Galactooligosaccharides, their preparation and use

Abstract

A β-galacto-oligosaccharide composition is disclosed, which comprises, by weight, 2.0-20 wt% of 3'-galactosyllactose, 2.0-20 wt% of 6'-galactosyllactose, 3.0-25.0 wt% of 4'-galactosyllactose, and at least 15 wt% of disaccharides gal-β-1,2-glc and gal-β-1,3-glc, and its production and use.

Description

[0001] This invention relates to novel galactooligosaccharides, their preparation, and their use in nutritional compositions.

[0002] β-galacto-oligosaccharides (GOS), also known as oligogalactosylactose, oligogalactose, or transgalacto-oligosaccharides (TOS), are important food ingredients.

[0003] GOS are complex mixtures of carbohydrates with varying chain lengths, bond types, and degrees of branching. Traditional GOS consist of galactose unit chains and terminal glucose units. Therefore, they have the general formula (Gal). n Glu. In each chain, the value of n ranges from 1 to 8, while the average value of n in traditional GOS is higher than 1. In other words, GOS contains disaccharides with the formula Gal-Glu and disaccharides with the formula (Gal). n Both are oligosaccharides of Glu (where n=2-8).

[0004] Due to its indigestible nature, GOS belongs to the prebiotic group. Prebiotics are defined as indigestible food components that beneficially influence the host by stimulating the growth and / or activity of beneficial bacteria in the colon. When added to infant formula, GOS is able to replicate the bifidobacterial effects of oligosaccharides (human milk oligosaccharides; HMOs) present in human milk in promoting bacterial colonization in the gut and protecting against pathogens. This ability significantly increases interest in its production and application in a variety of food and pharmaceutical processes. For example, GOS is present in commercially available food products for infants and adults, ranging from infant formula to foods for critically ill patients.

[0005] Human breast milk contains human motility organic compounds (HMOs) constructed from the following monosaccharides: D-glucose, D-galactose, L-fucose, sialic acid (N-acetylneuraminic acid), and N-acetylglucosamine. Examples of such HMOs include 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetrasaccharide (LNnT).

[0006] Other examples of HMOs are galactosyllactoses 3'-galactosyllactose (3'-GL), 4'-galactosyllactose (4'-GL), and 6'-galactosyllactose (6'-GL). These galactosyllactoses are also present in GOS, although their concentrations are highly dependent on the manner in which GOS are produced, such as in enzyme selection and reaction conditions.

[0007] GOS synthesis typically involves a number of galactosyl transfer processes catalyzed by β-galactosidase (β-D-galactosidase; EC 3.2.1.23).

[0008] On the one hand, β-galactosidase can use lactose as a galactosyl donor and lactose or intermediate GOS species as galactosyl acceptors to produce oligosaccharides; on the other hand, β-galactosidase can hydrolyze lactose and GOS species. The latter reaction mainly occurs in the later stage of the process, when the concentration of lactose substrate has decreased and the concentration of GOS species has increased.

[0009] Suitable examples of β-galactosidases are derived from the following: Bacillus circulans, Aspergillus oryzae, Aspergillus niger, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, and Papiliotrema terrestris.

[0010] The GOS yield and the composition of the resulting mixture depend in particular on the enzyme used. For example, fungal β-galactosidases derived from the genus *Aspergillus* primarily generate β1-6 bonds (thus producing GOS formulations mainly containing β1-6 bonds, which can be referred to as "6'-GOS"), with a maximum GOS yield of 30%-40% on a dry matter basis; while bacterial β-galactosidases derived from *Bacillus circinus* primarily generate β1-4 bonds (producing GOS formulations mainly containing β1-4 bonds, which can also be referred to as "4'-GOS"), with a maximum GOS yield of approximately 60%.

[0011] 3'-GL has been shown to improve intestinal barrier function (Salminen, S. et al., Nutrients, 2020, 12, 1952) and vaccine response (Toutounchi, NS et al., Nutrients, 2021, 13, 3190).

[0012] 6'-GL appears to not only have a high bifidogenicity, but also a high capacity to improve immunity by increasing the gene expression of TLR2 and TLR4, similar to other HMOs such as 3'-SL and 6'-SL (Asakuma et al., J. Appl. Glycosci., 2010, 57, 177-183).

[0013] 4'-GL has been shown to regulate gene expression in certain Bifidobacterium breve species (Shigehisa, Akira et al., Microbiology 161 (2015) 1463).

