Galacto-oligosaccharide preparation enriched in sialyllactose

Abstract

Process for the production of a galacto-oligosaccharide preparation enriched in sialyllactose starting from whey permeate, said process involving removal of ions, membrane filtration, and enzymatic conversion.

Description

[0001] GALACTO-OLIGOSACCHARIDE PREPARATION ENRICHED IN SIALYLLACTOSE

[0002] The invention relates to the field of nutritional ingredients. More in particular, it relates to a method for producing a galacto-oligosaccharides (GOS) preparation with a relatively high content of oligosaccharides that naturally occur in milk.

[0003] Various physiological functions of GOS have been reported, including the capacity to stimulate the growth of bifidogenic bacteria in the gut, to support normal gut transit, to contribute to natural defenses and to enhance mineral absorption. GOS has received particular attention for their prebiotic effects that promote the growth of Bifidobacterium, Lactobacillus, and other enteric bacteria. Therefore, GOS is commonly used in formula feeding (infant formula, follow-on formula, and young child formula).

[0004] Galacto-oligosaccharides (GOS) generally comprise a chain of galactose units and a terminal glucose unit, that arise through consecutive transgalactosylation reactions catalyzed by a beta-galactosidase. Some of the GOS components exist naturally in human breast milk and bovine colostrum. Typical GOS preparations mainly comprise di- to hexa-saccharides.

[0005] In general, GOS is produced from lactose by a transglycosylation reaction with a betagalactosidase enzyme (enzyme class EC.3.2.1 .23). Beta-galactosidase enzymes are produced in many microorganisms such as Bacillus circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Papiliotrema terrestris, and Lactobacillus fermentum. Beta-galactosidases differ in their three-dimensional structures, resulting in stereo- and regioselectivity of the glycosidic bonds that are formed. 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”), while a bacterial beta-galactosidase derived from Bacillus 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”). Moreover, beta-galactosidase produced by B. circulans possesses particularly strong transgalactosylation activity. As a result, GOS prepared by B. circulans beta-galactosidase is sold worldwide. The starting material for GOS production, lactose, is generally sourced from milk and obtained by crystallization from whey ultrafiltration permeate (herein referred to as “whey permeate”). This whey permeate results from ultrafiltration of whey. The lactose concentration in this whey permeate is generally in the range 75-90 wt% on dry matter.

[0006] Whey permeate as such can also be used as the lactose source for GOS production. And so can delactosed whey permeate (conventionally referred to as DLP or OPL), i.e. the mother liquor from the lactose crystallization, as disclosed in WO 2006 / 087391 , WO 2018 / 020473, and WO 2020 / 141032.

[0007] The process disclosed in WO 2006 / 087391 involves the enzymatic treatment with a beta-galactosidase of a lactose source obtained by concentration, lactose removal (e.g. crystallization), and demineralization (by reverse osmosis, nanofiltration, or ion exchange) of whey permeate.

[0008] Also WO 2020 / 141032 discloses GOS production starting from demineralized (e.g. by electrodialysis) delactosed whey permeate. In order to remove divalent anions like PO43' and citrate, this document proposes to add calcium in order to precipitate the corresponding calcium salts.

[0009] The process disclosed in WO 2018 / 020473 involves the precipitation of minerals from delactosed whey permeate by way of heat treatment, followed by ultrafiltration and nanofiltration.

[0010] One advantage of using whey permeate as the lactose source is that it allows valorization of one of the most abundant waste streams in the dairy industry. Another advantage is that this permeate contains a significant amount of oligosaccharides naturally present in milk, such as the sialyllactoses 3’-sialyllactose (3’-SL) and 6’- sialyllactose (6’-SL), thereby enabling the preparation of GOS containing such oligosaccharides. These oligosaccharides are also known to have significant health benefits by supporting resistance to pathogens, gut maturation, immune function, and cognitive development. The commercial production of such sialyllactoses currently involves fermentation reactions with genetically engineered microorganisms.

[0011] GOS can be added to formula feeding in concentrations of 2.4-8.0 g / L, preferably 3.0-

[0012] 7.5 g / L, most preferably 4.0-7.2 g / L. 3’-SL, the main sialyllactose in bovine milk, can be added to formula feeding in concentrations up to about 0.3 g / L.

