Galactooligosaccharide, its preparation and application

Abstract

Beta-galactooligosaccharide composition comprising 2.0-20 wt% 3' -galactosyllactose, 2.0-20 wt% 6'galactosyllactose, 3.0-25.0 wt% 4'-galactosyllactose, and a total amount of the disaccharides gal-β-1,2-glc and gal-β-1,3-glc of at least 15 wt%, all based on the total weight of oligosaccharides other than lactose in the composition, its production and use.

Description

[0001] GALACTOOLIGOSACCHARIDE, ITS PREPARATION AND APPLICATION

[0002] The present invention relates to a new galactooligosaccharide, its preparation, and its use in a nutritional composition.

[0003] Beta-galactooligosaccharide (GOS), also known as oligogalactosyllactose, oligogalactose, oligolactose or transgalactooligosaccharide (TOS), is an important food ingredient.

[0004] GOS is a complex mixture of carbohydrates with differing chain length, linkage type, and degree of branching. Conventional GOS comprises a chain of galactose units and a terminal glucose unit. Hence, it has the general formula (Gal)nGlu. In the individual chains n has a value in the range 1 -8, while the average value of n in conventional GOS is higher than 1 . In other words, GOS contains both disaccharides of the formula Gal-Glu and oligosaccharides of the formula (Gal)nGlu wherein n=2-8.

[0005] Because of its indigestible nature, GOS belongs to the group of prebiotics. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by stimulating the growth and / or activity of beneficial bacteria in the colon. When added to infant milk formulas, GOS is able to replicate the bifidogenic effect of the oligosaccharides present in human milk (Human Milk Oligosaccharides; HMOs) in facilitating bacterial colonization in the gut and protection from pathogens. This ability has significantly increased interest in its production and application in various food and pharmaceutical processes. For example, GOS occurs in commercially available food products for both infants and adults, ranging from infant formula to food for the critically ill.

[0006] The HMOs in human breast milk are built from the following monosaccharides: D- glucose, D-galactose, L-fucose, sialic acid (N-acetyl neuraminic acid), and N-aceyl glucosamine. Examples of such HMOs are 2'-fucosyl lactose (2'-FL), 3-fucosy I lactose (3-FL), 3’-siallyllactose (3’-SL), 6’-sialyllactose (6’-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT).

[0007] Further examples of HMOs are the galactosyllactoses 3’-galactosyllactose (3’-GL), 4’- galactosyllactose (4’-GL), and 6’-galactosyllactose (6’-GL). These galactosyllactoses are also present in GOS, although their concentration is highly dependent on the manner in which GOS is produced, e.g. in terms of enzyme selection and reaction conditions.

[0008] GOS synthesis typically involves a number of galactosyl transfer processes catalyzed by [3-galactosidase ([3-D-galactohydrolase; EC 3.2.1.23).

[0009] On the one hand, [3-galactosidases are able to produce oligosaccharides using lactose as galactosyl donor and lactose or the intermediate GOS species as galactosyl acceptor; on the other hand, [3-galactosidases are able to hydrolyse lactose and GOS species. The latter reaction mainly occurs at the late stages of the process when lactose substrate concentrations have been reduced and the concentration of GOS species has increased.

[0010] Examples of suitable beta-galactosidase enzymes are those derived from Bacillus circulans, Aspergillus oryzae, Aspergillus niger, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, and Papiliotrema terrestris.

[0011] The GOS yield and the composition of the mixture that is obtained depends, amongst others, on the enzyme used. For example, a fungal beta-galactosidase derived from Aspergillus predominantly produces [31 -6 bonds (thus resulting in a GOS preparation that predominantly comprises [31-6 bonds, which may be referred to as “6’-GOS”) with a maximum GOS yield of 30-40% on dry matter, whereas a bacterial betagalactosidase derived from Bacillus circulans predominantly produce [31 -4 bonds (resulting in a GOS preparation that predominantly comprises [31 -4 bonds, which may also be referred to as “4’-GOS”) with a maximum GOS yield around 60%.

[0012] 3’-GL has been shown to improve the gut barrier function (Salminen, S. et al., Nutrients, 2020, 12, 1952) and vaccination responsiveness (Toutounchi, N.S., et al., Nutrients, 2021 , 13, 3190).

