Purification of oligosaccharides from fermentation broth by using filtration

Abstract

A method for purifying oligosaccharides, preferably neutral human milk oligosaccharides (HMOs) or sialylated HMOs, from a fermentation broth, the method comprising: - providing a fermentation broth containing the target oligosaccharide, biomass, microbial cells, and carbohydrates other than the target oligosaccharide; - removing the microbial cells from the fermentation broth to provide a process stream; - subjecting the process stream to a first filtration step using a nanofiltration membrane to provide a filtrate containing the target oligosaccharide; - subjecting the filtrate to a second filtration step using a nanofiltration membrane to provide a retentate containing the target oligosaccharide; and - removing salts from the process stream using electrodialysis to provide a purified preparation of the target oligosaccharide.

Description

[Technical field]

[0001] 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 the desired oligosaccharides from the fermentation broth by using filtration methods. [Background technology]

[0002] Human milk is a complex mixture of sugars (carbohydrates), fats, proteins, vitamins, minerals and trace elements. The carbohydrate fraction is the most abundant solid fraction and can be further divided into lactose and more complex oligosaccharides, the so-called human milk oligosaccharides (HMOs).

[0003] Lactose is used as an energy source, but complex oligosaccharides are not metabolized by infants or adults. The complex oligosaccharide fraction represents up to 10% of the total carbohydrate fraction and probably consists of more than 150 different oligosaccharides. The presence and concentration of these complex oligosaccharides is unique to humans and therefore they are not found in significant amounts in the milk of other mammals such as domestic dairy animals. Although HMOs are only a minor fraction of total human milk, their highly beneficial effects on the development of breast-fed infants have become evident during the past few decades.

[0004] The most prominent HMO is 2'-fucosyllactose. Other prominent HMOs are 3-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose. Besides these neutral oligosaccharides, acidic HMOs are also found in human milk, such as 3'-sialyllactose, 6'-sialyllactose and sialyllactose-N-tetraose, e.g. LST-a, LST-b and LST-c (Table 1). Up to 20% of the total HMO content of human milk is acidic due to the presence of at least one sialic acid moiety. These structures are closely related to epitopes of epithelial cell surface glycoconjugates (Lewis blood group antigens), and the structural homology between HMOs and epithelial epitopes explains the protective ability of HMOs against bacterial pathogens (Weichert et al. 2013, Nutr. Res. 33:831; Weichert et al. 2016, J. Virol. 90:4843).

[0005] The presence of complex oligosaccharides in human milk has been known for a long time, and the physiological functions of HMOs have been the subject of medical research for 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). Specific functions have already been identified for some of the more abundant HMOs (Bode 2012, Glycobiology 22:147; Bode & Jantscher-Krenn 2012, Adv. Nutr. 3S:383; Morrow et al. 2004, J. Pediatr. 145:297). There is a great demand for including HMOs in food, especially infant nutrition products, due to their physiological ability to inhibit infectious agents (bacteria, viruses and bacterial toxins), their positive effects on brain development and their prebiotic functions. Table 1 provides an overview of HMOs that can be purified from fermentation broths by the methods disclosed herein.

[0006] [Table 1-1]

[0007] [Table 1-2]

[0008] [Table 1-3]

[0009] Table 1. List of human milk oligosaccharides that can be purified by the methods disclosed herein. The limited supply of individual HMOs and the inability to source these molecules in sufficient quantities have led to the development of methods based on chemical synthesis to produce them. However, chemical synthesis of HMOs, and in particular the large-scale production of HMOs in sufficient quantities for food applications, has proven to be extremely challenging. In particular, chemical synthesis of HMOs such as 2'-FL (WO2010 / 115935 A) requires the use of several hazardous chemicals, which can contaminate the final product.

[0010] Due to the shortcomings of chemical synthesis, several methods based on enzymes and fermentation have been developed (Miyazaki et al. 2010, Methods Enzymol. 480:511; Murata et al. 1999, Glycoconj. J. 16:189; Baumgartner et al. 2013, Microb. Cell Fact. 12:40; 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 contaminated with starting materials such as lactose as well as intermediates, unwanted by-products (e.g., from the side activities of certain glycosyltransferases) and substrates from the fermentation process, such as proteins, polypeptides, organic acids, and salts.

[0011] Many current methods for purifying individual oligosaccharides from mixtures are technically complex, difficult to scale, and uneconomical for food applications. Industrial-scale methods have been developed for purifying the disaccharides lactose and sucrose from whey and molasses, respectively, but these methods require multiple laborious crystallization steps and achieve low yields. However, both whey and molasses are "food-grade" starting materials and are far less complex and demanding (in terms of regulatory requirements) than fermentation broths containing recombinant bacteria or yeast. Gel filtration chromatography is the method of choice for the purification of complex oligosaccharides such as HMOs produced by microbial fermentation, but the disadvantages of this method include its lack of scalability and incompatibility with continuous processing. Thus, gel filtration chromatography is uneconomical and cannot be used to produce HMOs on an industrial scale.

