternatively, 150 to 300 Da; alternatively, 200 to 900 Da; alternatively, 200 to 800 Da; alternatively, 300 to 900 Da; alternatively, 300 to 800 Da; alternatively, 300 to 700 Da; alternatively, 300 to 500 Da; alternatively, 500 to 1000 Da; alternatively, 500 to 800 Da; alternatively, 500 to 700 Da; alternatively, 600 to 900 Da; or alternatively, 600 to 800 Da. As an example, subjecting the second composition to membrane filtration using a membrane with a molecular weight cut-off (MWCO) in the range of 300 to 800 Da encompasses: using a membrane with a 300 - 500 Da MWCO, using a membrane with a 500 - 700 Da MWCO, and using a membrane with a 600 - 800 Da MWCO.

[0061] In one aspect, the MWCO range of the membrane employed in step (f) is 200 to 800 Da, while in another aspect, the MWCO is 300 to 800 Da, and in another aspect, the MWCO is 300 to 700 Da, and in yet another aspect, the MWCO is 500 to 800 Da, and in yet another aspect, the MWCO is 500 to 700 Da.

[0062] The filtration temperature range at which step (f) can be carried out is 5 to 50 °C, but is not limited thereto. In one aspect, in step (f), the filtration temperature range can be 10 to 45 °C or 20 to 50 °C, while in another aspect, in step (f), the filtration temperature range can be 5 to 25 °C or 8 to 20 °C. In one aspect, the filtration temperature in step (f) can be at or below ambient conditions (e.g., 20 - 25 °C), so the filtration temperature can be less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 18 °C, or less than or equal to 15 °C; typical ranges include 8 - 18 °C and 10 - 15 °C.

[0063] The operating pressure for the membrane filtration step (f) is not particularly limited, but generally ranges from 100 to 1000 psig. In one aspect, the pressure at which step (f) is carried out ranges from 200 to 800 psig, while in another aspect, the pressure ranges from 300 to 650 psig, and in yet another aspect, the pressure ranges from 320 to 560 psig.

[0064] At this stage of the process (after step (f) and concentration / separation by membrane filtration), the concentrated GOS composition can generally contain 0.1 to 3 wt%, 0.5 to 2 wt% or 0.7 to 1.8 wt% of lactose, but is not limited thereto. Additionally or alternatively, the concentrated GOS composition can contain 10 to 20 wt%, 12 to 20 wt% or 14 to 18 wt% of GOS. Any combination of the above lactose and GOS amounts can be present in the concentrated GOS composition. However, the advantageous range of the weight ratio of GOS:lactose in the concentrated GOS composition can be from 6:1 to 45:1, from 7:1 to 35:1 or from 8:1 to 30:1, but the ratio is not limited to these ranges only.

[0065] Based on the carbohydrates in the concentrated GOS composition, the concentrated GOS composition can contain 0.5 to 6 wt%, 1 to 5.5 wt%, 2 to 6 wt% or 2.5 to 5 wt% of lactose. Additionally or alternatively, based on carbohydrates, the concentrated GOS composition can contain 24 to 50 wt%, 30 to 48 wt%, 33 to 50 wt% or 38 to 46 wt% of GOS.

[0066] Optionally, after step (f), the concentrated GOS composition can be heat-treated. In one aspect, the heat-treatment step can include pasteurization at a temperature in the range of 80 to 95 °C, with the pasteurization time ranging from less than 1 min to up to 15 min, such as 2 to 15 min. In another aspect, the heat-treatment step can include UHT sterilization for a time range of 1 to 10 sec at a temperature in the range of 135 to 145 °C. In yet another aspect, the heat-treatment step can include UHT sterilization for a time range of 0.05 to 1 sec at a temperature in the range of 148 to 165 °C, for example, in the range of 150 to 155 °C for a time range of 0.08 to 0.2 sec. Other suitable temperature and time conditions for pasteurization or sterilization are apparent from the present disclosure. Further, the present invention is not limited to the method or equipment used for carrying out the pasteurization / sterilization process, and any suitable technique and instrument can be used, whether in batch operation or continuous operation.