[0013] 6’-GL appears not only to have high bifidogenicity, but also a high capacity for improving the immunity through increasing the gene expression for TLR2 and TLR4, similar to other HMOs such as 3’-SL and 6’-SL (Asakuma et al., J. Appt. Glycosci., 2010, 57, 177-183).

[0014] 4’-GL has been shown to regulate the gene expression of certain Bifidobacterium breve species (Shigehisa, Akira, et al., Microbiology 161 (2015) 1463). Furthermore, Newburg et al., J. Nutri., 2016, 146, 358-367, observed that the three galactosyllactoses (3'-GL, 4'-GL, and 6'-GL) expressed in colostrum attenuated NF-KB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. This implies that these three galactosyllactoses may serve as strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation.

[0015] The object of the present invention is the provision of a beta-galactooligosaccharide composition that combines relatively high contents of the HMOs 3’-GL, 6’-GL, and 4’GL with a relatively high oligosaccharide content. The oligosaccharide content refers to the content of saccharides other than monosaccharides and lactose.

[0016] It is a further desire for the beta-galactooligosaccharide composition to contain a significant amount of the disaccharides gal-|3-1 ,2-glc and gal-|3-1 ,3-glc. Study has shown that these compounds are able to stimulate the production of mucin, which is critical 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 utilised very fast by the bifidobacterium, thereby suggesting that these DP2 GOS components are very bifidogenic and have high growth stimulating effect on bifidobacteria (Akkerman et al. (2022) Food & Function 13: 6510-6521 ).

[0018] An additional desire is a beta-galactooligosaccharide composition that contains a significant amount of oligosaccharide structures with a degree of polymerization (DP) of at least 5. The presence of such relatively long oligosaccharide chains contributes to a higher structural diversity, which is important because, depending on the specific microbiota, short and / or long galactosidase chains are the preferred prebiotics (see, e.g., Ladirat, S.E., et al., Bioactive Carbohydrates ad Dietary Fibre 3 (2014) 59, and Logtenberg, M. J., et al. Journal of agricultural and food chemistry, 2020, 68, 7800). Furthermore, DP>5 is beneficial for Bifidobacterium longum species (Barboza, Mariana, et al., Applied and environmental microbiology 75 (2009): 7319-7325).

[0019] The present invention therefore relates to a beta-galactooligosaccharide composition comprising 2.0-20.0 wt% 3’-galactosyllactose, 2.0-20.0 wt% 6’galactosyllactose, 3.0- 25.0 wt% 4’-galactosyllactose, and a total amount of the disaccharides gal-|3-1 ,2-glc and gal-|3-1 ,3-glc of at least 15 wt%, all based on the total weight of oligosaccharides other than lactose in the composition.

[0020] In a preferred embodiment, the present invention therefore relates to a betagalactooligosaccharide composition comprising 4.0-20.0 wt% 3’-galactosyllactose, 5.0-20.0 wt% 6’galactosyllactose, 3.0-25.0 wt% 4’-galactosyllactose, and a total amount of the disaccharides gal-|3-1 ,2-glc and gal-|3-1 ,3-glc of at least 15 wt%, all based on the total weight of oligosaccharides other than lactose in the composition.

[0021] In this specification, the term “oligosaccharides” refers to all saccharides with a degree of polymerisation of 2 or more. In other words, it excludes monosaccharides and includes disaccharides, including lactose.

[0022] The term “total weight of oligosaccharides other than lactose” therefore refers to the total weight of all saccharides with a degree of polymerisation of 2 or more, with the exception of lactose.

[0023] The 3’-galactosyllactose (3’-GL) content of the beta-galactooligosaccharide of the present invention is preferably in the range 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 beta-galactooligosaccharide of the present invention is preferably in the range 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 beta-galactooligosaccharide of the present invention is preferably in the range 5.0-15.0 wt% and most preferably 8.0-10.0 wt%.

[0026] These weight percentages are all based on the total weight of oligosaccharides other than lactose.

[0027] The 3’-GL, 4’-GL, and 6’-GL content can be determined by the fluorescent labelling method es described by J. C. Bigge et al., Analytical Biochemistry 230 (1995) 229- 238.