[0014] Furthermore, Newburg et al., J. Nutri. [Journal of Nutrition], 2016, 146, 358-367, observed that three galactosyllactoses (3'-GL, 4'-GL, and 6'-GL) expressed in colostrum attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and immature human intestine. This suggests that these three galactosyllactoses could serve as potent physiological anti-inflammatory agents in human colostrum and early milk, contributing to innate immune regulation.

[0015] The object of this invention is to provide a β-galacto-oligosaccharide composition that combines relatively high contents of HMO 3'-GL, 6'-GL, and 4'GL with a relatively high oligosaccharide content. Oligosaccharide content refers to the content of sugars other than monosaccharides and lactose.

[0016] Further research is expected to reveal that the β-galacto-oligosaccharide compositions contain significant amounts of the disaccharides gal-β-1,2-glc and gal-β-1,3-glc. Studies have shown that these compounds can stimulate the production of mucin, which is essential for the colonization of Bifidobacteria in the colon (Lammerts van Bueren et al., (2017) Scientific Reports 7:40478).

[0017] Furthermore, in vitro fermentation studies have shown that these DP2 species are utilized very rapidly by Bifidobacteria, indicating that these DP2 GOS components have a strong bifidogenic effect and a high growth-stimulating effect on Bifidobacteria (Akkerman et al. (2022) Food & Function 13: 6510-6521).

[0018] Another expectation is for β-galacto-oligosaccharide compositions containing significant amounts of oligosaccharide structures with a degree of polymerization (DP) of at least 5. The presence of such relatively long oligosaccharide chains contributes to greater structural diversity, which is important because short and / or long galactosidase chains are preferred prebiotics depending on the specific microbiome (see, for example, Ladirat, SE et al., Bioactive Carbohydrates and Dietary Fiber 3 (2014) 59 and Logtenberg, MJ et al., Journal of Agricultural and Food Chemistry, 2020, 68, 7800). Furthermore, DP>5 is beneficial to the Bifidobacterium longum species (Barboza, Mariana et al., Applied and Environmental Microbiology 75 (2009): 7319-7325).

[0019] Therefore, the present invention relates to a β-galacto-oligosaccharide composition comprising 2.0-20.0 wt% of 3'-galactosyllactose, 2.0-20.0 wt% of 6'-galactosyllactose, 3.0-25.0 wt% of 4'-galactosyllactose, and at least 15 wt% of disaccharides gal-β-1,2-glc and gal-β-1,3-glc in total weight of oligosaccharides other than lactose in the composition.

[0020] Therefore, in a preferred embodiment, the present invention relates to a β-galacto-oligosaccharide composition comprising 4.0-20.0 wt% of 3'-galactosyllactose, 5.0-20.0 wt% of 6'-galactosyllactose, 3.0-25.0 wt% of 4'-galactosyllactose, and at least 15 wt% of disaccharides gal-β-1,2-glc and gal-β-1,3-glc.

[0021] In this specification, the term "oligosaccharide" refers to all sugars with a degree of polymerization of 2 or greater. In other words, it excludes monosaccharides but includes disaccharides (including lactose).

[0022] Therefore, the term "total weight of oligosaccharides other than lactose" refers to the total weight of all sugars with a degree of polymerization of 2 or greater, excluding lactose.

[0023] The 3'-galactosyllactose (3'-GL) content of the β-galacto-oligosaccharide of the present invention is preferably in the range of 4.0-15.0 wt%, more preferably 4.0-10.0 wt%, and most preferably 5.0-8.0 wt%.

[0024] The 4'-galactosyllactose (4'-GL) content of the β-galacto-oligosaccharide of the present invention is preferably in the range of 5.0-20.0 wt%, more preferably 7.5-17.5 wt%, and most preferably 10.0-15.0 wt%.

[0025] The 6'-galactosyllactose (6'-GL) content of the β-galacto-oligosaccharide of the present invention is preferably in the range of 5.0-15.0 wt%, and most preferably in the range of 8.0-10.0 wt%.

[0026] These weight percentages are based on the total weight of oligosaccharides excluding lactose.

[0027] The contents of 3'-GL, 4'-GL and 6'-GL can be determined by the fluorescent labeling method described by JC Bigge et al., Analytical Biochemistry 230 (1995) 229–238.

[0028] In short, GOS samples were derivatized using a reagent solution of DMSO, acetic acid, o-aminobenzamide, and methylpyridine. Different concentrations of pure reference GL were derivatized and reduced using the same reagents to prepare calibration curves. The derivatized GOS and GL samples were analyzed by ultra-high performance liquid chromatography (using a gradient of acetonitrile and ammonium formate aqueous solution).