[0067] Typical UHT sterilization techniques include indirect heating, direct steam injection, direct steam infusion, etc. For indirect heating, the GOS composition does not come into direct contact with the heat source or heat medium, such as a heat exchanger. Due to heat transfer limitations, indirect heating requires a longer sterilization time. Advantageously, in some aspects of the present invention, direct UHT sterilization is used to heat-treat the GOS composition. In direct steam injection, high-temperature steam is injected into a tube or other container containing the GOS composition, thereby rapidly sterilizing the GOS composition. Direct steam injection is typically carried out continuously - a continuous flow of the GOS composition is combined with a continuous injection of steam. In direct steam infusion, the GOS composition is sprayed into a chamber containing steam, thereby rapidly and uniformly sterilizing the GOS composition. Similar to direct steam injection, direct steam infusion is typically carried out continuously. After the heat treatment step, the heat-treated GOS composition can be cooled to any suitable temperature, such as in the range of 5 to 40 °C or 10 to 30 °C.

[0068] The process for preparing galactooligosaccharides described herein can be carried out batchwise or continuously. The examples given below are batch experiments, but any step or any combination of steps in the process can alternatively be carried out continuously. Any suitable container (e.g., tanks, silos, etc.) can be used to carry out any step in the process, any combination of steps in the process, or all steps in the process. The process can further include the step of packaging (sterilely or otherwise) the GOS composition in any suitable container and under any suitable conditions, and illustrative and non-limiting examples of typical containers include cups, bottles, bags, or sachets, etc. The container can be made of any suitable material, such as glass, metal, plastic, etc., and various combinations thereof.

[0069] In some aspects, the process for preparing galactooligosaccharides can further include the following steps: (i) determining the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof in the first composition before step (d), and (ii) adjusting the first temperature, the first time period, the amount of the first β-galactosidase, or any combination thereof in step (a) based on the determined 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 can be adjusted, or any combination of these can be adjusted so that the amount of lactose in the first composition is reduced (e.g., reduced to a desired target amount).

[0070] Similarly, the process for preparing galactooligosaccharides may further include the following steps: (i) determining the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof in the second composition before step (f), and (ii) adjusting the second temperature, the second time period, the amount of the second β-galactosidase, or any combination thereof in step (d) based on the determined 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 period can be adjusted, the amount of the second β-galactosidase can be adjusted, or any combination of these can be adjusted to reduce the amount of lactose in the second composition (e.g., to a desired target amount).

[0071] Figure 1 Illustrative and non-limiting examples of a representative process 100 for preparing galactooligosaccharides (GOS) according to some aspects of the present invention are shown. First, a lactose-containing product 105, which contains 10-20 wt% lactose in Figure 1 is heated 110 to a temperature range of 35-55 °C and then contacted 115 with a first β-galactosidase that can be derived from Bifidobacterium bifidum, followed by incubation 120 at a suitable combination of temperature and time, such as incubation at 35-55 °C for 90 to 150 min. The resulting first composition is then heat-treated 125 at 85-105 °C for 5 to 10 min to inactivate the first β-galactosidase. After cooling 130 to a temperature of 30-45 °C, a second β-galactosidase, which can be derived from Kluyveromyces lactis, is added 135, followed by incubation 140 at a suitable combination of temperature and time, such as incubation at 30-45 °C for 90 to 150 min. The resulting second composition is then heat-treated 145 at 85-105 °C for 5 to 10 min to inactivate the second β-galactosidase, and then subjected to membrane filtration 150 to form a retentate fraction, which is a concentrated galactooligosaccharides (GOS) composition 155.

[0072] Galactooligosaccharides (GOS) composition

[0073] Illustrative and non - limiting examples of galactooligosaccharide (GOS) compositions according to the invention, based on carbohydrates, can contain from 0.5 to 6% by weight of lactose and from 24 to 50% by weight of GOS (DP3+). Another illustrative and non - limiting example of a GOS composition according to the invention contains lactose and GOS, and the weight ratio of GOS:lactose ranges from 6:1 to 45:1. Based on carbohydrates, another illustrative and non - limiting example of a GOS composition according to the invention can contain from 0.5 to 6% by weight of lactose and from 24 to 50% by weight of GOS, and wherein the weight ratio of GOS:lactose in the composition ranges from 6:1 to 45:1. Unless otherwise stated, these illustrative and non - limiting examples of GOS compositions according to the invention can also have any of the features and any combination listed below.