[0028] Briefly, the GOS samples are derivatized using a reagent solution of DMSO, acetic acid, anthranilamide, and methylpyridine. The individual pure reference GL of varying concentration is derivatized and reduced with the same reagents in order to prepare a calibration curve. The derivatized GOS and GL samples are analyzed with ultra performance liquid chromatography, using a gradient of acetonitrile and aqueous ammonium formate.

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

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

[0031] The beta-galactooligosaccharide composition according to the present invention can is obtainable by a process comprising the steps of: providing an aqueous lactose-containing feed containing an initial lactose concentration of 20-65 wt%, converting 25-55% of the initial lactose concentration in said feed with a first betagalactosidase enzyme at a temperature in the range 40-70°C to provide an intermediate feed, said first beta-galactosidase enzyme being derivable from Lactobacillus delbrueckii subspecies bulgaricus or Lactobacillus delbrueckii subspecies lactis, denaturing said first beta-galactosidase enzyme at a temperature in the range 65-95°C, adjusting the reaction temperature to 30-70°C, adding a second beta-galactosidase enzyme to the intermediate feed, said second beta-galactosidase enzyme being derivable from Bacillus circulans, converting lactose with the second beta-galactosidase at a temperature in the range 30-70°C, until at least 70 wt% of the initial lactose content has been converted, denaturing the second beta-galactosidase, removing enzyme residues. This process allows the provision of a GOS composition according to the present invention.

[0032] In addition, by allowing the GOS structures (DP3, DP2) formed by the first enzyme to be used by the second enzyme, new structures are formed that would not have been formed by using only one of the enzymes or by blending existing GOS compositions, thereby creating a very diverse GOS composition.

[0033] Furthermore, this process gives a high lactose conversion towards GOS structures compared to many other enzyme combinations and compared to the individual enzymes. The first enzyme used has a rather high GOS synthetic activity at high lactose concentrations, whereas the second enzyme has high GOS synthetic activity at low lactose concentration.

[0034] A further advantage is that the process can be performed as a one-pot process.

[0035] It should be noted that GOS production processes using two different enzymes have been disclosed before.

[0036] WO 2019 / 119102 discloses a process to produce GOS with two enzymes: an enzyme derived from a fungus, more in particular an Aspergillus, and an enzyme derived from a yeast, more in particular a Kluyveromyces. This is said to lead to a unique balance of DP2, DP3, DP4, and DP5 structures. As shown in the experiments below, both Aspergillus and Kluyveromyces enzymes give low GOS yields and leave a lot of lactose unconverted.

[0037] A. Botvynko et al., Biochemical and Biophysical Research Communications, 517 (2019) 762-766, discloses GOS production using different combinations of enzymes, either sequential or concurrent, and the effect of GOS yield. No information was provided on the individual oligosaccharides formed, nor was the combination of enzymes according to the present invention used.

[0038] EP 263700 also discloses a process for making GOS using two enzymes, but requires the second enzyme to hydrolyze the disaccharides (e.g. lactose) still present after the first enzyme reaction, thereby increasing the monosaccharide content and producing a sweet saccharide mixture with lower calorie increase than conventional sweeteners. KR100168718 discloses the production of GOS using Aspergillus oryzae and Bacillus circulans. As shown in the experiments below, the use of Aspergillus oryzae negatively affects the GOS yield and leaves a lot of lactose unconverted. The first step of the process of the present invention requires the provision of an aqueous lactose-containing feed with an initial lactose concentration of 20-65 wt%, preferably 30-65 wt%, most preferably 45-55 wt%.

[0039] This dispersion can be obtained by dissolving lactose crystals in water at high temperature, followed by adjusting the temperature to the desired reaction temperature.