[0013] It is therefore an object to provide a GOS preparation comprising, based on dry matter, 0.2-3.0 wt% sialyllactose, thereby allowing the direct introduction of this GOS preparation in formula feeding. Would the sialyllactose content be significantly higher than 3.0 wt%, direct introduction would run the risk of introducing more sialyllactose than allowed.

[0014] The above mentioned prior art processes require the use of delactosed whey permeate in order to obtain a sufficiently high sialyllactose content in the resulting GOS.

[0015] A disadvantage of delactosed whey permeate, however, is its high mineral content, requiring extensive demineralization and concentration steps. Furthermore, the sialyllactose content of the final GOS is difficult to control.

[0016] A further disadvantage of using delactosed whey permeate is that it contains nitrogencontaining compounds and compounds resulting in color formation, which cannot be removed with a conventional anion exchange column without losing sialyllactose.

[0017] It has now been found that GOS enriched in significant amounts of sialyllactose, such that the SL-enriched GOS can be introduced into formula feeding, can also be obtained from whey permeate instead of delactosed whey permeate.

[0018] The advantage of using whey permeate is that it has a much higher lactose content than delactosed whey permeate (75-90 wt% on dry matter versus 50-60 wt%) and a significantly lower mineral content.

[0019] The present invention relates to a process for the production of a galactooligosaccharide preparation with, based on dry matter, at least 35 wt% galactooligosaccharides other than lactose and a sialyllactose content in the range 0.2-3.0 wt%, comprising the steps of: i) providing a whey permeate with a lactose concentration in the range 75-90 wt% on dry matter, ii) removing mono- and multivalent ions from said whey permeate, thereby obtaining a demineralized whey permeate, iii) subjecting the demineralized whey permeate to membrane filtration using a membrane with a molecular weight cut-off in the range 400-2500 Da, thereby obtaining a retentate with a lactose concentration, based on dry matter, in the range 90-99.8 wt% and a sialyllactose content, relative to lactose, in the range 0.2-3.0 wt%, iv) adding a beta-galactosidase enzyme to the membrane filtration retentate in order to convert lactose into galacto-oligosaccharide, thereby forming the galactooligosaccharide preparation.

[0020] Whey permeate

[0021] The process of the present invention starts with whey permeate, i.e. the lactose-rich ultrafiltration permeate remaining after protein concentration from whey.

[0022] Whey results from separating (skimmed) milk into a casein-rich and a whey proteinrich fraction; either by renneting (i.e. cheese making), acidification, or microfiltration. Whey resulting from cheese making is referred to as cheese whey; whey resulting from acidification is referred to as acid whey; the permeate resulting from the microfiltration of milk is referred to as ideal whey.

[0023] Whey proteins are conventionally concentrated by submitting whey to ultrafiltration. This ultrafiltration conventionally uses a membrane with a molecular weight cut-off (MWCO) in the range 5-10 kDa. A major part of the water, lactose, sialyllactose, minerals, and vitamins passes the membrane and forms the whey permeate, whereas the proteins are retained by the membrane as a whey protein concentrate.

[0024] The whey permeate to be used in the process of the present invention can be a cheese whey permeate, an acid whey permeate, or an ideal whey permeate. Preferably, the whey permeate is a cheese whey permeate (CWP) or an acid whey permeate. Most preferably, it is a cheese whey permeate.

[0025] The lactose content of the whey permeate is in the range 75-90 wt%.

[0026] The sialyllactose content of whey permeate is generally around 0.1 -0.3 wt%, based on dry solids.

[0027] Demineralisation

[0028] The whey permeate is submitted to one or more demineralization steps order to remove monovalent and multivalent ions. The main multivalent ions are calcium and phosphate ions. This demineralisation can be conducted with electrodialysis, precipitation of the minerals, ion exchange, and / or nanofiltration. The demineralisation step should be conducted in a way that prevents removal of significant amounts of sialyllactose from the whey permeate.

[0029] Demineralisation preferably results in a whey permeate that is demineralized to an ash content of about 0.1 -5.0 wt% , more preferably 0.1 -4.0 wt%, even more preferably 0.1- 3.0 wt%, more preferably 0.1 -2.0 wt%, and most preferably 0.1 -1 .0 wt.%, based on dry matter.