[0029] Preferably, based on the dry matter content, the total oligosaccharide content excluding lactose, i.e. the content of sugars other than monosaccharides and lactose, is in the range of 30-75 wt%, more preferably 40-70 wt%, and most preferably 50-65 wt%.

[0030] The β-galacto-oligosaccharide composition preferably comprises at least 15 wt% of gal-β-1,2-glc and gal-β-1,3-glc based on the total weight of oligosaccharides other than lactose. The maximum content of gal-β-1,2 and gal-β-1,3-glc is preferably 30 wt%, more preferably 25 wt%, and most preferably 20 wt%.

[0031] The β-galacto-oligosaccharide composition according to the present invention can be obtained by a method comprising the following steps:

[0032] - Provide an aqueous lactose-containing feed with an initial lactose concentration of 20-65 wt%.

[0033] - At a temperature range of 40°C-70°C, 25%-55% of the initial lactose concentration in the feed is converted with a first β-galactosidase to provide an intermediate feed. The first β-galactosidase may be derived from Lactobacillus delbrueckii subspecies bulgaricus or Lactobacillus delbrueckii subspecies bulgaricus.

[0034] - The first β-galactosidase is denatured at a temperature ranging from 65°C to 95°C.

[0035] - Adjust the reaction temperature to 30°C-70°C.

[0036] - Add a second β-galactosidase to the intermediate feed, which may be derived from Bacillus circulatory systemicus.

[0037] - At a temperature range of 30°C-70°C, lactose is converted using this second β-galactosidase until at least 70 wt% of the initial lactose content is converted.

[0038] - This denatures the second β-galactosidase.

[0039] -Remove enzyme residues.

[0040] This method allows for the provision of GOS compositions according to the present invention.

[0041] Furthermore, by allowing the GOS structures (DP3, DP2) formed by the first enzyme to be used by the second enzyme, new structures are formed that cannot be formed by using only one of these enzymes or by blending existing GOS compositions, resulting in a very diverse range of GOS compositions.

[0042] Furthermore, compared to many other enzyme combinations and to single enzymes, this method produces high lactose conversion to GOS structures. The first enzyme used exhibits considerably high GOS synthetic activity at high lactose concentrations, while the second enzyme exhibits high GOS synthetic activity at low lactose concentrations.

[0043] Another advantage is that this method can be carried out in one pot.

[0044] It should be noted that the GOS production method using two different enzymes has been previously disclosed.

[0045] WO 2019 / 119102 discloses a method for producing GOS using two enzymes: one derived from fungi, more specifically *Aspergillus*, and the other from yeasts, more specifically *Kluyveromyces*. This is said to produce a unique balance of DP2, DP3, DP4, and DP5 structures. As shown in the following experiments, both the *Aspergillus* and *Kluyveromyces* enzymes yielded low GOS yields and left large amounts of unconverted lactose.

[0046] A. Botvynko et al., Biochemical and Biophysical Research Communications, 517 (2019) 762-766, disclose the production of GOS using different combinations of enzymes (sequentially or simultaneously), and the effect on GOS yield. No information is provided regarding the individual oligosaccharides formed, nor is the combination of enzymes according to the invention used.

[0047] EP 263700 also discloses a method for preparing GOS using two enzymes, but requires a second enzyme to hydrolyze disaccharides (e.g., lactose) that remain after the first enzyme reaction, thereby increasing the monosaccharide content and producing a mixture of sweet sugars with a lower caloric content than conventional sweeteners.

[0048] KR100168718 discloses the production of GOS using Aspergillus oryzae and Bacillus circulatoryus. As shown in the following experiments, the use of Aspergillus oryzae negatively impacts the GOS yield and leaves a large amount of unconverted lactose.

[0049] The first step of the method of the present invention requires providing an aqueous lactose-containing feed with an initial lactose concentration of 20-65 wt%, preferably 30-65 wt%, and most preferably 45-55 wt%.

[0050] This dispersion can be obtained by dissolving lactose crystals in water at high temperature, followed by adjusting the temperature to the desired reaction temperature.