[0040] Alternatively, the aqueous lactose-containing feed can be a lactose-containing whey permeate, such as cheese whey permeate (CWP) or a CWP that has been processed further to remove unwanted components and / or to enrich for desirable components. CWP is a lactose-rich effluent remaining after protein extraction from cheese whey, an abundant dairy waste. In all milk-producing countries, milk is primarily used to manufacture cheese. However, only approximately half of the solids present in milk are coagulated and recovered as cheese; the remaining half are recovered as whey. Whey contains mainly proteins, lactose, minerals and vitamins. Upon ultrafiltration (UF) of the whey, commercially valuable proteins are collected from the UF-retentate; the UF-permeate is the cheese whey permeate; also known as liquid permeate. This permeate contains mainly lactose, minerals and vitamins.

[0041] CWP can be used as the lactose-containing feed as such, i.e. without further treatment, or after demineralization. In one embodiment, the lactose-containing feed is a CWP that is demineralized to an ash content of up to about 4 wt.%, or a conductivity of up to about 4 mS. Demineralization may be performed by methods known in the art, including electrodialysis (ED), reverse osmosis (RO), nanofiltration (NF) or ion exchange technology.

[0042] In a further alternative embodiment, the lactose-containing feed may be delactosed whey permeate (DLP or OPL), which still contains lactose and protein, but also the minerals and vitamins originally present in the whey permeate. OPL generally also contains about 0.3-0.4% sialyl-lactose (2,3-sialylactose and 2,6- sialyl lactose) and possibly other valuable bovine milk oligosaccharides (bMOs) as well. Besides, it has been shown that bMOs contain as many oligosaccharides as found in human oligosaccharides (hMOs).

[0043] OPL contains relatively low amounts of calcium. Therefore, in order to remove multivalent anions such as PO43; citrate3extra calcium can be added to enhance the formation of insoluble calcium monohydrogen phosphate and calcium citrate, thus allowing for easy removal of salts by e.g. centrifugation. In a preferred embodiment, OPL is treated with lime (CaO / Ca(OH)2) to precipitate anions.

[0044] The first beta-galactosidase is derivable from Lactobacillus delbrueckii subspecies bulgaricus or Lactobacillus delbrueckii subspecies lactis, or has a strong similarity therewith. In a preferred embodiment, the first beta-galactosidase has been derived from Lactobacillus delbrueckii subspecies bulgaricus or Lactobacillus delbrueckii subspecies lactis, most preferably it has been derived from or Lactobacillus delbrueckii subspecies bulgaris.

[0045] Suitable enzymes have been disclosed in WO 2020 / 049016 and are available from IFF / Danisco as Bonlacta™. In a preferred embodiment, however, the enzyme is comprised in a micro-organism which endogenously expresses the enzyme. This allows cheaper and easier processing as it saves the effort of isolating the enzyme. The micro-organism, e.g. a strain of Lactobacillus delbrueckii subspecies bulgaricus or subspecies lactis, may be used as whole cells or as active part or fraction thereof, preferably a cell free extract.

[0046] A strain of Lactobacillus delbrueckii subspecies bulgaricus capable of producing a galactosidase enzyme activity for use in providing an oligosaccharide composition of the invention has been deposited under accession number DSM20080.

[0047] The first beta-galactosidase is added to the lactose-containing feed, in a preferred dosage of at least 2-100 LU / gram lactose, more preferably in the range 5-50 LU / gram lactose, even more preferably 10-40 LU / gram lactose, and most preferably 20-30 LU / gram lactose, depending on the desired reaction time. As used herein, one lactase unit (LU) is defined as the quantity of enzyme that liberates 1 pmole of galactose or O- nitrophenol per minute at the early stage ONPG (o-nitrophenyl-galactoside) hydrolysis at 40°C, pH 6.5.

[0048] The reaction with this first beta-galactosidase is then conducted at a preferred temperature in the range 40-70°C, preferably 40-65°C, more preferably 50-63°C, most preferably 55-60°C.

[0049] The reaction time preferably ranges from 0.1-50 hours, more preferably 2-24 hours, and most preferably 4-10 hours. The pH of the reaction mixture is preferred in the range 5.5-7.0, more preferably 5.8- 6.8, and most preferably 6.0-6.5.

[0050] After at least 25% and before more than 55% of the initial lactose concentration is converted, preferably after reaching 35-55%, most preferably 40-50% conversion of the initial lactose concentration, the reaction is stopped. This can be achieved by lowering the pH to 4 or less.

