WO2025114343A2

WO2025114343A2 - Process for the production of human milk oligosaccharides

Info

Publication number: WO2025114343A2
Application number: PCT/EP2024/083725
Authority: WO
Inventors: Bas Johan Henri KUIPERS; Brenda Maria Meijer; Linqiu Cao; Jacob HUISMAN; Divya Sony WADHWANI NANIKRAM
Original assignee: FrieslandCampina Nederland BV
Current assignee: FrieslandCampina Nederland BV
Priority date: 2023-11-29
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-29
Also published as: WO2025114343A3

Abstract

Process for the production of a human milk oligosaccharide (HMO)-containing preparation via a microbial fermentation reaction with glucose as carbon and/or energy source, involving enzymatic treatment with at least one carbohydrase in order to hydrolyse carbohydrate impurities originating from said glucose source.

Description

PROCESS FOR THE PRODUCTION OF HUMAN MILK OLIGOSACCHARIDES

The invention relates to a process for the production of human milk oligosaccharides, more in particular a method of the production of human milk oligosaccharides with improved purity.

Human milk contains various oligosaccharides (HMOs), which are important for a healthy development of infants. Many HMOs serve an important role in the development of a healthy intestinal microbiome, thereby enhancing the human body's defense mechanism against pathogens, the establishment of a particular intestinal flora, and stimulation of the immune system. Although HMOs are consumed by infants, it is accepted that the beneficial effects of these oligosaccharides will also apply later in life.

Numerous HMOs exist and more than 150 HMOs have been structurally characterized. Most HMOs contain a lactose moiety at the reducing end and a lot of them contain a fucose, N-acetyl glucosamine, and/or sialic acid moiety. The monosaccharides from which most HMOs are derived are D-glucose, D-galactose, N-acetylglucosamine, L- fucose, and sialic acid. The HMOs most abundantly present in human milk are fucosylated HMOs such as 2’-fucosy I lactose (2’-FL) and 3’-fucosyl lactose (3’-FL), sialylated HMOs such as 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL), and N- acetylated HMOs like lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

Fucosylated and N-acetylated HMOs, such as 2’-FL, 3-FL, LNT, and LNnT, are neutral, in the sense that they are not charged. Sialylated HMO’s, such as 3’-sialyllactose (3’- SL) and 6’-sialyllactose (6’-SL), are acidic/negatively charged and are generally prepared as a salt, conventionally the sodium salt.

The commercial production of HMOs and their addition to infant formula has gained increasing interest over the past years. Their production, however, remains a challenge. The most successful synthesis procedures are fermentation processes using recombinant microorganisms. Apart from the challenge of creating an optimal microorganism, also the purification of the HMO from the fermentation broth is a challenging task, since the final fermentation broth generally contains, apart from the desired HMO, various other, undesired oligosaccharides - intermediates, by-products, and starting substances - which need to be separated/removed from the desired HMO. Separation and purification steps generally applied in order to isolate the HMO from the fermentation broth include the removal of biomass, the removal of proteins and DNA (fragments), minerals, and small molecules, and concentration steps.

A review of fermentative synthesis routes towards HMOs is provided by K. Bych et al., Current Opinions in Biotechnology 56 (2019) 130-137.

Recombinant E-coli strains are generally the microorganisms of choice, together with lactose as the exogeneous sugar acceptor. Sucrose, glucose, and glycerol have been reported as primary carbon and/or energy sources. Each of these carbon and/or energy sources has its own advantages and disadvantages. The advantage of glucose and glycerol, in contrast to sucrose, is that they are readily metabolizabled by E. coli; the advantage of glucose compared to glycerol is its lower cost. A disadvantage of glucose compared to glycerol is, as will be further explained below, its limited purity. The produced HMO is purified from the fermentation broth by removal of the biomass, followed by various purification techniques, including anion- and cation-exchange chromatography, ultrafiltration, nanofiltration, reverse osmosis, electrodialysis, and/or activated carbon treatment.

As mentioned above, glucose is a suitable carbon and energy source in the fermentative production of HMOs. A widely available and inexpensive glucose source is starch. Starch can be obtained from various plant sources, including corn, wheat, rice, barley, potatoes, and cassava.