[0030] In one embodiment, the demineralisation is performed by electrodialysis, thereby removing both monovalent and multivalent ions.

[0031] In another embodiment, multivalent ions are replaced with monovalent ions in at least one cation exchange step in order to replace calcium ions with monovalent cations, such as sodium or potassium, and at least one anion exchange step in order to replace phosphate ions with monovalent anion, such as chloride, after which the monovalent ions can be removed by membrane filtration or further ion exchange steps.

[0032] Especially for the anion exchange step, it should be ensured that this step does not result in significant removal of sialyllactose. This can be achieved by performing the anion exchange with an anion exchange resin in the chloride form, preferably a geltype styrene-divinylbenzene or gel-type crosslinked acrylic anion exchange resin with tertiary amine functional groups, more preferably dimethylamine groups.

[0033] The ion exchange steps are preferably followed by a nanofiltration step using a membrane that permeates monovalent ions, such as sodium, potassium, and chloride, thereby further demineralizing and at the same time concentrating the whey permeate. Sialyllactose should remain in the nanofiltration retentate.

[0034] During this step, the whey permeate is preferably concentrated to a dry matter content in the range 10-25 wt%, preferably 15-20 wt%.

[0035] Any nanofiltration membrane suitable for demineralization purposes may be used for this step. In a preferred embodiment, the nanofiltration membrane has a molecular weight cut-off in the range 200-300 Da.

[0036] In a further preferred embodiment, the membrane is as poly(piperazine-amide) composite membrane. Said nanofiltration is preferably combined with diafiltration.

[0037] In addition to or instead of the membrane filtration step, monovalent ions can also be removed by cation and anion exchange steps. In a preferred embodiment, cation exchange is performed prior to anion exchange.

[0038] Apart from removing minerals, this ion exchange may also reduce the non-protein nitrogen (NPN) content of the demineralised whey permeate.

[0039] This cation exchange step is preferably performed using a strong cation exchange resin in the free acid form (H+form). The resin is preferably a styrene-divinylbenzene cation exchange resin, more preferably a macroporous or a gel-type styrene- divinylbenzene cation exchange resin. In a preferred embodiment, the cationexchange material comprises sulfonic acid functional groups. Most preferably, the cation-exchange material is a strong acid cation exchange resin having a styrene / divinylbenzene gel-type matrix and sulfonic acid functional groups.

[0040] In order to prevent the sialyllactose from binding to the anion exchange resin, the anion exchange resin should be a gel-type anion exchange resin (e.g. cross-linked polystyrene-divinylbenzene gel) with a moisture content of 30-48%, preferably 35-45%. At lower moisture contents, the demineralization capacity becomes too low; at high moisture contents, the affinity for sialyllactose increases. Due to the low moisture content and the dense network of a gel-type resin, the mobility of sialyllactose is lower than that of the abundantly present chloride anions. As a result, sialyllactose will remain in the liquid phase, whereas chloride anions are preferentially bound to the resin.

[0041] The moisture content is determined in the following manner: prior to measurement of the moisture content of the resin, adhering water is removed, for instance by wrapping the resin in a cloth and then subjecting it to centrifugation (centrifuge: 30 cm diameter; 3,000 rpm); the resin is then weighed, for instance in a weighing bottle; after which the resin is dried for 4 hours at a constant temperature of 105°C.; the resin is then cooled down in an exsiccator for 30 minutes; after which in turn the weight of the dry resin is determined; the moisture percentage (wt%)=[(weight loss after drying (g)) / (weight of the wet resin)]*100%.

[0042] The anion exchange resin preferably has strong anion exchange groups, preferably type II anion exchange groups. In order to remove any non-ionic organic components and / or undissociated molecules, such as non-protein nitrogen, it may be desired to submit the demineralised whey permeate to a column of absorbent material. This can be a conventional absorbent material such as activated carbon, but preferably is a cation-exchange type of material. This cation exchange material preferably has a macroporous structure with a higher porosity and a lower density of ionic groups than the cation exchange resin referred to above.

[0043] The porosity of this cation exchange material is preferably in the range 0.8 to 1.2 ml / g, more preferably 0.9 to 1 .1 ml / g, and most preferably 0.95 to 1 .05 ml / g. The BET surface area is preferably > 600 m2 / g, more preferably > 650 m2 / g, even more preferably > 670 m2 / g and most preferably > 700 m2 / g.