[0074] In one aspect, any GOS composition described herein can contain from 1 to 5.5% by weight of lactose or from 30 to 48% by weight of GOS, or can contain both from 1 to 5.5% by weight of lactose and from 30 to 48% by weight of GOS. In another aspect, the GOS composition can contain from 2 to 6% by weight of lactose, or from 33 to 50% by weight of GOS, or can contain both from 2 to 6% by weight of lactose and from 33 to 50% by weight of GOS. Additionally or alternatively, the GOS composition can contain from 2.5 to 5% by weight of lactose, or from 38 to 46% by weight of GOS, or can contain both from 2.5 to 5% by weight of lactose and from 38 to 46% by weight of GOS. The respective amounts of lactose and GOS in the composition are based on total carbohydrates. Additionally or alternatively, the weight ratio of GOS:lactose in the GOS composition can fall within the range of 7:1 to 35:1, 7:1 to 15:1, 8:1 to 30:1, or 8:1 to 12:1.

[0075] As will be readily appreciated by those skilled in the art, the GOS composition is not limited to lactose and GOS components. For example, the GOS composition can further comprise: glucose, galactose, and DP2 carbohydrates; alternatively, glucose and galactose; alternatively, glucose; alternatively, galactose; or alternatively, DP2 carbohydrates. In one aspect, the amount of galactose (or glucose) present in the GOS composition is greater than the amount of lactose. For example, the weight ratio of galactose:lactose in the GOS composition can fall within the range 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 weight ratio of glucose:lactose in the GOS composition can generally 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. Although not limited thereto, any GOS composition described herein can contain 5 to 20 wt% galactose, such as 6 to 15 wt% or 7 to 10 wt% galactose, and / or 15 to 30 wt% glucose, such as 18 to 26 wt% or 20 to 24 wt% glucose. These amounts are based on total carbohydrates.

[0076] Similarly, the GOS composition is not limited to carbohydrates and can also contain fats, proteins, and minerals. Although not limited thereto, the GOS composition can contain, for example, 0.01 to 2 wt% fat, and more typically, 0.03 to 1 wt% or 0.03 to 0.2 wt% fat. Additionally or alternatively, the GOS composition can comprise 0.3 to 3 wt% protein, and more typically, can comprise 0.4 to 1.5 wt% or 0.6 to 1.2 wt% protein. Additionally or alternatively, the GOS composition can contain 0.5 to 5 wt% minerals, and more typically, can comprise 1 to 3 wt% or 1.5 to 2.5 wt% minerals. Examples

[0077] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting the scope of the present invention. After reading the description herein, various other aspects, modifications, and their equivalents can be envisioned by those of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims.

[0078] Total solids (wt%) were determined by the procedure SMEDP 15.10 C using a CEM Turbo solids and moisture analyzer (CEM Corporation, Matthews, North Carolina). Ash is the residue remaining after ignition to constant weight at 550 °C in a suitable apparatus; this treatment at 550 °C generally eliminates all organic matter and the remaining material is mainly minerals (Standard Methods for the examination of dairy products, 17 th th Edition (2004), American Public Health Association, Washington DC). The ash test was performed using a Phoenix (CEM microwave oven) which heated the sample at 550 °C for 30 min. The mineral content (by weight %) is generally similar to the ash content (wt %), and thus the results of the ash test were used to quantify the total mineral content in the present disclosure. Protein content and fat content were determined by AOAC (Association of Official Analytical Chemists) methods.

[0079] The carbohydrate profile (glucose, galactose, lactose, DP2, DP3, DP4 and DP4+) was determined by high performance liquid chromatography (HPLC) using different detectors. Galactose, glucose, DP2, DP3, DP4 and DP4+ were quantified using HPLC-RI (refractive index). The HPAEC-PAD (pulsed amperometric detection) method was used to separate DP2 GOS from lactose.

[0080] Example 1

[0081] Figure 1 Summarized the process used in Example 1, and Table I summarizes the carbohydrate breakdown of certain starting, intermediate and final compositions. The starting lactose-containing product used in the example 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 (balance water).