Starch consists of a large number of glucose units joined by glycosidic bonds. Glucose is usually produced via enzymatic or acid hydrolysis of starch. The resulting starch hydrolysates generally contain about 95 wt% glucose (based on dry weight) and some residual oligosaccharides.

In order to minimize the amount of contaminating species in the resulting HMO, the components added to the fermentation broth should be very pure. High purity glucose - which means: at least 99 wt% glucose based on dry weight - is commercially available; as syrup and as powder. The powders are often made by crystallization and offer a very high purity (up to 99.9 wt%). Disadvantages of using powders are their high costs, the energy consumption and substantial carbon footprint associated with their production, and the extra handling step - dissolution of the powder - that has to be added to the production line.

High purity glucose sources, including those obtained by crystallization, however, still contain some oligosaccharide impurities, including non-fermentable oligosaccharides that cannot be metabolized by the microorganisms used to produce HMOs. These oligosaccharide impurities are therefore difficult to remove from the HMO.

Examples of such oligosaccharides are di- and trisaccharides of glucose, such as panose, nigerose (also known as sakebiose), gentiobiose, alpha-1 , 6-glucofuranosyl glucose, and beta-1 , 6-glucofuranosyl glucose.

Gentiobiose is a disaccharide of two glucopyranoses, connected via a (31 ,6 bond.

Nigerose is a disaccharide of two glucopyranoses, connected via a [31 ,3 bond.

Panose is a trisaccharide of three glucopyranoses, connected via a a1 ,4 and an a1 ,6 bond.

The presence of these impurities complicates the purification of the HMOs, especially of the human milk tri- and tetra-saccharides, since it is difficult to separate the above impurities from such HMOs based on molecular weight or size differences.

The object of the present invention is therefore to provide a process for the production of an HMO with increased purity in terms of oligosaccharide contaminants (non- fermentable sugars) originating from the glucose source. It is a further object to obtain such purified HMO without significant yield loss.

An additional object is the provision of an HMO production process that allows the use of less pure glucose sources, preferably glucose syrups, without compromising on HMO purity.

It has been found that the contaminants present in a glucose source can be eliminated from a fermentation broth or HMO-containing solution using one or more enzymes that selectively hydrolyse these contaminants.

In other words, the oligosaccharide contaminants are eliminated from the fermentation broth or the produced HMO without compromising on HMO yield. The present invention therefore relates to a process for the production of a human milk oligosaccharide (HMO)-containing preparation, the process comprising the following steps:

- forming an HMO in a fermentation broth via a microbial fermentation reaction with glucose as carbon and/or energy source, said glucose being introduced into the fermentation broth via a glucose source that comprises carbohydrate impurities,

- removing biomass from the fermentation broth to form a clarified fermentation broth,

- purifying and concentrating the clarified fermentation broth to form the HMO- containing preparation,

- optionally drying the HMO-containing preparation, the process comprising the step of enzymatically treating the fermentation broth, the clarified fermentation broth, and/or the HMO-containing preparation with at least one carbohydrase in order to hydrolyse at least part of the carbohydrate impurities originating from said glucose source.

The HMO-containing preparation resulting from the process of the present invention preferably has an HMO purity - that is: the purity of a single HMO or the purity of a combination of two or more HMOs - of at least 80 wt%, preferably, at least 85 wt%, and most preferably at least 90 wt%, based on dry matter, and determined by high- performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

It is noted that WO 2015/36138 discloses the addition of a glycosidase during or after the fermentative production of HMOs in order to hydrolyse unreacted lactose and/or side products formed during the fermentation. Disclosed glycosidases are galactosidases, glucosidases, N-acetyl glucosamidases, N-acetylohexoamidases, mannosidases, fucosidases, and sialidases. This document, however, only exemplifies the addition of a beta-galactosidase to remove unreacted lactose after the fermentation and subsequent biomass removal. Further, the fermentation reactions disclosed in that document use glycerol as carbon source, not glucose, meaning that this document neither addresses the problem underlying the present invention, nor its solution.

What is more, lactose is present in large amounts in a fermentation broth producing HMOs, meaning that a beta-galactosidase that is added according to this prior art document is confronted with a high substrate concentration. The present invention, on the other hand, relates to the removal of compounds that are only present in very small amounts, implying a low substrate concentration, which complicates the enzymatic reaction.