[0051] It is important to stop the reaction at this point since the enzyme will start to hydrolyse lactose and GOS species at low lactose substrate concentrations. The lactose conversion is determined by the brix change of the reaction mixture, relative to the initial lactose concentration.

[0052] The first beta-galactosidase is then denatured by subjecting the reaction mixture to a temperature in the range 65-95°C. The reaction mixture is kept at this temperature for a period of time sufficient to denature the enzyme, preferably for a time period of at least 10 minutes, more preferably 20-90 minutes, most preferably 30-60 minutes.

[0053] The temperature of the reaction mixture is subsequently adjusted to a temperature in the range 30-70°C, more preferably 40-65°C, even more preferably 50-60°C, most preferably 55-60°C.

[0054] The second beta-galactosidase is then added to the feed.

[0055] The second beta-galactosidase is derivable from Bacillus circulans or has a strong similarity therewith.

[0056] In a preferred embodiment, the second beta-galactosidase has been derived from Bacillus circulans. Even more preferably, the second beta-galactosidase has been derived from Bacillus circulans and has a molecular weight of 195 kDa (by SDS- PAGE).

[0057] A suitable enzyme may be derived from Bacillus circulans ATCC31382. Its isolation has been disclosed in EP2439270 A1 . It is available from Amano Enzymes under the name Biolacta N5.

[0058] The second beta-galactosidase is preferably added in a preferred dosage of at least 1.5 LU / gram lactose, more preferably in the range 2-10 LU / gram lactose, even more preferably 3-8 LU / gram lactose, and most preferably 4-6 LU / gram lactose. The reaction with the second beta-galactosidase is then conducted at a preferred temperature in the range 30-70°C, more preferably 40-65°C, even more preferably 50- 60°C, most preferably 55-60°C.

[0059] The reaction is continued until at least 70 wt%, preferably at least 75% of the initial lactose content has been converted,

[0060] The reaction time preferably ranges from 10-50 hours, more preferably 15-45 hours, and most preferably 24-36 hours.

[0061] The reaction is stopped by denaturing the second beta-galactosidase - for instance by heat treatment (e.g. 100°C for 15 minutes) and / or acidification, and enzyme residues are removed from the GOS-containing reaction mixture, for instance of ion exchange and / or microfiltration.

[0062] The resulting GOS may then be further purified. Conventional purification steps can be applied, such as removal of enzyme residues, demineralization, de-proteinization, removal of mono-sugar components, and / or decolouration (e.g. by treatment with activated carbon). In one embodiment, the GOS composition is subjected to a nanofiltration (NF) or ultrafiltration step (UF) to remove mono-sugars and any protein that may be present. A further concentration step, for example using NF or evaporation, may be performed to obtain a concentrated GOS preparation having a dry matter content of, e.g., at least 70 wt%, preferably at least 75 wt%.

[0063] Alternatively, only enzyme residues are removed from the reaction mixture, but no further purification or isolation of individual components is applied.

[0064] The GOS according to the present invention can be used in nutritional compositions. This can be nutritional compositions for pregnant women (MUM compositions), young children (formula milk), adolescents (13-20 years of age), or adults (>20 years of age). The nutritional composition can be used as a regular food composition, as nutritional therapy, as nutritional support, as medical food, as a food for special medical purposes, or as a nutritional supplement.

[0065] An example of a nutritional composition is formula milk. Formula milk includes infant formulas, follow-up formulas and growing-up formulas (also called young child formulas). Other examples of nutritional compositions are compositions for adults, such as patients or frail elderly or anyone else desiring to boost their immune system or gut health.

[0066] Infant formula, baby formula or just formula (American English) or baby milk, infant milk or first milk (British English), is a manufactured food designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottle-feeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water). The U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants by reason of its simulation of human milk or its suitability as a complete or partial substitute for human milk". Similarly, the Codex Alimentarius international food standards (WHO and FAO) defines infant formula as a breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding. The Codex Alimentarius describes the essential composition of an infant formula with amounts and specifications for the lipid source, protein source, carbohydrate source, vitamins and minerals.

[0067] In order to constitute the nutritional composition, in particular the formula milk, the GOS according to the invention - either as aqueous composition or a (spray)dried powder obtained from it - is blended with the further ingredients of the nutritional composition. In case of formula milk, these ingredients 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 ingredients.