[0082] In the first enzymatic step, lactose-containing product per liter is mixed with 3.6 g of β-galactosidase (from Bifidobacterium bifidum) in a stirred tank at a temperature of 55 °C for 120 min to form a first composition, so as to maximize the GOS yield. The first composition is gently mixed throughout the process to prevent the sedimentation of lactose and to increase the interaction between the enzyme in the tank and the structural units of GOS. Then enzyme inactivation is carried out at 95 °C for 7.5 min to minimize or stop the hydrolysis of the enzyme. If inactivation is not carried out at the appropriate time, the reaction equilibrium may shift towards the hydrolysis of GOS, thus reducing the GOS yield.

[0083] After the first enzymatic step, the temperature is lowered to 37 °C, and the first composition contains a mixture of GOS with different degrees of polymerization (DP), residual lactose, DP2, glucose and galactose (see Table I). In the second enzymatic step, the first composition is contacted with about 0.06 wt% of a different β-galactosidase (from Kluyveromyces lactis) at a temperature of 37 °C for 120 min to form a second composition, and this step reduces the amount of residual lactose by hydrolyzing it into glucose and galactose. The enzyme in this step has high hydrolytic activity and is highly selective for decomposing lactose and has a lower affinity for GOS. Gentle mixing is maintained throughout this step to increase the interaction between the second enzyme and the residual lactose. The temperature and time for the second enzymatic treatment step are usually selected to achieve a significant reduction in lactose (such as 65 - 85%), while maintaining almost all of the GOS (such as 95% or more).

[0084] Inactivation of the second enzyme is carried out at 95 °C for 7.5 min to minimize or stop the enzymatic decomposition of GOS. If inactivation is not carried out at the appropriate time, the GOS yield will be reduced. After the second enzymatic step, the temperature is lowered to 5 - 10 °C, and the second composition contains a mixture of GOS with different degrees of polymerization (DP), residual lactose, DP2, glucose and galactose (see Table I).

[0085] The second composition is subjected to membrane filtration at a temperature in the range of 10 - 20 °C and a pressure in the range of 360 - 560 psig to concentrate it into approximately twice the (high molecular weight) GOS, while permeating other (lower molecular weight) sugars, including DP2, lactose, glucose and galactose. A nanofiltration unit is used to form a GOS composition (retentate) and an NF permeate. The nanofiltration unit employs a polymeric membrane filter with an MWCO of 500 - 700 Daltons.

[0086] Advantageously, in the process of Example 1 as shown in Table I, the first composition contains 6.8 wt% of GOS and only 2.8 wt% of lactose (the weight ratio of GOS:lactose is equal to 2.4:1). Based on carbohydrates, the first composition contains 36 wt% of GOS and only 15 wt% of lactose, and the lactose contained in the first composition is 80 wt% less than the lactose present in the starting lactose-containing product.

[0087] Table I demonstrates that the amount of GOS relative to lactose in the second composition is surprisingly high. The second composition contains 6.5 wt% of GOS (only 0.2 - 0.3 wt% lower than in the first composition) and only 0.7 wt% of lactose (the weight ratio of GOS:lactose is equal to 9.3:1). Based on carbohydrates, the second composition contains 35 wt% of GOS and only 3.8 wt% of lactose, and the lactose contained in the second composition is 95 wt% less than the lactose present in the starting lactose-containing product.

[0088] After the membrane filtration step, the concentrated GOS composition unexpectedly has a relatively high amount of GOS relative to lactose, while the total amount of carbohydrates increases (higher solids%). The GOS composition contains 15.6 wt% of GOS (DP3+) and only 1.5 wt% of lactose (the weight ratio of GOS:lactose is equal to 10.3:1). Based on carbohydrates, the GOS composition contains 43 wt% of GOS and only 4 wt% of lactose. The GOS composition in Table I has 35 - 36 wt% solids and contains approximately 0.09 wt% of fat, 0.95 wt% of protein, and 2.1 wt% of minerals.