It is further noted that WO 2005/100583 and WO 2014/093312 disclose the fermentation of glucose into lactic acid and the simultaneous or subsequent enzymatic conversion of residual non-fermentable oligosaccharides resulting from the glucose source. The glucose source applied in these documents has a purity of 95%, which is significantly lower than preferred in the present invention, which means that the enzyme in the present invention is faced with a significantly lower substrate concentration. Furthermore, the target compound to be produced by these prior art fermentations is not an (oligo)saccharide, meaning that these documents do not face the difficulty of separating the oligosaccharide impurities from an oligosaccharide target compound of similar structure and size.

The fermentation reaction itself and the subsequent purification of the HMO can be performed according to the various methods disclosed in the prior art using a recombinant microorganism, such as recombinant bacteria or fungi, more preferably recombinant bacteria, most preferably recombinant E. coli.

The glucose source to be used as carbon and/or energy source in the process of the present invention preferably has a glucose purity of at least 97 wt%, more preferably at least 98 wt%, and most preferably at least 99 wt%, based on dry weight.

The impurities in the glucose source preferably comprise di- and/or trisaccharide impurities.

The glucose source has preferably been obtained from starch hydrolysis.

The glucose source is conventionally added to the fermentation broth as a solution, said solution being either prepared by dissolving glucose powder in water or is an - optionally diluted - glucose syrup. Said glucose syrup preferably has been produced in the absence of crystallization steps.

Prior to addition, the glucose source is preferably sterilized.

The glucose solution added to the fermentation broth preferably has a glucose concentration in the range 50-80 wt%, preferably 60-75 wt%. The room temperature viscosity of such highly concentrated solutions may be too high for the dosing equipment, which means that the glucose solution is preferably added to the fermentation broth at elevated temperature, e.g. 30-60°C, preferably 40-55°C.

The fermentation reaction may require the supply of air/oxygen to the fermentation broth. In order to allow optimal consumption of residual saccharide impurities, such air/oxygen supply can be continued for at least about 30 minutes after the last glucose addition.

After the fermentation reaction, the HMO is purified using several steps. First of all, biomass has to be removed. Separation of biomass can be accomplished by conventional methods, such as centrifugation, and/or microfiltration (MF). MF is a preferred clarification method. Such MF is preferably carried out with a membrane having a pore size of less than 1 pm, preferably of about 0.1 to about 0.2 pm. MF is particularly suitable to remove cell material (complete cells, fragments thereof) and other supramolecular debris. MF can be performed at about ambient temperature. Preferably, MF is performed at a temperature in the range of 20-75°C, more preferably 30-75°C, most preferably 35-70°C, such as 40-50°C or 60-70°C. A temperature in the range 60-70°C is advantageous for achieving a higher concentration factor of cell material during microfiltration, which has a positive effect on the yield of the HMO.

Prior to or during biomass removal, the fermentation broth can be subjected to a degassing step. Degassing is advantageous in that it reduces the risk of the formation of gas bubbles during subsequent purification steps.

Proteins, DNA (fragments), and/or endotoxins can be conveniently removed from the clarified fermentation broth by ultrafiltration (UF), e.g. using a membrane with a molecular weight cut-off of 5 kDa or less, more preferably about 1 -5 kDa, most preferably 1 -3 kDa.

Minerals are conveniently removed by applying ion exchange steps, such as a combination or anion and cation exchange treatments, optionally combined with electrodialysis.

Evaporation, nanofiltration, and reverse osmosis are examples of methods to concentrate the purified solution. Nanofiltration has the further advantage of removing small molecules, such as monosaccharides and residual salts. In addition to or instead of any of the above purification steps, additional purification steps may also be applied, such chromatography steps, optionally in the form of simulated moving bed chromatography.

The present invention requires the use of at least one carbohydrase enzyme in order to eliminate the impurities introduced via the glucose source. As explained above, these are very specific impurities that are generally hard to remove from the HMO with the conventional purification techniques applied in HMO purification.

The enzyme can be added at several moments during the HMO production process. In a first embodiment, the enzyme can be introduced in the fermentation broth; before, during, or after the fermentation reaction.

