RU2808729C2 - Purification of oligosaccharides from fermentation broth by filtration - Google Patents

Abstract

FIELD: organic chemistry; food industry.

SUBSTANCE: invention is aimed at purifying the target oligosaccharide from the fermentation broth through the use of filtration methods. A method of purifying a target oligosaccharide from a fermentation broth is disclosed, the said method includes the following: - providing a fermentation broth that contains the target oligosaccharide, biomass, microbial cells and carbohydrates other than the target oligosaccharide; - removing microbial cells from the fermentation broth, thereby obtaining a process stream; - subjecting the process stream to a first stage of filtration using a nanofiltration membrane, thereby obtaining a filtrate that contains the target oligosaccharide; - subjecting the filtrate to a second stage of filtration using a nanofiltration membrane, thereby obtaining a retentate that contains the target oligosaccharide; and - removing salts from the process stream using electrodialysis to thereby obtain a purified preparation of the target oligosaccharide, where the target oligosaccharide is a neutral human milk oligosaccharide (HMO) or sialylated HMO, wherein the said method does not include an ion exchange step, and wherein the solution of the purified the oligosaccharide is spray-dried after filter sterilization.

EFFECT: invention provides a simple, effective industrial purification of the target oligosaccharide, resulting in a product having a purity that makes it suitable for human consumption.

17 cl, 7 dwg, 1 tbl, 2 ex

Description

The present invention relates to the production of oligosaccharides by microbial fermentation on an industrial scale. More specifically, the present invention relates to the purification of a target oligosaccharide from a fermentation broth through the use of filtration methods.

BACKGROUND ART

Human breast milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The carbohydrate fraction is the most predominant solid phase fraction and can be further subdivided into lactose and more complex oligosaccharides, so-called human milk oligosaccharides (HMOs).

While lactose is used as an energy source, complex oligosaccharides are not metabolized by infants and adults. The complex oligosaccharide fraction accounts for up to 10% of the total carbohydrate fraction and is likely composed of more than 150 different oligosaccharides. The abundance and concentration of these complex oligosaccharides are specific to humans, and thus they are not found in large quantities in the milk of other mammals, such as domesticated dairy animals. Although HMOs represent only a minor fraction of all human breast milk, their highly beneficial effects on infant development have become evident over the past decades.

The most common HGM is 2'-fucosyllactose. Additional common HGMs are 3-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose. In addition to these neutral oligosaccharides, acidic HMOs can also be found in human breast milk, including 3'-sialyllactose, 6'-sialyllactose and sialyllacto-N-tetraoses such as LST-a, LST-b and LST-c (Table 1) . Up to 20% of the total HGM content of human breast milk is acidic due to the presence of at least one sialic acid group. These structures are closely related to epithelial cell surface glycoconjugate epitopes (Lewis blood group antigens), and the structural homology between OGM and epithelial epitopes explains the ability of OGM to protect against bacterial pathogens (Weichert et al. 2013, Nutr. Res. 33:831; Weichert et al. 2016 J Virol 90:4843).

The presence of complex oligosaccharides in human breast milk has been known for a long time, and the physiological functions of OGM have been the subject of medical research for many decades (Gura 2014, Science 345:747; Kunz & Egge 2017, In McGuire, McGuire & Bode (eds) Prebiotics and Probiotics in Human Milk, Elsevier, London, pp. 3-16). For some of the more common OMGs, specific functions have already been identified (Bode 2012, Glycobiology 22:147; Bode & Jantscher-Krenn 2012, Adv. Nutr. 3S:383; Morrow et al. 2004, J. Pediatr. 145:297). Due to their physiological ability to inhibit infectious agents (bacteria, viruses and bacterial toxins), their beneficial effects on brain development, and their prebiotic functions, there is a great need for the inclusion of HMOs in food products, particularly infant formula products. Table 1 provides an overview of HMOs that can be purified from fermentation broth by the methods disclosed herein.

The limited supply of individual HMOs and the inability to supply sufficient quantities of these molecules has led to the development of chemical synthesis-based methods for their preparation. However, the chemical synthesis of OGM has proven to be extremely difficult, especially large-scale production of OHM with sufficient quality for applications in the food industry. In particular, the chemical synthesis of OHMs such as 2'-FL (WO 2010/115935 A) requires several harmful chemicals that can contaminate the final product.

The disadvantages of chemical synthesis have led to the development of several enzymatic and fermentation-based methods (Miyazaki et al. 2010, Methods Enzymol. 480:511; Murata et al. 1999, Glycoconj. J. 16:189; Baumgartner et al. 2013, Microb. Cell Fact. M AO; Lee et al. 2012, Microb. Cell Fact. 1:48; Albermann et al. 2001, Carbohydr. Res. 334:97; US 7,521,212 B1; Fierfort & Samain 2008, J. Biotechnol. 134: 216). However, these processes result in complex mixtures of oligosaccharides, and the desired product is thus contaminated with starting materials such as lactose, as well as intermediates, unwanted by-products (for example, by-products resulting from side activities of certain glycosyltransferases) and substrates from the fermentation process, such as proteins, polypeptides, organic acids and salts.

Many current methods for purifying individual oligosaccharides from mixtures are technically complex, difficult to scale up, and uneconomical for food industry applications. Industrial-scale processes have been developed to purify the disaccharides lactose and sucrose from whey and molasses, respectively, but these processes involve multiple crystallization steps that are elaborate and achieve only low yields. However, whey and molasses are primarily “food” products and are by no means as complex and demanding (from a regulatory perspective) as fermentation broths containing recombinant bacteria or yeast. Gel chromatography is the best way to purify complex oligosaccharides such as OGMs produced by microbial fermentation, but disadvantages of this method include its lack of scalability and its incompatibility with continuous processing. Gel chromatography is thus unprofitable and cannot be used to obtain OGM on an industrial scale.

In theory, gel chromatography is the best way to purify complex oligosaccharides such as OGM produced by microbial fermentation, but disadvantages of gel chromatography include its lack of scalability and its incompatibility with continuous processing. Gel chromatography is thus unprofitable and cannot be used to obtain OGM on an industrial scale.

Having developed efficient methods and processes for the production of HMOs through microbial fermentation, the next logical step was to develop efficient downstream methods for isolating HMOs from the culture broth.