[0089] Table I Carbohydrate profile for Example 1 (each value in wt%)

[0090]

[0091] Examples 2 - 4

[0092] In Examples 2 - 4, representative first compositions have 18 - 21 wt% total solids, the total solids having the components and amounts shown in Table II, are treated with different amounts of a second enzyme (0.06 wt%, 0.1 wt%, 0.4 wt%), and incubated at a fixed temperature of 37 °C for different times (90 min, 120 min, 180 min) to determine the effect on lactose, GOS (DP3+), and the weight ratio of GOS:lactose in the second composition (total solids range is 18 - 21 wt%). Table II summarizes these results.

[0093] Higher enzyme loadings reduce the amounts of both lactose and GOS in the second composition and generally increase the GOS:lactose ratio. However, in the second composition, surprisingly high GOS:lactose ratios are generally accompanied by significantly lower amounts of GOS (e.g., lower GOS yields). Longer incubation time periods generally also reduce the amounts of both lactose and GOS in the second composition and generally increase the GOS:lactose ratio.

[0094] Table II. Carbohydrate profiles for Examples 2 - 4 (values in wt%)

[0095]

[0096] Examples 5 - 9

[0097] Examples 5 - 9 were conducted to determine the effect of the molecular weight cut-off (MWCO) of the polymeric nanofiltration membrane on the distribution of carbohydrates (sugars and GOS) in the NF retentate and NF permeate. In Examples 5 (MWCO of 200 Da) and 8 (MWCO of 600 - 800 Da), and the representative first composition before filtration (18.7 wt% solids, 0.05 wt% fat, 0.66 wt% protein, and 1.15 wt% minerals) has the components and amounts shown in Table III and is subjected to membrane filtration at temperatures in the range of 5 - 50 °C and pressures in the range of 200 - 500 psig. In Examples 6 (MWCO of 150 - 300 Da) and 7 (MWCO of 300 - 500 Da), and the representative first composition before filtration (21.2 wt% solids, 0.05 wt% fat, 0.66 wt% protein, and 1.22 wt% minerals) has the components and amounts shown in Table III and is subjected to membrane filtration at temperatures in the range of 5 - 45 °C and pressures in the range of 350 - 550 psig.

[0098] The NF permeates of Examples 5 - 7 contain very little GOS, so the NF retentate contains nearly all of the GOS present in the composition before filtration, i.e., very good GOS yields. However, these NF permeates also do not contain large amounts of sugars (DP2, glucose, galactose, lactose), indicating that many sugars are also in the NF retentate, i.e., both sugars and GOS are concentrated. Thus, these MWCOs are useful when GOS yield is very important but a significant GOS concentration relative to sugars is not required (note the GOS / sugar ratio in the NF retentate).

[0099] The NF permeate of Example 8 contains a significant amount of GOS, and the GOS / sugar ratio in the NF retentate is very high. The MWCO of Example 8 permeates a substantial amount of GOS, so the GOS yield in the NF retentate is lower, but a significant concentration of GOS relative to sugars is observed in the NF retentate.

[0100] Example 9 (MWCO of 500 - 700 Da) was carried out with different starting materials, and before filtration (19.3 wt% solids, 0.06 wt% fat, 0.4 wt% protein, and 1.07 wt% minerals) the representative second composition had the components and amounts shown in Table III, which was subjected to membrane filtration at a temperature in the range of 10 - 20 °C and a pressure in the range of 320 - 560 psig. This example had a good balance between the GOS yield in the NF retentate (relatively low GOS in the NF permeate) and the concentrated GOS relative to sugars in the NF retentate (GOS / ratio of the NF retentate to the composition before filtration). 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.

[0101]

Claims

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

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

3. The method according to claim 1 or 2, wherein the lactose-containing product comprises: 0.01 to 1% by weight, 0.01 to 0.5% by weight or 0.01 to 0.2% by weight of fat; and / or 0.1 to 2% by weight, 0.1 to 1.2% by weight or 0.2 to 1% by weight of protein; and / or 0.5 to 3% by weight, 0.7 to 2.5% by weight or 1 to 2% by weight of minerals.

4. The method according to any one of claims 1 to 3, wherein the lactose-containing product comprises the nanofiltration (NF) retentate portion of the ultrafiltration (UF) permeate portion of whole milk or skim milk.

5. The method according to claim 4, wherein the lactose-containing product comprises a concentrated NF retentate portion.