[0051] Alternatively, aqueous lactose-containing feedstock can be lactose-containing whey permeate, such as cheese whey permeate (CWP), or a CWP that has undergone further processing to remove unwanted components and / or enrich desired components. CWP is a lactose-rich waste liquid remaining after protein extraction from cheese whey and is a significant portion of dairy waste. In all milk-producing countries, milk is primarily used to manufacture cheese. However, only about half of the solids present in milk coagulate and are recycled as cheese; the remaining half is recycled as whey. Whey primarily contains protein, lactose, minerals, and vitamins. Following whey ultrafiltration (UF), commercially valuable proteins are collected from the UF-retentate; the UF-permeate is cheese whey permeate; also known as liquid permeate. This permeate primarily contains lactose, minerals, and vitamins.

[0052] CWPs can be used as lactose-containing feeds without further processing or after demineralization. In one embodiment, the lactose-containing feed is a CWP that has been demineralized to an ash content of up to about 4 wt.% or a conductivity of up to about 4 mS. Demineralization can be performed by methods known in the art, including electrodialysis (ED), reverse osmosis (RO), nanofiltration (NF), or ion exchange techniques.

[0053] In another alternative embodiment, the lactose-containing feed can be lactose-free whey permeate (DLP or OPL), which still contains lactose and protein, as well as the minerals and vitamins originally present in the whey permeate. OPL typically also contains about 0.3%–0.4% sialyl lactose (2,3-sialyl lactose and 2,6-sialyl lactose) and possibly other valuable bovine milk oligosaccharides (bMOs). Furthermore, it has been shown that bMOs contain as many oligosaccharides as those found in human oligosaccharides (hMOs).

[0054] OPL contains a relatively small amount of calcium. Therefore, in order to remove polyvalent anions, such as PO4, 3- citrate 3- Additional calcium can be added to enhance the formation of insoluble calcium hydrogen phosphate and calcium citrate, thereby allowing for easy removal of the salt by means of, for example, centrifugation. In a preferred embodiment, OPL is treated with lime (CaO / Ca(OH)2) to precipitate anions.

[0055] The first β-galactosidase may be derived from *Lactobacillus delbrueckii* subsp. bulgaricus or *Lactobacillus delbrueckii* subsp. lactis, or have a strong similarity to them. In a preferred embodiment, the first β-galactosidase is derived from *Lactobacillus delbrueckii* subsp. bulgaricus or *Lactobacillus delbrueckii* subsp. lactis, and most preferably, it is derived from *Lactobacillus delbrueckii* subsp. bulgaricus.

[0056] The appropriate enzyme has been disclosed in WO 2020 / 049016 and can be Bonlacta. TM Purchased from IFF / Danisco. However, in a preferred embodiment, the enzyme is contained in a microorganism that endogenously expresses the enzyme. This allows for cheaper and easier processing as it saves the work of isolating the enzyme. Microorganisms, such as strains of *Lactobacillus delbrueckii* subsp. bulgaricus or *Lactobacillus* subsp., can be used as whole cells or their active parts or components, preferably cell-free extracts.

[0057] A strain of *Lactobacillus delbrueckii* subsp. *bulgaricus* capable of producing galactosidase activity for providing the oligosaccharide compositions of the present invention has been deposited with accession number DSM20080.

[0058] Depending on the desired reaction time, the first β-galactosidase is added to the lactose-containing feed at a preferred dose in the range of at least 2-100 LU / g lactose, more preferably 5-50 LU / g lactose, even more preferably 10-40 LU / g lactose, and most preferably 20-30 LU / g lactose. As used herein, one lactase unit (LU) is defined as the amount of enzyme that releases 1 μmol of galactose or o-nitrophenol per minute at 40°C and pH 6.5 during the early stages of ONPG (o-nitrophenyl-galactoside) hydrolysis.

[0059] Then, the reaction with the first β-galactosidase is carried out at a preferred temperature in the range of 40°C-70°C, preferably 40°C-65°C, more preferably 50°C-63°C, and most preferably 55°C-60°C.

[0060] The reaction time is preferably in the range of 0.1-50 hours, more preferably 2-24 hours, and most preferably 4-10 hours.

[0061] The pH of the reaction mixture is preferably in the range of 5.5-7.0, more preferably 5.8-6.8, and most preferably 6.0-6.5.

[0062] The reaction is terminated after at least 25% conversion of the initial lactose concentration and before exceeding 55% conversion, preferably after reaching 35%-55% and most preferably 40%-50% conversion of the initial lactose concentration. This can be achieved by lowering the pH to 4 or lower.

[0063] Terminating the reaction at this point is important because the enzyme will begin hydrolyzing lactose and GOS species at low lactose substrate concentrations. Lactose inversion is determined by the change in Brix of the reaction mixture relative to the initial lactose concentration.