[0044] The absorbent material preferably is a styrene / divinyl benzene copolymer matrix of which the hydrophilicity is increased by the presence of sulphonic acid groups. In the process of the present invention it is used to absorb components, typically organic components, in particular non-cationic components, more in particular components that are neutral at the pH of the solution. An advantage of such material over absorbent materials like activated carbon is its inertness towards absorption of acidic oligosaccharides.

[0045] Membrane filtration

[0046] The demineralized whey permeate is then further concentrated to a dry matter content of at least 15 wt%, preferably 19-25 wt.% by membrane filtration using a membrane with a molecular weight cut-off in the range 400-2500, preferably 400-2000, more preferably 400-1500, even more preferably 400-1000, even more preferably 400-700, more preferably 400-600, and most preferably 400-500 Dalton. This concentration step not only leads to further concentration and demineralization of the whey permeate, it additionally allows lactose to pass the membrane, thereby increasing the sialyllactose / lactose ratio in the retentate.

[0047] This membrane filtration step should preferably be performed until the resulting retentate has a lactose content, based on dry matter, is in the range 90-99.8 wt% and the sialyllactose content, relative to lactose, in the range 0.2-3.0 wt%. This membrane filtration is preferably performed at a temperature in the range 5-60°C, preferably 10-50°C, most preferably 40-50°C. Since the solubility of lactose increases with temperature, higher filtration temperatures allow higher lactose concentrations.

[0048] On the other hand, temperatures in the range 5-15°C, more preferably 5-10°C, may be desired for reducing the growth of microorganisms.

[0049] The pH of the solution submitted to membrane filtration is preferably above 4.5, more preferably at least 5, more preferably at least 7-10, and most preferably 8.5-9.5, in view of sialyllactose stability and improved sialyllactose retention.

[0050] This membrane filtration may be combined with diafiltration. Diafiltration serves in a further reduction of multivalent ions (e.g. phosphate) and non-protein nitrogen (NPN).

[0051] Conversion to GOS

[0052] The nanofiltration retentate is then submitted to enzymatic treatment with a [3- galactosidase in order to convert lactose into galacto-oligosaccharides (GOS). The resulting product is a GOS preparation comprising a significant amount of sialyllactose.

[0053] It should be noted that also the nanofiltration permeate can be used as lactose source for GOS preparation. GOS prepared from the permeate will evidently not be enriched in sialyllactose.

[0054] The pH of the nanofiltration retentate is preferably adjusted to a value in the range 5- 7, preferably 5.5-6.5, most preferably 6.0-6.3. If the pH is already in this range, no action is required. If the pH is outside this range, it can be adjusted by the addition of acid or base, such as citric acid or NaOH.

[0055] Prior to conversion into galacto-oligosaccharide, the dry matter content of the nanofiltration retentate is preferably adjusted to at least 35 wt%, preferably at least 40 wt%, more preferably at least 45 wt%, and most preferably at least 50 wt%. This can be achieved by conventional techniques, such as membrane filtration or evaporation. If the dry matter content is already in this range, no action is required.

[0056] Suitable [3-galactosidase enzymes for use in the process of the present invention include those derived from, e.g., Bacillus circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, and Papiliotrema terrestris (Cryptococcus Papiliotrema terrestris). A preferred enzyme is [3-galactosidase produced by Bacillus circulans.

[0057] The enzymatic conversion is preferably conducted at a temperature of about 20-60°C, more preferably 40-60°C, most preferably 45-55°C.

[0058] The enzyme is preferably used in an amount of 0.60-2.5 LU / gram, preferably of 0.65- 2.0 LU / gram, most preferably 0.65-1.1 LU / gram.

[0059] A lactase unit (LU) is defined as the quantity of enzyme that liberates 1 micromole glucose per minute at the early stage of the reaction at 40°C, pH 6.0.

[0060] After the enzymatic reaction, which may take 6-50 hours, preferably 10-48 hours, even more preferably 18-24 hours, the enzyme may be denatured (e.g. by heating at about 95-100°C or adjusting the pH to about 3.5 or less) and the GOS preparation can be subjected to additional purification and concentration steps to obtain a syrup, which may optionally be dried, e.g. spray-dried, to form a powder.