6. The method according to any one of claims 1 to 5, wherein: Based on the amount of lactose in the lactose-containing product, the amount of the first β-galactosidase is 1 to 15% by weight, 1.5 to 6% by weight, 1.5 to 4% by weight, or 2 to 3% by weight; and / or Based on the total weight of the lactose-containing product, the amount of the first β-galactosidase is 0.05 to 1% by weight, 0.1 to 0.8% by weight, or 0.15 to 0.65% by weight; and / or The first β-galactosidase is derived from Bifidobacterium bifidum.

7. The method according to any one of claims 1 to 6, wherein: The first temperature range during step (a) is 20 to 70 °C, 25 to 65 °C, 30 to 70 °C, 30 to 60 °C, or 35 to 55 °C; and / or The first time period range for step (a) is 15 min to 10 h, 30 min to 8 h, 45 min to 6 h, 1 h to 4 h, or 90 min to 150 min.

8. The method according to any one of claims 1 to 7, wherein: The inactivation in step (b) includes subjecting the first composition to a temperature within the following range, or heat-treating the first composition at a temperature within the following range: 75 to 150 °C, 80 to 120 °C, 80 to 115 °C, or 85 to 105 °C; and / or The inactivation in step (b) includes subjecting the first composition to a time period within the following range at the temperature, or heat-treating the first composition at the temperature for a time period within the following range: 1 min to 6 h, 1 min to 30 min, 2 min to 20 min, or 4 min to 15 min.

9. The method according to 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. The method according to any one of claims 1 to 9, wherein before step (d), the first composition contains: 1 to 5% by weight, 1.5 to 4.5% by weight or 2 to 3.5% by weight of lactose; and / or 4 to 10% by weight, 5 to 9% by weight or 6 to 8.5% by weight of GOS; and / or a weight ratio of GOS:lactose of 1.6:1 to 3.5:1, 2:1 to 3:1 or 2.2:1 to 2.8:1; and / or 10 to 20% by weight, 12 to 18% by weight or 13 to 17% by weight of lactose (based on carbohydrates); and / or 30 to 44% by weight, 32 to 42% by weight or 34 to 40% by weight of GOS (based on carbohydrates); and / or 60 to 95% by weight, 70 to 90% by weight or 75 to 85% by weight less lactose than the lactose-containing product described in step (a).

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

12. The method according to any one of claims 1 to 11, wherein: The second temperature range during step (d) is 10 to 60 °C, 20 to 50 °C, 25 to 45 °C, or 30 to 45 °C; and / or The second time period range for step (d) is 15 min to 10 h, 30 min to 8 h, 45 min to 6 h, 1 h to 4 h, or 90 min to 150 min.

13. The method according to any one of claims 1 to 12, wherein: The inactivation in step (e) includes subjecting the second composition to a temperature within the following ranges, or heat-treating the second composition at a temperature within the following ranges: 75 to 150 °C, 80 to 120 °C, 80 to 115 °C, or 85 to 105 °C; and / or The inactivation in step (e) includes subjecting the second composition to a time period within the following ranges at the temperature, or heat-treating the second composition at the temperature for a time period within the following ranges: 1 min to 6 h, 1 min to 30 min, 2 min to 20 min, or 4 min to 15 min.

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

15. The method according to any one of claims 1 to 14, wherein before step (f), the second composition contains: 0.05 to 5% by weight, 0.05 to 1.5% by weight, 0.1 to 4% by weight, 0.1 to 1% by weight, 0.2 to 2% by weight or 0.2 to 0.9% by weight of lactose; and / or 4 to 15% by weight, 4 to 9% by weight, 5 to 12% by weight, 5 to 8% by weight, 5.5 to 9.5% by weight or 5.5 to 7.5% by weight of GOS; and / or a weight ratio of GOS:lactose of 4:1 to 40:1, 7:1 to 35:1 or 8:1 to 30:1; and / or 0.5 to 5% by weight, 1 to 5% by weight or 2 to 4% by weight of lactose (based on carbohydrates); and / or 20 to 45% by weight, 25 to 40% by weight or 32 to 39% by weight of GOS (based on carbohydrates); and / or 80 to 99% by weight, 85 to 98% by weight, 90 to 99% by weight, 92 to 99% by weight or 93 to 98% by weight less lactose than the lactose in the lactose-containing product described in step (a); and / or 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.8% by weight or less, 0.5% by weight or less or 0.3% by weight or less less GOS than the GOS in the first composition.