An advantage of this embodiment is that the resulting process does not require additional steps for removal of the enzyme in addition to the purification steps already required for isolating and purifying the HMO-containing preparation.

A further advantage of introducing the enzyme before or during the fermentation reaction is that the enzymatic reaction will not require additional processing time and the majority of the enzymatic reaction products (e.g. glucose) can be fermented by the microorganism.

According to this first embodiment, the enzyme may be present during the fermentation reaction - for instance from the start or, more preferably, during the last 36 hours, even more preferably during the last 24 hours, and most preferably during the last 12 hours - in order to ensure that the fermentable saccharides that are created upon the enzymatic hydrolysis can be fermented during the fermentation reaction.

According to the first embodiment, it is also possible to add the enzyme after the fermentation reaction has been completed, but before the removal of biomass. The most convenient way to apply this is to add the enzyme to a stirred and temperature controlled harvest tank in which the fermentation broth is collected prior to biomass removal. An advantage compared to enzyme addition during the fermentation reaction is that it allows the conditions (e.g. temperature, pH) of the fermentation broth to be adjusted to the optimal enzymatic conditions without negatively effecting the fermentation reaction. One advantage of adding the enzyme after the fermentation reaction or during the last part (e.g. the last 36, 24, or 12 hours) of the fermentation reaction is that the total carbohydrate content in the fermentation broth - in mol% - at that point in time is lower than at the start of the reaction, meaning that the concentration of the carbohydrate impurities, based on total carbohydrate content, will be higher than at the start of the reaction. This higher concentration implies a higher substrate concentration for the enzyme and therefore a higher enzymatic reaction rate. A further advantage of adding the enzyme after the fermentation reaction or during the last part (e.g. the last 36, 24, or 12 hours) of the fermentation reaction is that this addition would not require sterile enzyme addition, thereby making the enzyme addition less complicated and easier to perform than enzyme addition at the beginning of the fermentation reaction.

In a second embodiment, the enzyme is added to the clarified fermentation broth, i.e. the broth from which biomass has been removed. The enzyme can subsequently be removed from the clarified broth during the subsequent purification steps. If this subsequent purification involves ultrafiltration, the enzyme will be retained by the ultrafiltration membrane, allowing easy enzyme removal and further allowing all clarified broth to come into contact with the enzyme.

This embodiment allows the conditions (e.g. temperature, pH) of the clarified fermentation broth to be adjusted to the optimal enzymatic conditions with a broader window of opportunity than before biomass removal in view of the need to prevent microbial growth and foaming problems before biomass removal.

In a third embodiment, the enzyme is added to the HMO preparation, after purification and concentration. The enzyme can be contacted with the HMO preparation before an optional drying step or after an initial drying and subsequent dissolution in water. The drying can be performed in conventional ways, such as spray-drying or freeze-drying, preferably by spray-drying.

Advantages of this embodiment - compared to the first and second embodiment - are the even wider window of opportunities in terms of setting optimal enzymatic reaction conditions, higher concentration of the target impurities, and the lower level of potentially interfering compounds.

This embodiment can be effectively reduced to practice by contacting the HMO preparation with a (bed of) immobilized enzyme, followed by nanofiltration - optionally using diafiltration - of the so-treated HMO preparation in order to remove monosaccharides formed during the enzymatic reaction via the permeate. The use of immobilized enzyme allows easy separation of the enzyme from the liquid HMO preparation before submission to the nanofiltration membrane and prevents enzyme from ending up in the nanofiltration retentate. Alternatively, when the enzyme is not in immobilized form, the enzyme can be removed from the HMO preparation by thermal deactivation or by ultrafiltration, either before or after the nanofiltration.

The at least one enzyme to be used in the process of the present invention is a carbohydrase. Carbohydrases are enzymes that catalyse the hydrolysis of di-, oligo- and polysaccharides into smaller saccharides, preferably monosaccharides.

Preferred carbohydrases include endo-1 ,4-beta-glucanases, xylanases, alphaglucosidases, glucoamylases (including alpha- and beta-glucoamylases), and combinations thereof. Most preferred is a combination of alpha-transglucosidase and glycoamylase.