[0061] The enzyme can be used in powder form (e.g. freeze dried, vacuum dried, or spray dried) or liquid form (e.g. dissolved or dispersed in water, a phosphoric acid buffer solution, a tri-ethanol amine buffer solution, a tris-hydrochloric acid buffer solution, or a GOOD buffer solution).

[0062] In a specific embodiment, the enzyme is used in immobilized form. Various ways of enzyme immobilization are known in the art. They typically comprise a porous carrier onto which the beta-galactosidase is immobilized via covalent binding, via physical adsorption (charge-charge or van der Waals interaction), via gel encapsulation, or a combination thereof. Besides, carrier-free immobilized enzymes such as CLEC (crosslinked enzyme crystals) or CLEA (crosslinked enzyme aggregates) might be also applied.

[0063] Carriers that can promote direct covalent binding of the enzyme are preferred, in view of their ease of operation and absence of leakage into the reaction mixture. An example of a solid carrier is an activated acrylic polymer, preferably a functionalized polymethacrylate matrix. For example, a hexamethylenamino-functionalized polymethacrylate matrix (Sepabeads) or a microporous acrylic epoxy-activated resin, like Eupergit C 250L, can be used. The use of immobilized enzyme allows a repeated batch operating system involving several consecutive batches (‘cycles’) of GOS purification. It also allows for recycling of enzyme, which enables semi-continuous operation and multiple reuse of the enzyme.

[0064] In another specific embodiment, the reaction is conducted in an enzymatic membrane reactor.

[0065] The resulting product may be submitted to purification steps, evaporation, and / or (spray) drying. In a preferred embodiment, one of the purification steps involves a decolorization step using a cation exchange resin in the free acid form and an absorbent resin as defined above. Apart from removing colour, this step also adds to a further reduction of the ash content. In order to prevent the loss of sialyllactose at this stage, binding to an anion exchange resin should be prevented, either by not conducting an anion exchange step, or by applying an anion exchange step with a resin that does not bind sialyllactose as discussed above. More preferably, no anion exchange step is performed at this stage; not only does that prevent the introduction of additional anions in the product, the use of cation exchange in the absence of anion exchange also results in a pH reduction of the product that improves microbial stability. The latter thus discards the need for acid addition for achieving microbial stability.

[0066] The GOS preparation that is obtained from the process of the present invention contains at least 35 wt% preferably 40-90 wt%, most preferably 50-70 wt% galactooligosaccharides other than lactose, based on dry matter. The GOS preparation contains, on dry matter, 0.2-3.0 wt%, preferably 0.2-2.0 wt%, and most preferably 0.4- 1 .0 wt% sialyllactose.

[0067] Based on the total weight of oligosaccharides other than lactose, the GOS preparation preferably contains 0.2-5.0 wt%, preferably 0.3-4.0 wt%, more preferably 0.3-3.0 wt%, even more preferably 0.4-2.0 wt%, and most preferably 0.5-1 .0 wt% sialyllactose.

[0068] This sialyllactose content manly consist of 3’-sialyllactose and 6’-sialyllactose and can be determined with the aid of a chromatographic technique using an Acquity premier glycan BEH amide 130A 1.7 pm column and a fluorescence detector. The sialyllactose content is calculated as the sum of the content of 3'-sialyllactose and 6'-sialyllactose. Application

[0069] The sialyllactose-enriched galacto-oligosaccharide preparation resulting from the process of the present invention can be used in nutritional compositions for human subjects of any age. In one embodiment, the subject is an adolescent or an adult. An adolescent is herein defined as a person having an age of from 13 to 20 years. An adult is herein defined as a person having an age of 20 years or higher. In another embodiment, the subject is a child having an age of 3 years (36 months) to 13 years. In yet another embodimentm the subject is child having an age of 0 to 3 years, preferably having an age of 24 months or below, more preferably having an age of 18 months or below.

[0070] In one embodiment, the nutritional composition is a MUM composition for pregnant women, a growing up milk (GUM), a follow-up formula, or an infant formula.