WO 2015/049331 A discloses a method for purifying neutral HMOs. The method uses ion exchange, electrodialysis and simulated moving bed chromatography (SMB) to achieve continuous and efficient purification of large quantities of OGM. In contrast to chemical synthesis routes for obtaining neutral OHMs and their subsequent purification, the described method of fermentation and purification makes it possible to provide OHMs that do not contain harmful chemicals, such as heavy metals - trace elements and organic solvents. The purification process produces a highly purified OGM-based product in solid form (by spray drying) or as a concentrated syrup that can be used in food processing applications.

WO 2015/106943 A describes a simple method for the purification of neutral HMOs obtained by microbial fermentation. The method uses a combination of cation exchange, anion exchange and nanofiltration and/or electrodialysis, which makes it possible to effectively purify large quantities of neutral OHMs. Unlike earlier purification methods for neutral HMOs obtained through microbial fermentation, the method does not include chromatographic separation steps. The method results in the production of OGM in solid form as a spray-dried substance, as a crystalline substance or as a filter-sterilized concentrate. The resulting HMOs do not contain proteins and substances originating from the recombinant microbial strains used for production and are thus ideal for food, health food and feed (eg pet food) applications.

WO 2016/095924 A describes a method for purifying 2'-FL by crystallization. OGM was obtained through microbial fermentation, and after removing the biomass, it was concentrated through nanofiltration and electrodialysis. Finally, 2'-FL was selectively crystallized from an aqueous solution also containing 2',3-di-O-fucosyllactose (DFL) using acetic acid.

LNT and LNnT can be separated from carbohydrate by-products by selective coupling with activated carbon followed by elution with organic solvents and separation by gel chromatography (Priem et al. 2002, Glycobiology 12:235; Gebus et al. 2012, Carbohydr. Res. 361 :83;Baumgartner et al. 2014, ChemBioChem 15:1896). As stated above, gel chromatography is a convenient laboratory technique, but it cannot be effectively scaled up for industrial production.

BWO 2015/049331 A discloses the following sequence of operations for purifying LNT produced by bacterial fermentation to provide a clear, particulate-free solution: electrodialysis, nanofiltration, pseudo-moving bed (SMB) chromatography using a strong cation exchange resin, electrodialysis, ultrafiltration and second chromatography step with SMB.

Various methods have also been described for the isolation of acidic HMOs, especially sialylated lactoses or sialylated oligosaccharides.

US 2007/0020736 A discloses the production of 3'-SL with disialylated and trisialylated lactoses as by-products of a genetically modified strain of the bacterium Escherichia coli. 3′-SL accumulated in the culture broth to a concentration of ∼0.8 mM. 3'-SL was purified using the following steps: centrifugation, adsorption by passing the supernatant through charcoal and washing out water-soluble salts with distilled water, gradient elution in aqueous ethanol, and finally separation on a Biogel column and desalting. The yield from 1 liter of broth was 49 mg of 3'-SL.

Another disclosed method involves producing 3'-SL using a genetically modified E. coli strain and subsequent isolation by heat permeabilization of cells followed by centrifugation (Priem et al. 2002, Glycobiology 12:235). The substance in the supernatant was adsorbed on carbon, and the water-soluble salts were washed out with distilled water. After gradient elution in aqueous ethanol, 3'-SL was adsorbed onto a strong anion exchanger in its HCO ₃ ^- (bicarbonate ion) form and eluted using a linear gradient of sodium bicarbonate (NaHCO ₃ ). The latter was removed via cation exchange (using the resin in its acidic form), resulting in a 3'-SL recovery rate of 49%.

An alternative method, starting from the fermentation broth, involved the steps of heat permeabilization, centrifugation, adjusting the pH to 3.0 by adding a strong cation exchange resin in its acidic form and removing precipitated proteins by centrifugation (Fierfort et al. 2008, J, Biotechnol, 134:261 ). The pH of the supernatant was then adjusted to 6.0 by adding a weak anion exchanger in its basic form, and 3'-SL was coupled to the anion exchanger in the form of HCO ₃ ^- . After washing with distilled water followed by elution with a continuous NaHCO ₃ gradient, NaHCO ₃ was removed by cation exchange (using the resin in its acid form) until the pH dropped to 3.0. Finally, the pH was adjusted to 6.0 with NaOH. This purification strategy achieved a 3′-SL recovery rate of 59%.

The enzymatically produced 3'-SL was also separated from lactose by nanofiltration (Nordvang et al. 2014, Separ. Purif. Technol. 138:77). The authors of the invention showed that two different nanofiltration membranes, one with a molecular weight cutoff (MWCO) of 600-800 Da (membrane based on sulfonated polyethersulfone (SPES)) and the other with MWCO 1000-1400 Da (SPES membrane), could separate most of the 3'-SL from lactose after diafiltration. However, there was significant loss of 3'-SL during this process and the purity was low, requiring an additional ion exchange purification step.

WO 2010/106320 A2 describes a method for fortifying 3'-SL from whey. First, proteins are removed by ultrafiltration, and then the clarified whey filtrate is incubated with a 3'-SL capture ion exchange resin. After elution from the ion exchanger, the enriched 3'-SL fraction is concentrated by nanofiltration to demineralize the concentrate. After demineralization, 3'-SL is concentrated and dried, resulting in a final dry product containing 20 wt % 3'-SL. %.

WO 2018/020473 A describes a further method for enriching 3'-SL and 6'-SL from a liquid source. Both OGMs were isolated from lactose crystallization mother liquor through the steps of solution heating, enzymatic treatment, and additional ultrafiltration and nanofiltration. After enrichment, the content of 3'-SL and 6'-SL was 10-30 wt. % dry weight.

Based on this prior art, the objective was to propose a method for the purification of target oligosaccharides, in particular neutral HMOs or sialylated HMOs, which are obtained through microbial fermentation, wherein said method is easy to scale up and is suitable for the production of said target oligosaccharides in industrial and production scale, and which can result in a product having a purity that makes the product suitable for human consumption.

It must be emphasized that microbial fermentation broths, particularly those containing recombinant microorganisms (bacteria or eukaryotic microorganisms such as baker's yeast) are much more complex than dairy product streams. The composition of whey, for example, is as follows: approximately 94% water, 4-5% lactose, 0.5-1% protein and only a few certain minerals like calcium, potassium and phosphorus, along with some vitamins, a simple matrix that only that is concentrated and demineralized in dairy product streams. In contrast, the matrix of the Sugar solution obtained from the recombinant microbial fermentation process is very complex, firstly, primarily based on the requirement to legally separate the recombinant biomass and inactivate it in accordance with government regulations. The resulting clarified broth is an indeterminate matrix of different salts and ions, also containing heavy metals and trace elements. The problem of such a liquid, moreover, is the removal of cell debris, membrane fragments such as lipids, proteins, molecules originating from the metabolism of microbial cells, and mainly DNA. Isolation of oligosaccharide produced by a recombinant process aid, such as genetically modified bacteria, is thus an even greater challenge compared to whey and milk product streams, since the diversity of contaminants within the broth is very high in terms of molecular weight, charged molecules (single-charged and multi-charged) and coloring molecules.