16. The method according to any one of claims 1 to 15, wherein: Step (f) includes nanofiltrating the second composition using a polymer membrane; and / or Step (f) includes diafiltrating the second composition using a nanofiltration membrane.

17. The method according to any one of claims 1 to 16, wherein the second composition is subjected to membrane filtration using a membrane with a molecular weight cut-off (MWCO) in the range of 150 to 1000 Da, 150 to 900 Da, 150 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. The method according to any one of claims 1 to 17, wherein step (f) is carried out under the following conditions: A filtration temperature in the range of 5 to 50 °C, 10 to 45 °C, 20 to 50 °C, 5 to 25 °C or 8 to 20 °C; and / or A pressure in the range of 100 to 1000 psig, 200 to 800 psig, 300 to 650 psig or 320 to 560 psig.

19. The method according to any one of claims 1 to 18, wherein the concentrated GOS composition contains: 0.1 to 3% by weight, 0.5 to 2% by weight or 0.7 to 1.8% by weight of lactose; and / or 10 to 20% by weight, 12 to 20% by weight or 14 to 18% by weight of GOS; and / or A weight ratio of GOS:lactose of 6:1 to 45:1, 7:1 to 35:1 or 8:1 to 30:1; and / or 0.5 to 6% by weight, 1 to 5.5% by weight, 2 to 6% by weight, or 2.5 to 5% by weight of lactose (based on carbohydrates); and / or 24 to 50% by weight, 30 to 48% by weight, 33 to 50% by weight, or 38 to 46% by weight of GOS (based on carbohydrates).

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

21. The method according to any one of claims 1 to 20, the method further comprising the following steps: (i) Determining the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof in the first composition before step (d); and (ii) Based on the determined compositional characteristics of the first composition, adjusting the first temperature, the first time period, the amount of the first β-galactosidase, or any combination thereof in step (a).

22. The method according to any one of claims 1 to 21, the method further comprising the following steps: (i) Determining the amount of lactose, the amount of GOS, the GOS:lactose ratio, or any combination thereof in the second composition before step (f); and (ii) Based on the determined compositional characteristics of the second composition, adjusting the second temperature, the second time period, the amount of the second β-galactosidase, or any combination thereof in step (d).

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

24. A composition, the composition comprising: Lactose at 0.5 to 6% by weight based on carbohydrates and GOS at 24 to 50% by weight based on carbohydrates; and / or A weight ratio of GOS:lactose of 6:1 to 45:

1.

25. The composition according to claim 23 or 24, wherein the composition comprises: 1 to 5.5% by weight of lactose and / or 30 to 48% by weight of GOS; or 2 to 6% by weight of lactose and / or 33 to 50% by weight of GOS; or 2.5 to 5% by weight of lactose and / or 38 to 46% by weight of GOS.

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

1.

27. The composition according to any one of claims 23 to 26, wherein: The composition further comprises galactose in a weight ratio of galactose:lactose in the range 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; and / or The composition further comprises glucose in a weight ratio of glucose:lactose in the range of 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 Based on the total carbohydrates, the composition further comprises 5 to 20 wt%, 6 to 15 wt%, or 7 to 10 wt% of galactose; and / or Based on the total carbohydrates, the composition further comprises 15 to 30 wt%, 18 to 26 wt%, or 20 to 24 wt% of glucose.

28. The composition according to any one of claims 23 to 27, wherein the composition further comprises: 0.01 to 2% by weight, 0.03 to 1% by weight or 0.03 to 0.2% by weight of fat; and / or 0.3 to 3% by weight, 0.4 to 1.5% by weight or 0.6 to 1.2% by weight of protein; and / or 0.5 to 5% by weight, 1 to 3% by weight or 1.5 to 2.5% by weight of minerals.

29. The composition according to any one of claims 23 to 28, wherein the composition is prepared by the method according to any one of claims 1 to 22.

30. The method according to any one of claims 1 to 22, wherein the composition prepared is the composition according to any one of claims 23 to 28.

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