Examples of suitable carbohydrases for use in the process of the present invention are:

- Alpha-glucoamylases, which mainly catalyse the hydrolysis of a-1 ,4 bonds, but are also capable of catalysing the hydrolysis of a-1 ,3 and a-1 ,6 bonds. Examples of such glucoamylases are summarized in P. Kumar, et al., Critical reviews in biotechnology 29 (2009) 225-255. Preferred commercial glucoamylases are those originating from Bacillus licheniformis (e.g. Termamyl® 120Lm type T, and Liquozyme® Supra 2.2X; both from Novozymes), Rhizopus oryzae (e.g. Gluczyme AF6 from Amano Enzyme), or Aspergillus niger (e.g. Gluczyme SD from Amano Enzyme).

- Alpha-glucosidases, e.g maltases, which catalyse the hydrolysis of terminal, nonreducing ( 1 — >4)-linked a-D-glucose residues with the release of D-glucose. Examples of suitable alpha-glucosidases are those originating from Aspergillus niger (e.g. transglucosidase L-500 from IFF, Transglucosidase L from Amano Enzyme, and Distilase@l-400, Optidex L-400, G-ZYME®G990 4X or G-ZYME®490 from IFF/Genencor), genetically modified Trichoderma reesei (e.g. Fermgen®l-400 from IFF/Genencor), or Aspergillus aculeatus (e.g. Viscozyme from Novozymes).

- Endo-1 ,4-beta-glucanases, such as those originating from Trichoderma reesei (e.g. Optimash BG and Accellerase 1000 from IFF/Genencor; Celluclast 1.5L, Cellic Ctec-1 and Cellic Ctec-2 from Novozymes), and Geosmithia emersonii, also known as Talaromyces emersonii (e.g. Optimash TBG from DuPont/Genencor). Further examples are Cellulase DS and Cellulase AP3 from Amano Enzyme.

The enzyme(s) can be used as such, or in immobilized form. Various ways of enzyme immobilization are known in the art. They typically comprise a porous carrier onto which the enzyme is immobilized via covalent binding, via physical absorption (chargecharge or van der Waals interaction), via gel encapsulation, or a combination thereof. Examples of suitable solid carriers are activated acrylic polymers, preferably functionalized polymethacrylate matrices such as hexamethylenamino-functionalized polymethacrylate matrices (Sepabeads) or macroporous acrylic epoxy-activated resins like Eupergit C 250L.

Besides, carrier-free immobilized enzymes such as CLEC (cross-linked enzyme crystals) or CLEAs (cross-linked enzyme aggregates) might be also applied.

The enzymatic reaction is preferably performed at a temperature in the range 10-60°C, preferably 20-50°C, most preferably 25-40°C. The enzymatic reaction is preferably performed at a pH in the range 4.5-7.0, preferably 5.0-6.5, most preferably 5.5-6.0.

The enzyme is preferably added in an amount of 0.1 -100 ll/gram HMO, preferably 1- 50 ll/gram HMO, most preferably 10-25 ll/gram HMO.

HMOs that can be produced via the process of the present invention include neutral and acidic HMOs containing fucosyl, N-acetyl glucosamine, and/or sialyl groups.

Examples of neutral HMOs are N-Acetyllactosamine (LacNAc), lacto-N-biose (LNB), lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N- hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (para-LNH), para- lacto-N-neohexaose (para-LNnH), 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LnNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-neofucopentaose V (LnNFP V), F-LNnH, lacto-N-difucohexaose I (DF-LNH I), lacto-N-difucohexaose II (DF-LNH II), para-lacto- N-difucohexaose (DF-para-LNH) and para-lacto-N-difuconeohexaose (DF-para- LNnH). Examples of acidic HMOs are 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), fucosylsialyllactose (FSL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c) and disyalyllacto-N-tetraose (DS-LNT), fuco- sialyllacto-N-tetraose a (F-LST-a), fuco-sialyllacto-N-tetraose b (F-LST-b), and fuco- sialyllacto-N-tetraose c (F-LST-c).

Preferably, the HMO is selected from neutral tri- and tetrasaccharides, most preferably from the group consisting of 2’-fucosy I lactose (2’-FL), 3-fucosy I lactose (3-FL), lacto-N- tetraose (LNT), and lacto-N-neotetraose (LNnT).