SUMMARY OF THE INVENTION

The present invention provides a method for purifying a target oligosaccharide produced by batch or continuous fermentation from a culture broth or a fermentation broth produced by microbial fermentation using recombinant fermentation strains. Previously described purification strategies often used expensive ion exchange steps (requiring both cation and anion exchangers). Ion exchangers cannot operate continuously because they require regeneration. The culture broth contains the target oligosaccharide, biomass, media components, salts and contaminants such as other acids and pigments.

During the purification process, the culture broth can be subjected to the following purification steps to obtain the target oligosaccharide:

1) Separation of microbial cells from the culture broth through microfiltration;

2) Separation of proteins from the clarified culture broth (representing the process stream) by means of ultrafiltration;

3) The first stage of nanofiltration to remove peptides and high-molecular-weight (HMW) impurities;

4) Second stage of nanofiltration to remove water and salts;

5) Treatment with activated carbon to remove pigments and other impurities;

6) Electrodialysis or diafiltration using a nanofiltration membrane to completely remove contaminant salts;

7) Removing water through reverse osmosis.

Additionally, another electrodialysis step could be introduced before the activated carbon treatment to reduce the amount of contaminant ions. To obtain the target oligosaccharides in solid form after purification in aqueous solution, the substance can be either spray dried or granulated.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

In FIG. 1 is a process diagram of an illustrative embodiment of the method of the invention.

In FIG. 2 is a process diagram of yet another illustrative embodiment of the method of the invention.

In FIG. 3 is a process diagram of yet another illustrative embodiment of the method of the invention.

In FIG. 4 is a process diagram of yet another illustrative embodiment of the method of the invention.

In FIG. 5 is a process diagram of yet another exemplary embodiment of the method of the invention.

In FIG. Figure 6 shows an HPLC (high performance liquid chromatography) chromatogram of a clarified fermentation broth containing 3-fucosyllactose.

In FIG. 7 shows an HPLC chromatogram of 3-fucosyllactose after purification from a fermentation broth by an exemplary embodiment of the method of the invention.

DETAILED DESCRIPTION

According to the invention, the term "purity" refers to chemical purity and defines the degree to which a substance such as 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or other target oligosaccharide (Table 1) , does not dilute or mix with foreign matter. Therefore, chemical purity is an indicator of the relationship between the monocomponent substance and any by-products/impurities. Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

In a composition containing a target oligosaccharide, the purity of the compound can be determined by any suitable method known to one skilled in the art, such as high performance liquid chromatography (HPLC). The appropriate detector can be selected from the group consisting of an electrochemical detector, a refractive-index detector, a mass spectrometer (MS), a diode-array detector (DAD), and a diode-array detector. nuclear magnetic resonance (NMR). In HPLC, for example, purity can be determined by calculating the ratio of the area under a target peak (representing the amount of the target oligosaccharide) to the sum of the areas under all peaks (representing both the amount of the target oligosaccharide and compounds other than that substance in the same chromatogram). However, it follows from the above that all impurities can be analyzed using the selected HPLC method. Otherwise, a mass balance approach is required, namely, determining the absolute amount of the desired product. In this approach, pure substances are used as a reference to quantify purity, which is then assessed relative to the dry matter obtained from the product (desired product plus any impurities). This mass balance approach can also be used to determine purity according to the invention.

According to the invention, "culture broth" or "fermentation broth" refers to any liquid after fermentation containing 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or any other target oligosaccharide (Table 1) for the cleaning. The terms "culture broth", "fermentation broth" and "culture medium" are used as symptoms in this document. The culture broth contains the target oligosaccharide that must be purified, as well as biomass (eg, biological cells and cell debris), media components, salts, and additional contaminants such as other acids and pigments. The biological cells contained in the culture broth are biological cells that produce the target oligosaccharide intracellularly and secrete the compound into the liquid culture medium. Biological cells may contain or consist of genetically modified biological cells, such as genetically modified E. coli cells. Genetic modification may include or consist of modification to produce a target oligosaccharide, especially during the growth phase of said biological cells.

The term "biomass", as used herein, refers to the collection of biological cells present in the fermentation broth at the end of the fermentation stage. Biomass includes microbial cells that produced the target oligosaccharide, cells derived from the microorganism that may have lost their ability to produce the target oligosaccharide during the fermentation step, and any other cells that happen to be present in the fermentation broth at the end of the fermentation step. Therefore, essentially all biological cells that are present in the fermentation broth at the end of the fermentation stage are separated from the fermentation broth such that the clarified fermentation broth, known as the "process stream" (any solution including or containing the target oligosaccharides that are to be purified) contains essentially or no cells.

The biomass preferably contains biological cells that produce the target oligosaccharide, preferably bacterial cells that produce the target oligosaccharide, and more preferably recombinant bacterial cells that produce the target oligosaccharide, most preferably recombinant E. coli cells that produce the target oligosaccharide.

Biomass and/or microbial cells can be removed from the fermentation broth by centrifugation and/or filtration.

In centrifugation methods suitable for removing biomass from a culture broth, the biomass is obtained as a sediment and the supernatant as a clarified process stream, which is subjected to additional treatments. In suitable filtration methods for removing biomass from the culture broth, the filtrate becomes a clarified process stream. The preferred filtration method for biomass removal is microfiltration and/or ultrafiltration, the latter having the ability to remove even smaller particles than microfiltration and also larger molecules. Microfiltration and ultrafiltration can be carried out by dead-end filtration (the process stream flows perpendicular to the filter) or cross-flow filtration (the process stream flows parallel to the filter).

Microfiltration is a physical separation method in which a liquid containing particles is passed through a medium, said medium containing either a porous substance containing channels to retain the particles (depth filtration) and/or a membrane with a specific pore size allowing the passage of the particles. molecules that are smaller than the specified pore size (membrane filtration). The term "microfiltration", as used herein, refers to a physical separation method where biological cells (and cell debris) are removed from the fermentation broth, leaving a (clarified) process stream.