[0064] The first β-galactosidase is then denatured by subjecting the reaction mixture to a temperature in the range of 65°C to 95°C. The reaction mixture is held at this temperature for a period of time sufficient to denature the enzyme, preferably at least 10 minutes, more preferably 20-90 minutes, and most preferably 30-60 minutes.

[0065] The temperature of the reaction mixture is then adjusted to a range of 30°C-70°C, more preferably 40°C-65°C, even more preferably 50°C-60°C, and most preferably 55°C-60°C.

[0066] Then the second β-galactosidase was added to the feed.

[0067] The second β-galactosidase may be derived from Bacillus circulans or have a strong similarity to it.

[0068] In a preferred embodiment, the second β-galactosidase is derived from Bacillus circulatoryus. Even more preferably, the second β-galactosidase is derived from Bacillus circulatoryus and has a molecular weight of 195 kDa (by SDS-PAGE).

[0069] Suitable enzymes can be derived from Bacillus Circulatingus ATCC31382. Its isolation has been disclosed in EP2439270 A1. It can be obtained under the name Biolacta N5 from Amano Enzymes Co., Ltd.

[0070] The second β-galactosidase is preferably added at a preferred dose in the range of at least 1.5 LU / g lactose, more preferably in the range of 2-10 LU / g lactose, even more preferably in the range of 3-8 LU / g lactose, and most preferably in the range of 4-6 LU / g lactose.

[0071] Then, the reaction with the second β-galactosidase is carried out at a preferred temperature in the range of 30°C-70°C, more preferably 40°C-65°C, even more preferably 50°C-60°C, and most preferably 55°C-60°C.

[0072] Continue the reaction until at least 70 wt%, preferably at least 75%, of the initial lactose content has been converted.

[0073] The reaction time is preferably in the range of 10-50 hours, more preferably 15-45 hours, and most preferably 24-36 hours.

[0074] The reaction is terminated by denaturing the second β-galactosidase, for example by heat treatment (e.g., at 100°C for 15 minutes) and / or acidification, and by removing enzyme residues from the reaction mixture containing GOS, for example by ion exchange and / or microfiltration.

[0075] The resulting GOS can then be further purified. Conventional purification steps can be applied, such as removal of enzyme residues, demineralization, deproteinization, removal of monosaccharide components, and / or decolorization (e.g., by treatment with activated charcoal). In one embodiment, the GOS composition is subjected to a nanofiltration (NF) or ultrafiltration (UF) step to remove monosaccharides and any proteins that may be present. Additional concentration steps can be performed, for example, using NF or evaporation, to obtain a concentrated GOS formulation with a dry matter content of, for example, at least 70 wt%, preferably at least 75 wt%.

[0076] Alternatively, only enzyme residues are removed from the reaction mixture, but no further purification or separation of the individual components is performed.

[0077] The GOS according to the present invention can be used in nutritional compositions. This can be a nutritional composition for pregnant women (MUM composition), a nutritional composition for infants (formula milk), a nutritional composition for adolescents (aged 13-20 years), or a nutritional composition for adults (aged >20 years). The nutritional composition can be used as a regular food composition, as a nutritional therapy, as a nutritional support, as a medical food, as a food for special medical purposes, or as a nutritional supplement.

[0078] Examples of nutritional compositions are formula milk. Formula milk includes infant formula, follow-up formula, and growing-up formula (also known as toddler formula). Other examples of nutritional compositions are compositions intended for adults, such as patients or frail elderly individuals, or anyone else wishing to enhance their immune system or gut health.

[0079] Infant formula, neonatal formula, or just formula (American English) or newborn milk, infant milk, or first milk (British English) are processed foods designed and marketed for feeding newborns and infants under 12 months of age. They are typically prepared from powder (mixed with water) or liquid (with or without additional water) for bottle or cup feeding. The U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food that claims or represents, due to its mimicry of human milk or its suitability as a complete or partial substitute for human milk, for a specific dietary purpose solely as a food for infants." Similarly, the Codex Alimentarius International Food Standards (WHO and FAO) defines infant formula as a specially manufactured substitute for breast milk that itself meets the nutritional needs of infants in the first few months after birth until the introduction of appropriate complementary feeding. The Codex Alimentarius describes the essential components of infant formula, including the amounts and specifications of lipid sources, protein sources, carbohydrate sources, vitamins, and minerals.

[0080] To form a nutritional composition, particularly a formula milk, the GOS according to the invention (as an aqueous composition or a (spray-dried) powder obtained therefrom) is blended with other components of the nutritional composition. In the case of formula milk, these components include at least one protein source, at least one lipid source, vitamins, and minerals. Preferably, the aqueous composition is added to a liquid blend of said components.