[0068] The lipid source for use in formula milk may be any lipid or fat suitable for use in formula milk. Preferred fat sources include milk fat, safflower oil, egg yolk lipid, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm oleic, high oleic sunflower oil and high oleic safflower oil, and microbial fermentation oil containing long-chain, polyunsaturated fatty acids. In one embodiment, anhydrous milk fat is used. The lipid source may also be in the form of fractions derived from these oils such as palm olein, medium chain triglycerides, and esters of fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, and the like. Small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils may be added. The fat source preferably has a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15:1 ; for example about 8:1 to about 10:1 . In a specific aspect, the infant formula comprises an oil mix comprising palmitic acid esterified to triacylglycerols, for example wherein the palmitic acid esterified in the sn- 2 position of triacylglycerol is in the amount from 10% to 60% by weight of total palmitic acid and palmitic acid esterified in the sn-1 / sn-3 position of triacylglycerol is in the amount of from 30% to 80% by weight of total palmitic acid.

[0069] Examples of protein sources include milk, preferably bovine milk, whey protein sources like whey protein concentrate and serum protein concentrate, and various plant proteins. The proteins may be hydrolyzed or unhydrolysed.

[0070] Examples of vitamins and minerals that are preferably present in formula milk are 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, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form.

[0071] If necessary, the nutritional composition may contain emulsifiers and stabilisers such as soy lecithin, citric acid esters of mono- and di-glycerides, and the like. It may also contain other substances which may have a beneficial effect such as lactoferrin, nucleotides, nucleosides, probiotics, and the like. Suitable probiotics include Lactobacteria, Bifidobacterium lactis such as Bifidobacterium lactis Bb12, Streptococcus thermophilus, Lactobacillus johnsonii La1, Bifidobacterium longum BL999, Lactobacillus rhamnosus LPR, L rhamnosus GG, Lactobacillus reuteri, Lactobacillus salivarius. Such prebiotics are commercially available.

[0072] EXAMPLES

[0073] Determination of galactosyllactose contents

[0074] To an Eppendorf vial was added 200 pl of a labeling reagent solution prepared by mixing 1.633 ml DMSO (100%), 0.7 ml acetic acid (100%), 46 mg anthranilamide, and 102 mg 2,2-methylpyridine borane complex. Subsequently, 20 pl of the sample was added and mixed well under vortex. The solution was spinned down in a microcentrifuge for 2 or 3 seconds at a very low speed. The solution was then heated at 65°C for 2 hours while shaking at 400 rpm in a heat block followed by cooling on ice for 5 to 10 minutes, spinning down for 2 or 3 seconds at a very low speed in a microcentrifuge, and adding 700 pl of aceton itrile / water (70 / 30) and mixing well under vortex. The samples were filtered through 0.2 pm PVDF microfilter and analysed by LIPLC with fluoresce detector.

[0075] This LIPLC applied a TSK Gel Amide-80 guard (3.2 15 mm, 3 pm) and analytical (4.6 x 150 mm, 3 pm) columns (Tosoh Bioscience, Stuttgart, Germany). Detection was performed by a Shimadzu RF-10Axl fluorescence detector using Aex=330 nm and Aex=420 nm. Eluent A was 100% acetonitrile; eluent B was 100 mmol / L ammonium formate, pH 4.4. A 3 pL aliquot of the labelled solution was injected onto the guard cartridge under gradient conditions (summarized below) at a flow rate of 0.5 ml / min.

[0076] Table 1 - Gradient conditions:

[0077] Example 1

[0078] A 180 ml plastic reactor vessel was filled with a reaction mixture (100 g) comprising 50 wt% lactose, 1 ml 1.0 M potassium phosphate buffer (pH 6.5), and water. The reaction mixtures were stirred in a water bath (50°C) for at least half an hour.

[0079] Subsequently, 20 LU of enzyme was added to start the conversion. The reaction was allowed to continue for 24 hours and stopped by adding 1.5% (v / v) 1 M HCI and denaturing the enzyme at 95°C for 15 minutes.