Suitable membranes for biomass removal by microfiltration may have a pore size of at least 0.2 μm and could be hollow fiber or spiral wound membranes. Alternatively, biomass removal could be achieved by microfiltration using membranes with MWCO of 100-1000 kDa, preferably 150-500 kDa, to remove biomass, additional cellular debris and large proteins. For example, membranes such as TRISEP® DS MVP20 (0.2 μm pore size) (Microdyn-Nadir GmbH, Wiesbaden, Germany), which is coiled to provide a compact design and better efficiency, can be used to separate cells from the culture broth. Also, spiral wound membranes with a pore size of 0.05-0.1 µm, like TRISEP® DS MP005, which is a module containing a PES (polyethersulphone) membrane and a nominal pore size of 0.05 µm, can be used for separation of biomass and proteins.

In addition, hollow fiber modules like the FS10-FC FUS1582 (Microdyn-Nadir GmbH), a hollow fiber filter module using a PES membrane (5 m ² ) with a MWCO of 150 kDa, can be used as an alternative.

In summary, the present invention can utilize the following options for removing biomass from the fermentation broth:

i) Collection by centrifugation. Insoluble components are removed from the culture broth in one step. Advantage: rapid removal of insoluble components.

ii) Collection by microfiltration. Insoluble components and large molecules above a certain size are removed from the culture broth in a single step. Spiral wound membranes or cross-flow hollow fiber filters can be used for microfiltration with MWCO of 500 kDa or greater. Advantage: Rapid removal of insoluble components and large soluble molecules above a certain size.

iii) Collection by centrifugation combined with microfiltration: Insoluble components and large molecules above a certain size are removed from the culture broth in two steps. Spiral wound membranes or cross-flow hollow fiber filters can be used for microfiltration with MWCO of 500 kDa or greater. Advantage: Rapid removal of insoluble components and large molecules above a certain size without clogging microfiltration membranes.

Ultrafiltration is a form of membrane filtration that is not fundamentally different from microfiltration. In ultrafiltration, forces generated by pressure and concentration gradients result in the removal of particles and large soluble molecules by passing a liquid containing such particles and large soluble molecules through a semi-permeable membrane. This ensures that particles and large soluble molecules are retained in the so-called retentate, while water and low-molecular-weight (LMW) solutes, such as the resulting neutral and acidic oligosaccharides, pass through the membrane into the permeate (filtrate). ). Ultrafiltration membranes are defined by their MWCO, which describes the maximum molecular weight of a soluble molecule that can pass through the membrane into the permeate. Any particles, as well as molecules larger than MWCO, are unable to pass through the membrane and are retained in the retentate. Ultrafiltration can be applied in cross-flow mode, where the fluid flow is parallel to the membrane surface, or in “dead-end filtration” mode, where the fluid flow is perpendicular to the membrane surface.

The clarified process stream containing the target oligosaccharide produced typically contains significant amounts of undesirable impurities, including (but not limited to) monovalent ions, divalent ions, amino acids, polypeptides, organic acid proteins, nucleic acids, monosaccharides and/or oligosaccharides.

Suitable membranes for removing most proteins by ultrafiltration have a MWCO of at least 50 kDa, preferably at least 30 kDa, more preferably 10 kDa, and could be hollow fiber or spiral wound membranes. For example, membranes such as SPIRA-CEL® DS UP010 (MWCO is 10 kDa) or SPIRA-CEL DS UP005 (MWCO is 5 kDa), both marketed by Microdyn-Nadir GmbH, are helix wound to provide a compact design and better efficiency, and these membranes can be used to separate remaining protein from cell-free culture broth.

In addition, hollow fiber modules such as ROMICON® HF UF Cartridge PM10 (Koch Membranes Systems, Wilmington, USA), which are PES membranes with area up to 12 m ² and MWCO 10 kDa, can be used as an alternative.

Nanofiltration is a membrane filtration method in which the membrane contains pores in the nanometer range (1-10 nm). The pore size of nanofiltration membranes is smaller than the pore size of microfiltration and ultrafiltration membranes, but larger than the pore size of membranes used for reverse osmosis. Nanofiltration membranes are primarily made from thin films of polymers such as polyethylene terephthalate or metals such as aluminum with pore densities of 1-10 ⁶ pores per cm ² .

Nanofiltration is used in the target oligosaccharide purification process to remove LMW impurities such as peptides and salts and to increase the concentration of oligosaccharides in the clarified process stream or eluate.

The molecular weight of most OGMs ranges from 400 to 1200 Da. At least two nanofiltration steps are required to remove HMW impurities (greater than 1200 Da), such as peptides and pigments, and LMW impurities (less than 400 Da), such as salts from the process stream.

After ultrafiltration, all molecules greater than 5 kDa or greater than 10 kDa (depending on the membrane used) must be removed from the process stream. To remove impurities such as peptides and pigments that are below a given threshold but still larger than the target oligosaccharide, a first nanofiltration step could be carried out using MWCO, which allows the oligosaccharide to pass into the permeate while the HMW impurities remain in retentate.

To separate most of the desired oligosaccharide from HMW impurities and to increase the yield of the desired oligosaccharide, the process should be continued until more than 70%, preferably more than 80%, more preferably more than 90% of the product passes through the membrane. Additionally, a diafiltration step could be included to increase the yield of oligosaccharide in the clarified process stream.

After this first nanofiltration step, the only impurities in the process stream containing the target oligosaccharide should have the same molecular weight as or less than the given oligosaccharide, and could include monovalent ions, divalent ions, amino acids, organic acids, monosaccharides and/or oligosaccharides.

Membranes suitable for the first stage of nanofiltration include thin film composite membrane materials - polyamide or polypiperazine, achieving size separation in the range of 400-1200 Da. Examples include HYDRACoRe70 membranes (MWCO is 720 Da) or HYDRACoReSO (MWCO is 1000 Da), both from Hydranautics membranes (Oceanside, USA), NADIR® NP010P (MWCO is 1000-1500 Da) or NADIR® NP030P (MWCO is 500-1000 Yes) (Microdyn-Nadir GmbH). Such membranes enable high intensity flow. Additional examples of suitable nanofiltration membranes include Trisep® SBNF (MWCO is 2000 Da), cellulose acetate nanofiltration membrane (Microdyn-Nadir GmbH) and GE-Series membranes (MWCO is 1000 Da) from Suez Water Technologies & Solutions (Ratingen, Germany ).