[0081] The lipid source for formula milk can be any lipid or fat suitable for use in formula milk. Preferred fat sources include milk fat, safflower oil, egg yolk lipids, rapeseed oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palmitoleic acid, high-oleic sunflower oil and high-oleic safflower oil, and microbially fermented oils containing long-chain polyunsaturated fatty acids. In one embodiment, anhydrous milk fat is used. The lipid source can also be a fraction of esters derived from these oils, such as palm oil extract, medium-chain triglycerides, and fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, caproic acid, etc. Small amounts of oils containing large amounts of pre-formed arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oils, can be added. The fat source preferably has an n-6 to n-3 fatty acid ratio of about 5:1 to about 15:1; for example, about 8:1 to about 10:1. In certain aspects, infant formula milk contains an oil mixture containing palmitic acid esterified to triacylglycerol, for example, wherein the amount of palmitic acid esterified at the sn-2 position of the triacylglycerol is 10% to 60% of the total palmitic acid by weight, and the amount of palmitic acid esterified at the sn-1 / sn-3 position of the triacylglycerol is 30% to 80% of the total palmitic acid by weight.

[0082] Examples of protein sources include milk (preferably cow's milk), whey protein sources (such as whey protein concentrate and serum protein concentrate), and various plant proteins. Proteins can be hydrolyzed or unhydrolyzed.

[0083] Examples of vitamins and minerals preferably found in formula milk include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in the form of salts.

[0084] If necessary, the nutritional composition may contain emulsifiers and stabilizers, such as soy lecithin, citrates of monoglycerides and diglycerides. The nutritional composition may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, nucleosides, and probiotics. Suitable probiotics include lactic acid bacteria, Bifidobacterium lactis such as Bifidobacterium lactis Bb12, Streptococcus thermophilus, Lactobacillus johnsonii La1, Bifidobacterium longum BL999, Lactobacillus rhamnosus LPR, Lactobacillus rhamnosus GG, Lactobacillus reuteri, and Lactobacillus salivarius. Such prebiotics are commercially available. Example

[0085] Determination of galactosyllactose content

[0086] Add 200 μl of labeled reagent solution (prepared by mixing 1.633 ml DMSO (100%), 0.7 ml acetic acid (100%), 46 mg o-aminobenzamide, and 102 mg 2,2-methylpyridineborane complex) to an Eppendorf vial. Then add 20 µl of sample and mix thoroughly under vortex. Centrifuge the solution at a very low speed for 2 or 3 seconds. Then heat the solution at 65°C for 2 hours while shaking at 400 rpm in a heating block, followed by cooling on ice for 5 to 10 minutes, centrifuging at a very low speed for 2 or 3 seconds, and adding 700 µl of acetonitrile / water (70 / 30) and mixing thoroughly under vortex. Filter the sample through a 0.2 μm PVDF microfilter and analyze by UPLC with a fluorescence detector.

[0087] This UPLC employed a TSK Gel Amide-80 guard column (3.2 x 15 mm, 3 μm) and an analytical column (4.6 x 150 mm, 3 μm) (Tosoh Bioscience, Stuttgart, Germany). Detection was performed using a Shimadzu RF-10Axl fluorescence detector (λex = 330 nm and λex = 420 nm). Eluent A was 100% acetonitrile; eluent B was 100 mmol / L ammonium formate, pH 4.4. Under gradient conditions (summarized below), aliquots of 3 μL of the labeled solution were loaded onto the guard column at a flow rate of 0.5 ml / min.

[0088] Table 1 - Gradient Conditions:

[0089]

[0090] Example 1

[0091] Add 100 g of the reaction mixture containing 50 wt% lactose, 1 ml of 1.0 M potassium phosphate buffer (pH 6.5), and water to a 180 ml plastic reactor vessel. Stir the reaction mixture in a water bath (50°C) for at least half an hour.

[0092] Subsequently, 20 LU of enzyme was added to initiate the conversion. The reaction was allowed to proceed for 24 hours and terminated by adding 1.5% (v / v) 1 M HCl and denaturing the enzyme at 95°C for 15 minutes.

[0093] The sample was analyzed as described above, and the sugar composition and GL content were estimated by peak percentage. The positions of each GL on the chromatogram were determined by using the corresponding GL reference.

[0094] These results indicate that the GOS yield and lactose conversion obtained using enzymes derived from Kluyveromyces lactis and Aspergillus oryzae were significantly lower than those achieved using other enzymes.