[0080] The samples were analyzed as explained above and the sugar composition and GL content was estimated by the peak percentage. The individual GL position on the chromatogram was determined by the use of the corresponding GL reference. These results show that the GOS yield and lactose conversion obtained with enzymes originating from K. lactis and A. oryzae is significantly lower than that achieved with other enzymes.

[0081] Table 2 - Evaluation of different enzymes

[0082] 1oligosaccharides other than actose

[0083] Example 2

[0084] Three glass 250 ml bioreactors were provided, identified as Reactors 1 -3.

[0085] Each of these reactors contained a reaction mixture comprising 52.63 gram crystalline lactose hydrate (Lactopure, FrieslandCampina), 47.37 gram demi-water, and 1 ml 1.0 M potassium phosphate buffer (pH 6.5). The buffer to concentration relative to total water volume was 20 mM.

[0086] The reaction mixtures were heated in a water bath (50°C) while stirring. Once the temperature of the reaction mixture reached 50°C, a first beta-galactosidase enzyme (Bonlacta®, 17308 ll / gram enzyme formulation) was added in order to reach a total enzyme dose of 60 ll / gram lactose in the reactor.

[0087] The reaction was monitored by following the Brix change (using a Brix Refractometer), according to the time schedule defined in Table 3. The reaction was continued until a lactose conversion of 20% (Brix value of 45°; Reactor 1 ), a lactose conversion of 30% (Brix value 40°; Reactor 2), or a lactose conversion of 40% (Brix value 35°; Reactor 3) was reached. Reaching a lactose conversion of 20% required 10 minutes; reaching a lactose conversion of 30% required 40 minutes; reaching a lactose conversion of 40% required 75 minutes.

[0088] Once the desired lactose conversion was reached, the reaction was subjected to thermal denaturation at 95°C in a water bath for 15 minutes in order to denature the enzyme.

[0089] The reaction mixtures were subsequently cooled down to 58°C, followed by addition of the second beta-galactosidase enzyme (Biolacta® N5; originating from Bacillus circulans ATCC 31382, ex-Amana Enzymes) at an enzyme dosage 5.0 LU / gram initial lactose content. The reactions were continued with this second enzyme for 49 hours, after which the enzyme was denatured by adding 1 ,5%(v / v) of 1 M HCI.

[0090] The Brix value and the pH of the reaction m ixtures at the start and end of the enzymatic reactions are presented in Table 3. As shown in this table, the pH slightly decreased during the reaction with Biolacta N5.

[0091] Table 3

[0092] Aliquots of samples were taken at different time intervals. The samples were denatured by adding 1 ,5%(v / v) of 1 .0 M HCI and subsequent heating at 95°C for 15 minutes. The samples taken were analyzed by HPAEC-PAD HPLC using a CarboPac PA-1 column for the sugar composition (Table 4) and the fingerprint profile of GOS. The GOS content (wt%) is defined as 100%-galactose%-glucose%-lactose%-allolactose%- lactulose% and the oligosaccharide content is defined as the oligosacccharide content excluding lactose.

[0093] The concentrations of 3’-GL, 4’-GL, and 6’-GL were estimated as described above by the peak percentage using the analytic reference of each individual GL. The GL- content on the total oligosaccharide content was calculated using the oligosaccharide content of Table 4 and summarized in Table 5.

[0094] It can be observed that GOS prepared according to the process of the present invention has a significantly higher content of 3’-GL and 6’-GL and similar to even higher 4’-GL content than Vivinal® GOS. But apart from that, the fingerprint profile, i.e. the set of peaks and the peak intensities, of the GOS according to the invention was quite similar to that of Vivinal® GOS.

[0095] In addition, the 4’-GL and gal-|3-1 ,2-glc + gal-|3-1 ,3-glc contents resulting from the process of the present invention were significantly higher than that of commercial Bimuno® GOS.

[0096] Table 4 - Sugar composition, in wt% on dry matter, of different samples

[0097] 1oligosaccharides other than lactose Table 5 - 3’-GL, 4’-GL, and 6’-GL, and gal-|3-1 ,2-glc + gal-|3-1 ,3-glc content in different samples, in wt% based on oligosaccharide1

[0098] 1oligosaccharides other than lactose

[0099] Example 3

[0100] Example 2 was repeated, with the exception that the second enzyme (Biolacta N5) was used in a concentration of 10 LU / gram initial lactose, and the reaction time was shortened to 24 hours after addition of the second beta-galactosidase.