In an additional and/or alternative embodiment, the first nanofiltration stage achieves the following parameters:

i) the recovery after filtration of the target oligosaccharide should be greater than 70%, preferably greater than 80%, more preferably greater than 90%.

ii) the concentration of the target oligosaccharide in the process stream should be less than 50% (w/v), preferably less than 40% (w/v), more preferably less than 30% (w/v), preferably less than 20% ( w/v), more preferably less than 10% (w/v).

iii) the step should be carried out at a temperature of less than 80°C, preferably less than 50°C, more preferably 4-40°C (particularly relevant for nanofiltration).

iv) the step should be carried out at a pressure of 500-5000 kPa, preferably at a pressure of 1000-4000 kPa, more preferably at a pressure of 1500-3000 kPa.

In a preferred embodiment of the method, the process stream containing the target oligosaccharide is concentrated and desalted after the first nanofiltration step by using a second nanofiltration step. The target oligosaccharide can also be concentrated by vacuum evaporation (eg, using a falling film evaporator or plate evaporator) or reverse osmosis. The disadvantage of both of these methods is that the process stream is concentrated, but not desalted. Thus, nanofiltration is a preferred method because it can achieve both concentration and desalting, for example by using a membrane with a size separation limit of less than 2 nm.

A method for purifying a target oligosaccharide includes the step of subjecting the clarified process stream from the first nanofiltration step to at least one additional nanofiltration step to remove salts, smaller molecules, and water. Preferably, the eluate from the first nanofiltration (either filtrate or permeate) is directly subjected to a second nanofiltration step to remove salts and smaller molecules from the clarified process stream.

Membranes suitable for first stage nanofiltration include polyamide or polypiperazine thin film composite membrane materials achieving size separations in the range of 150-300 Da, such as Filmtec™ NF270 membranes (Dow Chemical Company, Midland, USA) and Trisep® XN45 or TS40 (Microdyn Nadir GmbH). Such membranes enable high intensity flow. Additional examples include Trisep® 4040-XN45-TSF membranes (Microdyn-Nadir GmbH) or GE4040F30 and GH4040F50 (Suez Water Technologies & Solutions).

Nanofiltration effectively removes significant amounts of salts and LMW impurities from the process stream containing the target oligosaccharide prior to electrodialysis. Nanofiltration is also effective in removing LMW contaminants after the ultrafiltration step, where removal of such contaminants is beneficial for concentrating and demineralizing the target oligosaccharide solution. The use of nanofiltration to concentrate the target oligosaccharide results in lower energy and processing costs and better product quality due to more limited thermal exposure.

The method concentrates a target oligosaccharide from an aqueous solution, where the concentration of the target oligosaccharide in the aqueous solution is 20% or less, 10% or less, or 5% or less for concentration.

In an additional and/or alternative embodiment, the concentration through nanofiltration should achieve the following parameters:

i) the oligosaccharide concentration is greater than 100 g/L, preferably greater than 200 g/L, more preferably greater than 300 g/L, most preferably greater than 400 g/L;

ii) the amount of salt in the purified solution should be less than 10 wt. %, preferably less than 5%, more preferably less than 2%; and/or the conductivity should be 0.5-10.0 mS/cm ² , preferably 1-8 mS/cm ² , more preferably 1.5-4.0 mS/cm ² .

iii) the step should be carried out at a temperature of less than 80°C, preferably less than 50°C, more preferably 4-40°C (particularly relevant for nanofiltration).

iv) the step should be carried out at a pressure of 500-5000 kPa, preferably at a pressure of 1000-4000 kPa, more preferably at a pressure of 1500-3000 kPa.

Pigments in the clarified solution and/or purified solution can be removed by treatment with activated carbon. The advantage of pigment removal using activated carbon is that both electrically charged and electrically uncharged (neutral) pigments can be removed.

Activated carbon, also called activated charcoal, is a form of carbon that is processed to form small, low-volume pores that increase the surface area available for adsorption. Typically, only 1 g of activated carbon has a surface area greater than 30.00 m ² as determined by gas adsorption, due to its high degree of microporosity.

Color imparting impurities such as Maillard products, riboflavin and other LMW impurities tend to adsorb to the surface of the activated carbon particles. In contrast to the resulting oligosaccharides, the amount of color imparting substances is much smaller and/or exhibits hydrophobic behavior in most cases. As a consequence of this fact, pigmented contaminants have a high rate of removal from the process stream. Water-soluble substances such as oligosaccharides bind more weakly and can be eluted by washing with water, leaving pigments adsorbed on the surface.

In an additional and/or alternative embodiment, the method for purifying a target oligosaccharide further includes at least one step in which the clarified process stream or eluate from the previous purification step is treated with activated carbon to remove pigments.

In a preferred embodiment, the activated carbon treatment should be carried out as follows:

i) after removing water and salt in the second stage of nanofiltration; and/or

ii) after removal of proteins by ultrafiltration or after removal of remaining salts by electrodialysis (alternatively, a diafiltration step can be introduced).

Suitable activated carbon for removing neutral oligosaccharides, pigments and other contaminants includes, but is not limited to, granular activated carbon such as Norit GAC830EN (Cabot Corporation, Boston, USA) and Epibon Y 12 x 40 spezial (Donau Carbon, Frankfurt, Germany ), or powdered activated carbon such as Norit DX1, Norit SA2 (Cabot Corporation) and Carbopal MB 4 (Donau Carbon).

Electrodialysis combines dialysis and electrolysis and can be used to separate and concentrate ions in solutions based on their selective electromigration across a semipermeable membrane. Applications of industrial electrodialysis date back to the early 1960s, when the method was used to demineralize cheese whey for inclusion in infant formula. Additional applications include pH adjustment of beverages such as wines, grape must, apple juice and orange juice.

Desalination of brackish water to produce drinking water and demineralization of whey to produce infant formula are the most widespread applications of electrodialysis today. The basic principle of electrodialysis involves an electrolytic cell containing a pair of electrodes immersed in an electrolyte to conduct ions, connected to a direct current generator. The electrode connected to the positive pole of the forward current generator is the anode, and the electrode connected to the negative pole is the cathode. The electrolyte solution then maintains the flow of electric current, which is obtained as a result of the movement of negative and positive ions towards the anode and cathode, respectively. Membranes used for electrodialysis are essentially sheets of porous ion exchange resins with negatively or positively charged groups, and are thus described as cationic or anionic membranes, respectively. Ion exchange membranes typically consist of a polystyrene matrix bearing a suitable functional group (such as a sulfonic acid in the case of cationic membranes or a quaternary ammonium group in the case of anionic membranes) cross-linked to divinylbenzene.