The HMO-containing preparation produced with the process according to the present invention may contain one or a combination of two or more of these HMOs.

The HMO-containing preparation produced with the process of the present invention can be used in nutritional compositions. This can be nutritional compositions for pregnant women (MUM compositions), infants or young children (e.g. 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.

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 the immune system or gut health.

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.

In order to constitute the nutritional composition, in particular the formula milk, the HMO-containing preparation produced 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.

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.

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.

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.

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.

EXAMPLES

Example 1

Several commercially available 99 wt% pure glucose syrups (from Roquette, CG chemikalien and SDA food) were analyzed using HPAEC-PAD, using a CarboPac PA1 (Thermo Scientifc) analytical anion exchange column with a CarboPac PA1 (Thermo Scientifc) guard column and the following gradient: A: 100 mM NaOH, B: 600 mM NaOAc in 100 mM NaOH, C: Milli-Q water, and D: 50 mM NaOAc.

The most prominent di- and oligosaccharide impurities were detected in these syrups: maltose (retention time 26.6 min), isomaltose (retention time: 22.8 min), gentiobiose (retention time 22.2 min), nigerose (retention time: 25.1 min), panose (retention time 32.7 min), [3-6’-glucofuranosyl glucose (beta-6-GFG; retention time 33.2 min), a-6’- glucofuranosyl glucose (alpha-6-GFG; retention time 31.7 min), and a three unidentified components with retention times 24.0 (X8), 29.0 (X9), and 40.0 (X15).

These sugars are difficult to separate from 2’-FL with conventional separation methods like ion exchange and membrane filtration.

Example 2

An accurate volume of a recombinant E. coli K12 culture was transferred to several shake flasks that are grown in parallel to form the starter culture for a seed fermenter. The seed fermenter was transferred to the main fermenter when a predefined culture density was reached. Induction of 2'-FL production - after an initial phase of batch and fed batch growth of the culture on 99 wt% pure glucose to build biomass - was accomplished by the addition of the amino acid L-tryptophan as inducer.

The culture was then grown in fed batch under a regime where constant feeds of glucose syrup and lactose were provided, and during this phase 2'-FL was excreted into the medium. After 90 hours of fermentation, the glucose feed was stopped (the lactose feed was stopped earlier) which ended the fermentation. Aeration was stopped 30 min later to allow for consumption of residual sugars.

Biomass was subsequently removed from the fermentation broth by microfiltration. The microfiltration permeate was subsequently submitted to several purification and concentration steps, including ultrafiltration and anion- and cation-exchange and finally spray-dried.

In order to analyse the glucose source-originating carbohydrate impurities in the 2’-FL, 150 gram of the 2’-FL powder was slowly added to 50 gram water, heated to 50°C under stirring for 30 minutes, and cooled to 4°C. After the centrifugation at 400 rpm and 9°C for 15 minutes, 121 gram paste and 89 gram mother liquor of brix 65.8 were obtained. The mother liquor, enriched in the impurities, was analysed with HPAC-PAD.

This analysis showed that the maltose that was present in the glucose syrup had been fermented during the fermentation reaction, whereas the other predominant peaks were still detected in the fermentation broth.

Example 3

Samples of the mother liquor prepared in Example 2, containing 5 wt% 2’-FL,were submitted to enzymatic hydrolysis with the following enzymes:

Transglucosidase L (ex-Amano) - an alpha-glucosidase

Glucoamylase SD (ex-Amano) - a glucoamylase

Glucozyme AF6 (ex-Amano) - a glucoamylase

Optidex L (ex-IFF) - an alpha-glucosidase

Transglucosidase L-2000 (ex-IFF) - an alpha-glucosidase To 10 gram mother liquor, 20 mM sodium phosphate buffer, pH 6.5, enzyme was added in a concentration of 20 ll/gram dry matter.

The samples were placed in a thermal block and thermostated at 40°C with a shaking speed of 1000 rpm. After 48-hour reaction, 15 pl 1.5 M HCI was added to 1 ml of the reaction sample and the acidified sample was heated at 95°C for 15 minutes in order to denature the enzyme. The samples were analyzed by the HPEAC-PAD.