[0101] Compared to Example 2, higher yields in shorter time frames were reached, with higher gal-|3-1 ,2-glc + gal-|3-1 ,3-glc contents, but at the expense of 4’-GL. The similarity between the GOS produced in reactors 1 -3 and Vivinal® GOS was determined by selecting nine DP2-DP4 peaks or peak pairs in the fingerprint profile and calculating the peak percentage of each peak relative to the same peak in the Vivinal® fingerprint profile. The averages of these nine relative peak areas - the similarity index - were 89.6 (GOS obtained in Reactor 1 ), 89.6 (GOS obtained in Reactor 2), and 94.2 (GOS obtained in Reactor 3).

[0102] Table 6 - Sugar composition, in wt% on dry matter, of different samples 1oligosaccharides other than lactose Table 7 - 3’-GL, 4’-GL, and 6’-GL, and gal-|3-1 ,2-glc + gal-|3-1 ,3-glc content in different samples, in wt% based on oligosaccharide1

[0103] 1oligosaccharides other than lactose

Claims

CLAIMS1. Process for producing a beta-galactooligosaccharide composition comprising 2.0-20.0 wt% 3’-galactosyllactose, 2.0-20.0 wt% 6’galactosyllactose, 3.0-25.0 wt% 4’-galactosyllactose, and a total amount of the disaccharides gal-|3-1 ,2-glc and gal-|3-1 ,3-glc of at least 15 wt%, all based on the total weight of oligosaccharides other than lactose in the composition, the process comprising the steps of:- providing an aqueous lactose-containing feed containing an initial lactose concentration of 20-65 wt%,- converting 25-55% of the initial lactose concentration in said feed with a first beta-galactosidase enzyme at a temperature in the range 40-70°C to provide an intermediate feed, said first beta-galactosidase enzyme being derivable from Lactobacillus delbrueckii subspecies bulgaricus or Lactobacillus delbrueckii subspecies lactis,- denaturing said first beta-galactosidase enzyme at a temperature in the range 65-95°C,- adjusting the reaction temperature to 30-70°C,- adding a second beta-galactosidase enzyme to the intermediate feed, said second beta-galactosidase enzyme being derivable from Bacillus circulans,- converting lactose with the second beta-galactosidase at a temperature in the range 30-70°C, until at least 70 wt% of the initial lactose content has been converted,- denaturing the second beta-galactosidase,- removing enzyme residues.

2. Beta-galactooligosaccharide composition obtainable by the process of claim 1 .

3. Beta-galactooligosaccharide composition according to claim 2 comprising 4.0- 20.0 wt% 3’-galactosyllactose, 5.0-20.0 wt% 6’-galactosyllactose, 3.0-25.0 wt% 4’-galactosyllactose, and a total amount of the disaccharides gal-|3-1 ,2-glc and gal-|3-1 ,3-glc of at least 15 wt%, all based on the total weight of oligosaccharides other than lactose in the composition.

4. Beta-galactooligosaccharide composition according to claim 2 or 3 wherein the 3’-galactosyllactose (3’-GL) content is in the range 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 mono sugars and lactose in the composition.

5. Beta-galactooligosaccharide composition according to any one of claims 2-4 wherein the 4’-galactosyllactose (4’-GL) content is in the range 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 mono sugars and lactose in the composition.

6. Beta-galactooligosaccharide composition according to any one of claims 2-5 wherein the 6’-galactosyllactose (6’-GL) content is in the range 5.0-15.0 wt% and most preferably 8.0-10.0 wt%, based on the total weight of oligosaccharides excluding mono sugars and lactose in the composition.

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

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

9. Nutritional composition comprising the beta-galactooligosaccharide composition of any of claims 2-8, additionally comprising one or more proteins, probiotics, lipids, and / or further carbohydrates.

10. Nutritional composition according to claim 9, said nutritional composition being an infant formula, follow-up formula, or growing up milk.

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