The electrolyte may be an aqueous solution containing, for example, sodium chloride, sodium acetate, sodium propionate and/or sulfamic acid. The electrolyte surrounds the cathode and anode and allows current to flow in the cell. The electrodialysis package is then assembled such that the anionic and cationic membranes are parallel, as in a filter press, between two electrode banks, such that the stream to be ion-depleted is well separated from the stream to be ion-enriched. These two solutions are also called dilute solution (to be ion-depleted) and concentrate (to be ion-enriched).

The central part of the electrodialysis process is the membrane stack, which consists of several anion-exchange and cation-exchange membranes separated by spacers installed between two electrodes. By applying direct current, anions and cations will migrate through the membranes towards the electrodes, forming a (desalted) dilute solution stream and a concentrate stream.

The pore size of ion exchange membranes used for electrodialysis is small enough to prevent diffusion of product from the dilute solution stream into the concentrate stream due to the large difference in concentration between the two streams.

Electrolysis is used to remove ions from aqueous solutions while neutral and acidic oligosaccharides remain in the process stream. An important advantage of electrodialysis is that recombinant DNA molecules can be completely removed from a solution containing the target oligosaccharide. In addition, the amount of salt in the process stream can be significantly reduced through electrodialysis. Indeed, sodium chloride can be completely removed from the product stream. This has the advantage that a solution containing the target oligosaccharide can be provided which is devoid of salts like sodium chloride, thereby preventing any negative effect of the salt in the final product, eg baby food.

After nanofiltration and activated carbon treatment, most of the salt and smaller organic impurities, like organic acids, should be removed from the process stream. To ensure effective removal of remaining salts and small, charged organic substances, an electrodialysis step is carried out.

Electrodialysis can be carried out until the process stream reaches a stable conductivity of 0.05-1.0 mS/ ^cm2 , preferably 0.1-0.5 mS/ ^cm2 , more preferably 0.2-0.4 mS/cm2 cm ² . In addition, electrodialysis can be carried out until the salt concentration drops to a concentration of less than 10.0 g/L, preferably less than 5.0 g/L, more preferably less than 1.0 g/L, most preferably less than 0.2 g/l.

In the case of neutral oligosaccharides, the electrodialysis step should be run under acidic or neutral pH conditions, preferably pH 3-8, more preferably pH 4-7. For acid oligosaccharides, the electrodialysis step should be run under neutral pH conditions, preferably pH 6-8, more preferably pH 6.5-7.5 due to the instability of acid oligosaccharides under acidic conditions. The pH of the acid oligosaccharide solution must be monitored during electrolysis and adjusted with NaOH if necessary.

A reverse osmosis step can be used instead of nanofiltration to concentrate the target oligosaccharide. Reverse osmosis is a membrane filtration method that concentrates particles greater than 0.1 nm in the retentate process stream while removing water. Reverse osmosis thus concentrates the process stream but does not achieve desalination.

The method may concentrate the target oligosaccharide in an aqueous or organic solvent, where the concentration of the target oligosaccharide is 20% (w/v) or less, 10% (w/v) or less, or 5% (w/v) or less to concentration.

In an additional and/or alternative embodiment, the concentration step should achieve the following parameters:

i) the oligosaccharide concentration is greater than 300 g/L, preferably greater than 400 g/L, more preferably greater than 500 g/L, most preferably 600 g/L.

ii) the step should be carried out at a temperature of less than 80°C, preferably less than 50°C, more preferably less than 4-40°C (particularly relevant for reverse osmosis).

iii) the step should be carried out at a pressure of 500-5000 kPa, preferably at a pressure of 1000-4000 kPa, more preferably at a pressure of 1500-3000 kPa.

In an additional and/or alternative embodiment of the method, the solution containing the neutral or acidic OHM is concentrated by evaporation in a vacuum (for example, using a rotary evaporator or plate evaporator), and it must achieve the following parameters:

i) the oligosaccharide concentration is greater than 300 g/L, preferably greater than 400 g/L, more preferably greater than 500 g/L, most preferably greater than 600 g/L.

ii) the step should be carried out at a temperature of less than 80°C, preferably less than 50°C, more preferably 20-50°C, even more preferably 30-45°C, most preferably 35-45°C (particularly relevant in the case of evaporation in a vacuum).

To remove any possible contaminants and endotoxins produced by microbes, the concentrated target oligosaccharide is sterilized by filtration through an ultrafiltration membrane.

In a preferred embodiment of the invention, the purified oligosaccharide solution is passed through a 10 kDa or smaller filter module, such as a 5 kDa or 3 kDa MWCO filter. Suitable ultrafiltration membranes for endotoxin removal have a MWCO of at least 10 kDa, preferably at least 5 kDa, and could be spiral wound or hollow fiber ultrafiltration membranes. Examples of spiral wound membranes include SPIRA-CEL® DS UP010 (MWCO is 10 kDa) or DS UP005 (MWCO is 5 kDa) membranes (Microdyn-Nadir GmbH) and Dairy-Pro® HPHT UF-5K (MWCO is 5 kDa) from Koch Membranes Systems. Examples of hollow fiber modules include ROMICON® HF UF Cartridge PM5 (MWCO is 5 kDa) from Koch Membranes Systems or Microzoa™ off-the-shelf membranes (MWCO is 3 or 6) from Pall Cooperation (Port Washington, USA).

In a preferred embodiment, the purified oligosaccharide solution is spray dried after filter sterilization. The solution can be spray dried using hot air to remove preferably at least 85 wt. % or more preferably at least 90 wt. % water. The solution can be spray dried using any conventional spray drying system, preferably coupled with a fluid bed dryer.

The purified solution can be spray dried to achieve a concentration of the target oligosaccharide of 5-60 wt. %, preferably 10-50 wt. %, more preferably 15-45 wt. %. The inlet temperature can be set in the range of 110-150°C, preferably 120-140°C, more preferably 125-135°C. The outlet temperature can be set in the range of 60-80°C, preferably 65-70°C.