[0012] Theoretically, gel filtration chromatography is the best method for the purification of complex oligosaccharides such as HMOs produced by microbial fermentation, but the disadvantages of gel filtration chromatography include its lack of scalability and incompatibility with continuous processing, etc. Therefore, gel filtration chromatography is uneconomical and cannot be used to produce HMOs on an industrial scale.

[0013] Following the development of an efficient method for the production of HMOs by microbial fermentation, the next logical step was to develop an efficient downstream process for the isolation of HMOs from the culture broth. WO2015 / 049331 A discloses a method for purifying neutral HMOs. The method utilizes ion exchange, electrodialysis and simulated moving bed (SMB) chromatography to achieve continuous and efficient purification of large amounts of HMOs. Unlike chemical synthesis routes for producing neutral HMOs and their subsequent purification, the described fermentation and purification method allows the provision of HMOs free of harmful chemicals such as trace heavy metals and organic solvents. The purification method provides a high-purity HMO product that can be used for food applications as a solid (by spray drying) or concentrated syrup.

[0014] WO2015 / 106943 A describes a simple method for purifying neutral HMO produced by microbial fermentation. The method allows for efficient purification of large amounts of neutral HMO by using a combination of cation exchange, anion exchange and nanofiltration and / or electrodialysis. Unlike earlier purification methods for neutral HMO produced by microbial fermentation, the method does not include a chromatographic separation step. The method obtains solid HMO as a spray-dried material, as a crystalline material, or as a filter-sterilized concentrate. The obtained HMO is free of proteins and materials derived from the recombinant microbial strain used for production, making it ideal for food, medical food and feed (e.g., pet food) applications.

[0015] WO2016 / 095924 A describes a method for the purification of 2'-FL by crystallization. HMO was produced by microbial fermentation and, after removal of the biomass, concentrated by nanofiltration and electrodialysis. Finally, 2'-FL was selectively crystallized with acetic acid from an aqueous solution that also contained 2',3-di-O-fucosyllactose (DFL).

[0016] LNT and LNnT can be separated from the carbohydrate by-products by selective binding to activated carbon followed by elution with organic solvents and separation by gel filtration chromatography (Priem et al. 2002, Glycobiology 12:235; Gebus et al. 2012, Carbohydr. Res. 361:83; Baumgartner et al. 2014, ChemBioChem 15:1896). As mentioned above, gel filtration chromatography is a convenient laboratory-scale method but cannot be efficiently scaled up for industrial production.

[0017] WO2015 / 049331 A discloses a sequence of operations for purifying LNT produced by bacterial fermentation to provide a clear, particle-free solution: electrodialysis, nanofiltration, simulated moving bed chromatography (SMB) using a strong cation exchange resin, electrodialysis, ultrafiltration and a second SMB chromatography step.

[0018] Various methods have also been described for the isolation of acidic HMOs, in particular sialylated lactose or sialylated oligosaccharides. US2007 / 0020736 A discloses the production of 3'-SL with di- and tri-sialylated lactose as a by-product of a genetically engineered E. coli strain. 3'-SL accumulated in the culture broth to a concentration of ~0.8 mM. 3'-SL was purified using the following steps: centrifugation, adsorption of the supernatant through activated charcoal and washing off the water-soluble salts with distilled water, gradient elution with aqueous ethanol, and finally separation and desalting on a Biogel column. The yield from 1 L of broth was 49 mg of 3'-SL.

[0019] Another disclosed method involves the production of 3'-SL using a genetically engineered E. coli strain followed by heat permeabilization of the cells and subsequent isolation by centrifugation (Priem et al. 2002, Glycobiology 12:235). The material in the supernatant was adsorbed onto activated charcoal and the water-soluble salts were washed away with distilled water. After gradient elution with aqueous ethanol, 3'-SL was purified by HClO3. - The 3'-SL was adsorbed onto a strong anion exchanger in the (bicarbonate) form and eluted with a linear gradient of sodium bicarbonate (NaHCO3), the latter of which was removed by cation exchange (using the resin in the acid form) to give 49% recovery of 3'-SL.

[0020] An alternative procedure starting from the fermentation broth included the steps of heat permeabilization, centrifugation, adjustment of the pH to 3.0 by addition of a strong cation exchange resin in its acidic form, and removal of precipitated proteins by centrifugation (Fierfort et al. 2008, J. Biotechnol. 134:261). The pH of the supernatant was then adjusted to 6.0 by addition of a weak anion exchanger in its basic form, and 3'-SL was eluted with HCO3 - The 3'-SL was bound to an anion exchanger in the acid form. After washing with distilled water and subsequent elution with a continuous NaHCO3 gradient, NaHCO3 was removed by cation exchange (using the acid form of the resin) until the pH was lowered to 3.0. Finally, the pH was adjusted to 6.0 with NaOH. This purification method achieved a recovery efficiency of 59% for 3'-SL.