[0071] Such nutritional compositions may further comprise a protein source, a digestible carbohydrate source, and / or a lipid source. Protein sources are known in the art, particularly for employment in infant formula, and include dairy proteins (whey proteins, casein) and / or plant protein sources (e.g. pea, faba bean, canola, soy proteins). Examples of digestible carbohydrate sources are disaccharides such as lactose and saccharose, monosaccharides, such as glucose, and maltodextrins, starch, and carbohydrate sources having a prebiotic effect. Suitable lipid sources include mono-, di-, and triglycerides, phospholipids, sphingolipids, fatty acids, and esters or salts thereof. The lipids may have an animal, vegetable, microbial or synthetic origin. Of particular interest are polyunsaturated fatty acids (PUFAs) such as gamma linolenic acid (GLA), dihomo gamma linolenic acid (DHGLA), arachidonic acid (AA), stearidonic acid (SA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and conjugated linoleic acid (CLA). Examples of suitable vegetable lipid sources include sun flower oil, high oleic sun flower oil, coconut oil, palm oil, palm kernel oil, soy bean oil, etc. Examples of suitable lipid sources of animal origin include milkfat, for example anhydrous milkfat (AMF), cream, etc. In a preferred embodiment, a combination of milkfat and lipids of vegetable origin are used.

[0072] The nutritional composition may further comprise probiotic bacteria, such as include bacteria of the genus Bifidobacteria (e.g. B. breve, B. longum, B. infantis, B. bifidum), Lactobacillus (e.g. L Acidophilus, L paracasei, L johnsonii, L plantarum, L reuteri, L rhamnosus, L casei, L lactis), and Streptococcus (e.g. S. thermophilus). B. breve and B. longum are especially suitable probiotics. Further, the composition may contain one or more conventional micro ingredients, such as vitamins, antioxidants, minerals, free amino acids, nucleotides, taurine, carnitine and polyamines. Examples of suitable antioxidants are BHT, ascorbyl palmitate, vitamin E, alpha and beta carotene, lutein, zeaxanthin, lycopene and phospholipids.

[0073] EXAMPLE

[0074] Cheese whey permeate with a dry matter content of 6.6 wt%, a lactose concentration of 83 wt% on dry matter, and a sialyllactose content of 0.2 wt% on dry matter was used as the starting material.

[0075] This cheese whey permeate was demineralized by removing mono- and multivalent ions.

[0076] First of all, multivalent ions were removed by passing the whey permeate at 10°C over a weak base anion exchange resin in the chloride form (XA3112; having a crosslinked acrylic gel structure matrix, tertiary amine functional groups, and a moisture holding capacity of 56-64%), followed by passing it over a strong acid cation exchange resin in the sodium form (XA 2033; having a styrene divinylbenzene matrix, sulphonate functional groups, and a moisture holding capacity of 43-45%). This resulted in exchange of the phosphate and calcium ions for chloride and sodium ions, respectively. When about 25 bed volumes (BV's) of whey permeate were applied, about 90% of the calcium ions and about 50-60% of the phosphate ions were found to be removed.

[0077] The resulting demineralised product was then submitted to nanofiltration using a Dow Filmtec™ NF-2540 membrane (DOW; a thin-film poly(piperazine-amide) composite membrane rejecting organics with a molecular weight above 200 Da), in order to remove monovalent ions. A concentrate was obtained through continuous recirculation of the demineralised product over the membrane at 10°C and a trans membrane pressure of 20-25 bar. As a result, the dry matter content in the concentrate increased to 17% and the lactose content increased from 83% to 93% on dry matter.

[0078] The nanofiltration retentate (10°C) was subsequently passed over a cation exchange resin in the free acid form (XA2041 Na; a macroporous strong cation resin with a styrene-divinylbenzene copolymer matrix, SO3 functional groups, and a moisture holding capacity of 48 ± 6 %), an anion exchange resin in the free base form (LIBA150; a strong anion exchange resin with styrene-divinylbenzene matrix, trimethyl ammonium groups functional groups, and a moisture holding capacity of 39-45%), and an absorbent resin (XA5072 H; a cation exchange resin with a styrene divinylbenzene copolymer matrix, SO3 functional groups, an average surface area of 700 m2 / g, and a moisture content 54 ± 3 %).

[0079] The demineralized product, with a pH of 8.9, was submitted to membrane filtration using 1000 Da NF membrane at a controlled temperature of 10°C and a pressure of 8 bar.