After spray drying, the purified target oligosaccharide may have the following properties:

i) solid granule shape; and/or

ii) glass transition temperature 60-90°C, preferably 62-88°C, more preferably 64-86°C, as determined by differential scanning calorimetry; and/or

iii) particle size 5-500 μm, preferably 10-300 μm, determined by laser diffraction; and/or

iv) an average particle size of 10-100 µm, preferably 20-90 µm, more preferably 30-80 µm, most preferably 40-70 µm, determined by laser diffraction; and/or

v) an amorphous state, preferably an amorphous state without characteristic peaks of a crystalline substance observed by X-ray powder diffraction; and/or

vi) moisture content 10 wt. % or less, preferably 8 wt. % or less, more preferably 5 wt. % or less.

The purified target oligosaccharide can be used for nutritional applications, preferably medical or dairy nutrition (eg, cereal products), and more preferably in infant nutrition or medicine, preferably in the prevention or treatment of gastrointestinal disorders.

In one embodiment, the target oligosaccharide is a neutral oligosaccharide or sialylated oligosaccharide, preferably a human milk oligosaccharide. The target oligosaccharide may be a neutral OGM or a sialylated OGM. In a further embodiment and/or alternative embodiment, the target oligosaccharide is selected from the group of human milk oligosaccharides consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto- N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N -tetraose, sialyllacto-N-tetraose c, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose and fucosyl-LST b.

The present invention will be described with respect to specific embodiments and with reference to drawings, but the invention is not limited thereto, but only by the claims. In addition, the terms first, second, and the like in the specification and claims are used to distinguish between like elements and not necessarily to describe sequence, in time, space, rank, or any other manner. It should be understood that the terms used in this manner are interchangeable in appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

It should be noted that the term “comprising” as used in the claims should not be considered limited to the means listed below; it does not exclude other elements or stages. Thus, it should be considered to determine the presence of the claimed features, integers, steps or components referred to, but it does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that for the purposes of the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to “one embodiment” or “embodiment” means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in different places throughout this specification do not necessarily all refer to the same embodiment, but may. Moreover, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Likewise, it should be understood that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in one single embodiment, figure, or description thereof for the purpose of simplifying the disclosure and aiding in understanding one or more of the various aspects of the invention. This manner of disclosure, however, should not be taken to imply that the claimed invention requires more features than are expressly listed in each claim. Rather, as reflected in the claims below, aspects of the invention are comprised of less than all of the features of the one disclosed embodiment set forth above. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing alone as a separate embodiment of the invention.

In addition, while some embodiments described herein include some, but not all, of the features included in other embodiments, it is understood that combinations of features of different embodiments are within the scope of the invention and form different embodiments, as will be appreciated by those skilled in the art. areas. For example, in the claims below, any of the claimed embodiments can be used in any combination.

In addition, some of the embodiments are described herein as a method or combination of method elements that may be implemented by a computer system processor or other means of performing a function. Thus, a processor with the necessary instructions for implementing such method or method element constitutes means for implementing the method or method element. In addition, the apparatus embodiment described herein is an example of a means of implementing the function performed by the apparatus for the purpose of carrying out the invention.

Numerous specific details are set forth in the descriptions and graphics provided herein. However, it will be understood that embodiments of the invention may be practiced without these specific details. In other examples, well-known methods, structures and techniques have not been shown in detail so as not to obscure the understanding of this description.

The invention will now be described by way of detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be put together in accordance with the knowledge of those skilled in the art without deviating from the true spirit or technical idea of the invention, the invention being limited only by the terms of the appended claims.

Example 1: Purification of 3-fucosyllactose obtained from bacterial fermentation

OGM-3-fucosyllactose was produced by bacterial fermentation, and bacteria were removed from the fermentation broth by filtration. To remove bacterial cells, the fermentation broth was filtered by microfiltration using a polyethersulfone membrane having a nominal pore size of 0.05 μm (NADIR® MP005; Microdyn-Nadir, Wiesbaden) and subsequent ultrafiltration using a hollow fiber module (ULTRADYN FS-10-FS FUS-1582, MWCO 150 kDa; Microdyn-Nadir, Wiesbaden, Germany).

The cell-free broth was then processed through diafiltration with a nanofiltration step to remove salts and smaller molecules, increasing the purity of 3-FL. The reverse osmosis system, type RO40404 (Aqmos GmbH, Rodgau, Germany), was equipped with Filmtec™ NF270 nanofiltration modules. The inlet pressure was set at 800 kPa and the solution was treated three times with an equal volume of reverse osmosis water to increase the concentration by halving the original volume. The 3-FL solution was then passed through a FS-10-FS FUS018110 kDa hollow fiber nanofiltration module (Microdyn-Nadir) to remove proteins and peptides.

Ions similar in size to 3-FL were removed from the process stream by electrodialysis using a PCCell P15 system (PCCell GmbH, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane stack containing a CEM:PCSK cation exchange membrane and a CEM:PcAcid60 anion exchange membrane size separation limit is 60 Da. The conductivity of the initial solutions ranged from 8 to 11 mS/cm ² and electrodialysis continued until the conductivity dropped to 0.5 mS/cm ² .

The 3-FL solution was then treated with powdered activated carbon to remove pigments. The solution was stirred for 2 hours with Norit DX1 activated carbon, which was then removed by filtration.

Another round of electrodialysis was performed using the same setup as described above. The conductivity of the initial solution was 1.0-1.5 cS/cm ² , and electrodialysis continued until the conductivity dropped to 0.3 mS/cm ² .

After electrodialysis, the 3-FL solution was concentrated by reverse osmosis using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau, Polch, Germany) equipped with a CSM RE8040BE reverse osmosis module. The solution was concentrated until the flow rate of the filtration system fell below 50 L/h. The dry matter after concentration was 20-25 wt. %. For spray drying (see Example 2), the 3-FL solution was further concentrated using a Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach, Germany) to 45 wt. % dry matter. The highly concentrated 3-FL solution was filter sterilized to remove endotoxins by passing it through a 5 kDa Spira-Cell WY UP005 2440 C ultrafiltration membrane (Microdyn-Nadir).

HPLC chromatograms illustrating the purification of 3-fucosyllactose from clarified fermentation broth by this method are shown in FIG. 6 and Fig. 7. In FIG. 6 shows the HPLC chromatogram of the clarified fermentation broth, while FIG. 7 shows an HPLC chromatogram of the sterile filtered process stream obtained from the clarified fermentation broth, resulting in the HPLC chromatogram shown in FIG. 6. Comparison of the chromatogram data reveals that most of the peaks in the HPLC chromatogram (each peak representing at least one compound) of the clarified fermentation broth are not present in the HPLC chromatogram of the sterile filtered process stream.