[0095] Table 2 - Evaluation of different enzymes

[0096]

[0097] 1 Oligosaccharides other than lactose

[0098] Example 2

[0099] Three 250 ml glass bioreactors were provided, labeled reactors 1-3.

[0100] Each of these reactors contains a reaction mixture comprising 52.63 g of crystalline lactose hydrate (Lactopure, FrieslandCampina), 47.37 g of demineralized water, and 1 ml of 1.0 M potassium phosphate buffer (pH 6.5). The concentration of the buffer relative to the total water volume is 20 mM.

[0101] The reaction mixture was heated in a water bath (50°C) with stirring. Once the temperature of the reaction mixture reached 50°C, the first β-galactosidase (Bonlacta®, 17308 U / g enzyme preparation) was added to bring the total enzyme dose in the reactor to 60 U / g lactose.

[0102] The reaction was monitored by tracking changes in Brix value (using a Brix refractometer) according to the timetable defined in Table 3. The reaction was continued until 20% lactose conversion was achieved (Brix value 45°; reactor 1), 30% lactose conversion (Brix value 40°; reactor 2), or 40% lactose conversion (Brix value 35°; reactor 3).

[0103] It takes 10 minutes to reach 20% lactose conversion; 40 minutes to reach 30% lactose conversion; and 75 minutes to reach 40% lactose conversion.

[0104] Once the desired lactose inversion is achieved, the reaction is subjected to heat denaturation at 95°C in a water bath for 15 minutes to denature the enzyme.

[0105] The reaction mixture was then cooled to 58°C, followed by the addition of a second β-galactosidase (Biolacta® N5; derived from Bacillus Circulatingus ATCC 31382, from Amano Enzyme Products Co., Ltd.) at a dosage of 5.0 LU / g initial lactose content. The reaction was continued with this second enzyme for 49 hours, after which the enzyme was denatured by adding 1.5% (v / v) 1 M HCl.

[0106] The Brix values ​​and pH of the reaction mixture at the start and end of the enzymatic reaction are presented in Table 3. As shown in the table, the pH decreased slightly during the reaction with Biolacta N5.

[0107] Table 3

[0108]

[0109] Equal aliquots of samples were taken at different time intervals. The samples were denatured by adding 1.5% (v / v) 1.0 M HCl followed by heating at 95°C for 15 minutes. The sugar composition (Table 4) and GOS fingerprint of the samples were analyzed using a CarboPac PA-1 column by HPAEC-PAD HPLC. GOS content (wt%) was defined as 100% - galactose% - glucose% - lactose% - isolaxose% - lactulose%, and oligosaccharide content was defined as the oligosaccharide content excluding lactose.

[0110] Using the analytical references for each GL, the concentrations of 3'-GL, 4'-GL, and 6'-GL were estimated by peak percentage as described above. The GL content based on the total oligosaccharide content was calculated using the oligosaccharide content in Table 4 and summarized in Table 5.

[0111] It can be observed that the GOS prepared according to the method of the present invention has significantly higher contents of 3'-GL and 6'-GL than Vivinal® GOS, and the 4'-GL content is similar to or even higher than that of Vivinal® GOS. However, apart from this, the fingerprint spectrum (i.e., peak set and peak intensity) of the GOS according to the present invention is very similar to that of Vivinal® GOS.

[0112] Furthermore, the content of 4'-GL and gal-β-1,2-glc + gal-β-1,3-glc produced by the method of the present invention is significantly higher than that of commercially available Bimuno® GOS.

[0113] Table 4 - Sugar composition of different samples (in wt% based on dry matter)

[0114]

[0115] 1 Oligosaccharides other than lactose

[0116] Table 5 - Contents of 3'-GL, 4'-GL, and 6'-GL, as well as gal-β-1,2-glc + gal-β-1,3-glc in different samples (based on oligosaccharides) 1 (in wt%)

[0117]

[0118] 1 Oligosaccharides other than lactose

[0119] Example 3

[0120] Example 2 was repeated, except that the second enzyme (Biolacta N5) was used at a concentration of 10 LU / g of initial lactose, and the reaction time was shortened to 24 hours after the addition of the second β-galactosidase.

[0121] Compared to Example 2, higher yields were achieved in a shorter timeframe with higher gal-β-1,2-glc + gal-β-1,3-glc content, but at the expense of 4'-GL content.