[0080] This membrane filtration increased the dry matter content from 11.4 to 14.2 wt%, reduced the ash content from 0.74 to 0.03 wt% on dry matter, increased the sialyllactose content from 0.18 to 0.6 wt% on dry matter, and increased the lactose content from 95 to 96 wt% on dry matter.

[0081] The retentate was concentrated by evaporation to a dry matter content of 53 wt%. This concentrated solution was used to make GOS via enzymatic reaction.

[0082] The pH of the solution was set to 6.3, kept constant during reaction by adding a citrate buffer, and a Bacillus c / rcu / ans-originating beta-galactosidase enzyme (2.7 Lll / g) was added. After 42 h reaction at 58°C, the solution was heated to 130°C to inactivate the enzyme and the pH of the resulting GOS syrup was adjusted to 3.8 with citric acid. The GOS syrup was concentrated to 75 wt% dry matter by evaporation.

[0083] It contained 61 wt% galacto-oligosaccharides other than lactose, 16.1 wt% lactose, and 0.58 wt% sialyllactose; all on dry matter. The ash content was 0.15 wt%.

Claims

CLAIMS1 . Process for the production of a galacto-oligosaccharide preparation with, based on dry matter, at least 35 wt% galacto-oligosaccharides other than lactose and sialyllactose content in the range 0.2-3.0 wt%, comprising the steps of: i) providing a whey permeate with a lactose concentration in the range 75- 90 wt% on dry matter, ii) removing mono- and multivalent ions from said whey permeate, thereby obtaining a demineralized whey permeate, iii) subjecting the demineralized whey permeate to membrane filtration using a membrane with the molecular weight cut-off in the range 400- 2500 Da, thereby obtaining a retentate with a lactose concentration, based on dry matter, in the range 90-99.8 wt% and a sialyllactose content, relative to lactose, in the range 0.2-3.0 wt%, iv) adding a beta-galactosidase enzyme to the retentate of step iii) in order to convert lactose into galacto-oligosaccharide, thereby forming the galacto-oligosaccharide preparation.

2. Process according to claim 1 wherein the galacto-oligosaccharide preparation contains, based on dry matter, 40-90 wt%, preferably 50-70 wt% galactooligosaccharides other than lactose.

3. Process according to claim 1 or 2 wherein the galacto-oligosaccharide preparation contains, based on dry matter, 0.2-2.0 wt%, preferably 0.4-1 .0 wt% sialyllactose.

4. Process according to any one of the preceding claims wherein the galactooligosaccharide preparation contains, based on the total weight of oligosaccharides other than lactose, 0.2-5.0 wt%, preferably 0.3-4.0 wt%, more preferably 0.3-3.0 wt%, even more preferably 0.4-2.0 wt%, and most preferably 0.5-1 .0 wt% sialyllactose.

5. Process according to any one of the preceding claims wherein the sialyllactose is combination of 3’-siallylactose and 6’-sialyllactose.

6. Process according to any one of the preceding claims wherein step ii) comprises electrodialysis.

7. Process according to any one of the preceding claims wherein step ii) comprises a cation exchange step replacing calcium ions with monovalent cations, preferably sodium and / or potassium ions.

8. Process according to any one of the preceding claims wherein step ii) comprises an anion exchange step replacing phosphate ions with monovalent anions, preferably chloride ions.

9. Process according to any one of the preceding claims wherein step ii) comprises nanofiltration over a membrane with a molecular weight cut-off in the range 200- 300 Da.

10. Process according to any one of the preceding claims wherein step ii) comprises anion exchange chromatography using a strongly basic gel-type anion exchange resin with a water holding capacity of 30-48% in the free base form.11 . Process according to any one of the preceding claims wherein the membrane used in step iii) has a molecular weight cut-off in the range 400-2000, more preferably 400-1500, even more preferably 400-1000, even more preferably 400-700, more preferably 400-600, and most preferably 400-500 Dalton.

12. Process according to any one preceding claims wherein the [3-galactosidase enzyme is derived from a microorganism selected from the group consisting of Bacillus circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, and Papiliotrema terrestris (Cryptococcus Papiliotrema terrestris), preferably Bacillus circulans.