Example 2: Preparation of 3-fucosyllactose in solid form by spray drying

The 3-FL solution obtained by filtration and electrodialysis was concentrated to 45 wt. % and filter sterilized to remove any bioburden and endotoxins as described in Example 1. The highly concentrated and sterile 3-FL solution was then spray dried using an LTC-GMP spray dryer (Nubilosa, Konstanz, Germany). Solution 3-FL, 45 wt. %, was passed through the nozzles of a spray dryer at 130°C and 350 kPa, and the flow was adjusted to maintain the exhaust temperature at 66-67°C. Using these setups, a spray-dried powder containing less than 5% moisture was obtained. Moisture content was determined by Karl Fischer titration.

Claims (37)

1. A method for purifying a target oligosaccharide from a fermentation broth, including: - providing a fermentation broth that contains the target oligosaccharide, biomass, microbial cells and carbohydrates other than the target oligosaccharide; - removing microbial cells from the fermentation broth, thereby obtaining a process stream; - subjecting the process stream to a first stage of filtration using a nanofiltration membrane, thereby obtaining a filtrate that contains the target oligosaccharide; - subjecting the filtrate to a second stage of filtration using a nanofiltration membrane, thereby obtaining a retentate that contains the target oligosaccharide; And - removing salts from the process stream using electrodialysis, thereby obtaining a purified preparation of the target oligosaccharide, where the target oligosaccharide is a neutral human milk oligosaccharide (HMO) or sialylated HMO, wherein said method does not include an ion exchange step, and wherein the purified oligosaccharide solution is spray dried after filter sterilization. 2. The method of claim 1, wherein the microbial cells are removed from the fermentation broth by subjecting the fermentation broth to at least one centrifugation step and/or at least one filtration step. 3. The method of claim 1 or 2, wherein at least one filtration step is microfiltration, preferably microfiltration using a membrane that has a molecular weight cutoff of about 500 kDa, more preferably a molecular weight cutoff of about 150 kDa. 4. The method of claim 3, wherein the process stream is subjected to at least one ultrafiltration step using a membrane having a molecular weight cutoff of about 50 kDa, preferably a molecular weight cutoff of about 30 kDa, more preferably a molecular weight cutoff 10 kDa. 5. Method according to any one of paragraphs. 1-4, wherein the nanofiltration membrane used in the first filtration step has a molecular weight cutoff of from about 700 daltons to about 3000 daltons, preferably a molecular weight cutoff of from about 1000 to about 2000 daltons. 6. Method according to any one of paragraphs. 1-5, wherein the nanofiltration membrane used in the second filtration step has a molecular weight cutoff of from 100 daltons to 1000 daltons, preferably a molecular weight cutoff of from about 150 daltons to about 500 daltons, more preferably a molecular weight cutoff from about 200 to about 300 daltons. 7. Method according to any one of paragraphs. 1-6, wherein the target oligosaccharide is an OGM selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N -neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I , lacto-N-difucohexaose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaoses, difucosyl-lacto-N-neohexaoses, 3'-sialyllactoses, 6'-sialyllactoses, lacto-N-sialylpentaoses a, lacto-N-sialylpentaoses b, lacto-N-sialylpentaoses c, fucosyl-lacto-N-sialylpentaoses a, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-tetraose, disialyl-lacto-N-fucopentaose, sialyl lacto-N-tetraose a, sialyl lacto-N-tetraose, sialyl lacto-N -tetraose c, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyllactose, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose, fucosyl-LST b and lacto-N-neodifucohexaose I. 8. Method according to any one of paragraphs. 1-7, in which the process stream is subjected to a second stage of filtration such that: i) the amount of salt in the retentate is less than 10% by weight, preferably less than 5% by weight, more preferably 2% by weight or less and/or ii) the conductivity is from 0.5 to 10.0 mS/cm ² , preferably from 1 to 8 mS/cm ² , more preferably from 1.5 to 4.0 mS/cm ² . 9. Method according to any one of paragraphs. 1-8, wherein the process stream is subjected to a concentration step, preferably concentration through reverse osmosis and/or an additional nanofiltration step, preferably through a nanofiltration step using a nanofiltration membrane that has a molecular weight cutoff in the range of 200 to 300 Da. 10. The method according to claim 9, in which the concentration step is carried out - by nanofiltration at a temperature of less than 80°C, preferably less than 50°C, more preferably from 4°C to 45°C, more preferably from 10°C to 40°C, even more preferably from 15°C to 30°C, most preferably 15°C to 20°C; and/or - by reverse osmosis at a temperature of from 20°C to 50°C, more preferably from 30°C to 45°C, most preferably from 35°C to 45°C; and/or - at a pressure from more than 500 kPa to less than 5000 kPa, preferably at a pressure from more than 1000 kPa to less than 4000 kPa, more preferably at a pressure from more than 1500 to less than 3000 kPa. 11. The method of claim 9 or 10, wherein the process stream is subjected to a concentration step such that the concentration of the target oligosaccharide is 100 g/L or more, preferably 150 g/L or more, more preferably 200 g/L or more. 12. Method according to any one of paragraphs. 1-11, wherein the process stream is subjected to a dye removal step, preferably by treating the process stream with activated carbon. 13. The method according to claim 12, in which the stage of removing dyes is carried out i) before or after the diafiltration stage; ii) before or after the process stream concentration step; and/or iii) before or after the electrodialysis stage. 14. Method according to any one of paragraphs. 1-13, wherein the electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions. 15. Method according to any one of paragraphs. 1-14, in which the process stream, after removing the salts contained therein, i) contains an amount of salt that is less than 1 wt%, preferably less than 0.5 wt%, more preferably less than 0.2 wt%; and/or ii) has a conductivity of 0.05 to 1.0 mS/cm ² , preferably 0.1 to 0.5 mS/cm ² , more preferably 0.2 to 0.4 mS/cm ² . 16. Method according to any one of paragraphs. 1-15, wherein the process stream is spray dried, preferably spray dried i) at an inlet temperature in the range of 110-150°C, preferably 120-140°C, more preferably 125-135°C; and the outlet temperature may be in the range of 60-80°C, preferably 65-70°C; and/or ii) at a concentration of the target oligosaccharide in the process stream of 5-60 wt%, preferably 10-50 wt%, more preferably 15-45 wt%. 17. Method according to any one of paragraphs. 1-16, wherein the purity of the target oligosaccharide in the purified preparation is 80 wt.% or more, preferably 85 wt.% or more, more preferably 90 wt.% or more, based on the dry matter of the purified preparation.

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