A significant decrease in especially isomaltose, nigerose, and unidentified compound X9 was achieved with the alpha-glucosidases.

These enzymes did not reduce the 2’-FL content in the samples, but they did increase the glucose concentration with 2-3 wt%. Since the lactose concentration did not change, this increase in glucose concentration was not due to lactose hydrolysis and must be due to hydrolysis of the glucose-originating impurities.

Since glucose can be easily removed from 2’-FL (e.g. by nanofiltration), the enzymatic treatment according to the present invention is therefore able to increase the 2’-FL purity with at least 2 percentage point; in the present case from 94 wt% to at least 96 wt%.

Table 1 - effect of different enzymes on carbohydrate impurities in 2’-FL mother liquor; compared to untreated mother liquor

++++ 70-80% reduction in peak height +++ 50-60% reduction in peak height ++ 30-40% reduction in peak height + 10-20% reduction in peak height Example 4

Example 2 was repeated, except that 43 hours after inocculation carbohydrase was added (20 ll/gram dry matter). After 90 hours of fermentation, the glucose feed was stopped (the lactose feed was stopped earlier) which ended the fermentation. Aeration was stopped 30 min later to allow for consumption of residual sugars.

Biomass was subsequently removed from the fermentation broth and the supernatant was analysed with HPAEC-PAD.

Table 2 - effect of different enzymes added during fermentation compared to the same experiment without enzyme addition

++++ 70-80% reduction in peak height +++ 50-60% reduction in peak height ++ 30-40% reduction in peak height + 10-20% reduction in peak height

Example 5

Example 2 was repeated, except that after the fermentation and before biomass removal, carbohydrase was added (20 ll/gram dry matter). After 24 at 30°C, biomass was removed from the fermentation broth and the supernatant was analysed with HPAEC-PAD. Table 3 - effect of different enzymes added during fermentation compared to same experiment without enzyme addition

++++ 70-80% reduction in peak height

+++ 50-60% reduction in peak height ++ 30-40% reduction in peak height

Claims

1. Process for the production of a human milk oligosaccharide (HMO)-containing preparation, the process comprising the following steps:

2. Process according to claim 1 wherein the glucose source has a glucose purity of at least 97 wt%, preferably at least 98 wt%, most preferably at least 99 wt%, based on dry weight.

3. Process according to claim 1 or 2 wherein the glucose source has been obtained by starch hydrolysis.

4. Process according to any one of the preceding claims wherein the glucose source has the form of a syrup or has been obtained by diluting a glucose syrup, said syrup being produced in the absence of crystallization steps.

5. Process according to any of claims 1 -3 wherein the glucose source has been obtained by dissolving a glucose powder in water.

6. Process according to any one of the preceding claims wherein the impurities originating from said glucose source are di- and trisaccharides, preferably built from glucose moieties, preferably from glucopyranose rings, and more preferably are selected from maltose, isomaltose, nigerose, panose, [3-6’-glucofuranosyl glucose, a-6’-glucofuranosyl, and combinations thereof.

7. Process according to any one of the preceding claims wherein the carbohydrase is selected from the group consisting of glucoamylases, alpha-glucosidases, endo- 1 ,4-beta-glucanases, and combinations thereof, preferably a combination of glucoamylase and alpha-glucosidase.

8. Process according to any one of the preceding claims wherein the fermentation broth is aerated for a least 30 minutes after the last glucose addition.

9. Process according to any one of the preceding claims wherein biomass is removed from the fermentation broth by microfiltration.

10. Process according to any one of the preceding claims wherein the clarified fermentation broth is purified and concentrated by ultrafiltration, ion exchange, nanofiltration and/or reverse osmosis steps.

11 . Process according to any one of the preceding claims wherein the at least one carbohydrase is added to the fermentation broth during the last 36, preferably the last 24, most preferably the last 12 hours of the main fermentation towards the HMO.

12. Process according to any one of claims 1 -10 wherein the at least one carbohydrase is added to the clarified fermentation broth.

13. Process according to any one of claims 1 -10 wherein the HMO-containing preparation is contacted with the carbohydrase, after which the enzymatically treated HMO-containing preparation is submitted to nanofiltration.

14. Process according to any one of the preceding claims wherein the carbohydrase is in immobilized form.