[0122] The similarity between GOS produced in reactors 1-3 and Vivinal® GOS was determined by selecting nine DP2-DP4 peaks or peak pairs from the fingerprint spectrum and calculating the percentage of each peak relative to the same peak in the Vivinal® fingerprint spectrum. The average of the nine relative peak areas (similarity index) was 89.6 (GOS obtained in reactor 1), 89.6 (GOS obtained in reactor 2), and 94.2 (GOS obtained in reactor 3).

[0123] Table 6 - Sugar composition of different samples (in wt% based on dry matter)

[0124]

[0125] 1 Oligosaccharides other than lactose

[0126] Table 7 - Contents of 3'-GL, 4'-GL, and 6'-GL, as well as gal-β-1,2-glc + gal-β-1,3-glc in different samples (based on oligosaccharides) 1 (in wt%)

[0127]

[0128] 1 Oligosaccharides other than lactose.

Claims

1. A method for producing a β-galacto-oligosaccharide composition comprising, based on the total weight of oligosaccharides other than lactose, 2.0-20.0 wt% of 3'-galactosyllactose, 2.0-20.0 wt% of 6'-galactosyllactose, 3.0-25.0 wt% of 4'-galactosyllactose, and at least 15 wt% of disaccharides gal-β-1,2-glc and gal-β-1,3-glc, the method comprising the following steps: - Provide an aqueous lactose-containing feed with an initial lactose concentration of 20-65 wt%. - At a temperature range of 40°C-70°C, 25%-55% of the initial lactose concentration in the feed is converted with a first β-galactosidase to provide an intermediate feed. The first β-galactosidase may be derived from *Lactobacillus delbrueckii* subsp. bulgaricus or *Lactobacillus delbrueckii* subsp. *lactosporum*. - The first β-galactosidase is denatured at a temperature ranging from 65°C to 95°C. - Adjust the reaction temperature to 30°C-70°C. - Add a second β-galactosidase to the intermediate feed, which may be derived from Bacillus circulatory systemicus. - At a temperature range of 30°C-70°C, lactose is converted using this second β-galactosidase until at least 70 wt% of the initial lactose content is converted. - This denatures the second β-galactosidase. -Remove enzyme residues.

2. A β-galacto-oligosaccharide composition obtainable by the method of claim 1.

3. The β-galacto-oligosaccharide composition of claim 2, wherein the β-galacto-oligosaccharide composition comprises 4.0-20.0 wt% of 3'-galactosyllactose, 5.0-20.0 wt% of 6'-galactosyllactose, 3.0-25.0 wt% of 4'-galactosyllactose, and at least 15 wt% of disaccharides gal-β-1,2-glc and gal-β-1,3-glc.

4. The β-galacto-oligosaccharide composition of claim 2 or 3, wherein the 3'-galactosyllactose (3'-GL) content is in the range of 4.0-15.0 wt%, preferably 4.0-10.0 wt%, and most preferably 5.0-8.0 wt%, based on the total weight of oligosaccharides excluding monosaccharides and lactose in the composition.

5. The β-galacto-oligosaccharide composition according to any one of claims 2-4, wherein the 4'-galactosyllactose (4'-GL) content is in the range of 5.0-20.0 wt%, preferably 7.5-17.5 wt%, and most preferably 10.0-15.0 wt%, based on the total weight of oligosaccharides excluding monosaccharides and lactose in the composition.

6. The β-galacto-oligosaccharide composition according to any one of claims 2-5, wherein the 6'-galactosyllactose (6'-GL) content is in the range of 5.0-15.0 wt%, and most preferably 8.0-10.0 wt%, based on the total weight of oligosaccharides excluding monosaccharides and lactose in the composition.

7. The β-galacto-oligosaccharide composition according to any one of claims 2-6, wherein the total content of oligosaccharides other than lactose is in the range of 30-75 wt%, more preferably 40-70 wt%, and most preferably 50-65 wt%, based on dry matter content.

8. The β-galacto-oligosaccharide composition according to any one of claims 2-7, wherein the total amount of the disaccharides gal-β-1,2-glc and gal-β-1,3-glc is 15-30 wt%, more preferably 15-25 wt%, and most preferably 15-20 wt%, based on the total weight of the oligosaccharides other than lactose.

9. A nutritional composition comprising a β-galacto-oligosaccharide composition as described in any one of claims 2-8, the nutritional composition further comprising one or more proteins, probiotics, lipids and / or additional carbohydrates.

10. The nutritional composition of claim 9, wherein the nutritional composition is infant formula, follow-up formula, or growing-up formula.

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