[0021] Enzymatically produced 3'-SL has also been separated from lactose by nanofiltration (Nordvang et al. 2014, Separ. Purif. Technol. 138:77). The authors showed that most of the 3'-SL could be separated from lactose after diafiltration using two different nanofiltration membranes, one with a molecular weight cut-off (MWCO) of 600-800 Da (sulfonated polyethersulfone (SPES) membrane) and the other with a MWCO of 1000-1400 Da (SPES membrane). However, there was a significant loss of 3'-SL during this process, and the purity was low, requiring an additional ion-exchange purification step.

[0022] WO2010 / 106320 A2 describes a method for concentrating 3'-SL from whey. First, proteins are removed by ultrafiltration, then the clarified whey permeate is incubated with an ion exchange resin to capture the 3'-SL. After elution from the ion exchange material, the concentrated 3'-SL fraction is concentrated by nanofiltration and the concentrate is desalted. After desalting, the 3'-SL is concentrated and dried to obtain a final dry product with a 3'-SL content of 20 wt%.

[0023] WO2018 / 020473 A describes an additional method for concentrating 3'-SL and 6'-SL from liquid feedstocks. The two HMOs were isolated from the mother liquor of lactose crystallization by heating the solution, enzymatic treatment and additional ultrafiltration and nanofiltration steps. After concentration, the content of 3'-SL and 6'-SL was 10-30 wt% of the dry mass.

[0024] Starting from this prior art, it was an object to provide a method for purifying target oligosaccharides, in particular neutral HMOs or sialylated HMOs, produced by microbial fermentation, which purification method is easy to scale up, suitable for commercial or industrial scale production of said target oligosaccharides, and which can result in a product with a purity that allows it to be made into a product suitable for human consumption.

[0025] It must be emphasized that microbial fermentation broths, especially those containing recombinant microorganisms (bacteria or eukaryotic microorganisms such as baker's yeast), are much more complex than product streams derived from dairy products. For example, whey has a composition of ~94% water, 4-5% lactose, 0.5-1% protein and very few well-defined minerals such as calcium, potassium and phosphorus as well as some vitamins, a simple matrix that is only concentrated and desalted in dairy process streams. In contrast, the matrix of sugar solutions obtained from recombinant microbial fermentation processes is highly complex, starting with the legal requirements to separate the recombinant biomass and inactivate it according to government regulations. The resulting clarified broth is an ill-defined matrix that also contains various salts and ions and heavy metals and trace elements. Moreover, a challenge with such liquids is the removal of cell debris, membrane fragments, e.g. lipids, proteins, molecules derived from microbial cell metabolism, and especially DNA. The recovery of oligosaccharides produced with the help of recombinant processes such as genetically modified bacteria is therefore even more difficult compared to whey and dairy streams. This is because the contaminants in the broth are very diverse in terms of molecular weight, charged molecules (single and multiply charged) and colored molecules. [Prior art documents] [Patent documents]

[0026] [Patent Document 1] International Patent Application Publication No. 2010 / 115935 A [Patent Document 2] U.S. Patent No. 7,521,212 B1 [Patent Document 3] International Patent Application Publication No. 2015 / 049331 A [Patent Document 4] International Patent Application Publication No. 2015 / 106943 A [Patent Document 5] International Patent Application Publication No. 2016 / 095924 A [Patent Document 6] International Patent Application Publication No. 2015 / 049331 A [Patent Document 7] US Patent Application Publication No. 2007 / 0020736 A [Patent Document 8] International Patent Application Publication No. 2010 / 106320 A2 [Patent Document 9] International Patent Application Publication No. 2018 / 020473 A [Non-patent literature]

[0027] [Non-Patent Document 1] Weichert et al. 2013, Nutr. Res. 33:831 [Non-Patent Document 2] Weichert et al. 2016, J. Virol. 90:4843 [Non-Patent Document 3] Gura 2014, Science 345:747 [Non-Patent Document 4] Kunz & Egge 2017, In McGuire, McGuire & Bode (eds.) Prebiotics and Probiotics in Human Milk, Elsevier, London, pp. 3-16 [Non-Patent Document 5] Bode 2012, Glycobiology 22:147

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[0028] The present invention provides a method for purifying target oligosaccharides produced by fermentation in a batch or continuous manner from a culture or fermentation broth obtained by microbial fermentation using a recombinant fermentation strain. The aforementioned purification methods often use expensive ion exchange steps (requiring both cation and anion exchangers). Ion exchangers cannot be operated continuously since they require regeneration. The culture broth contains the target oligosaccharides, biomass, medium components, salts, and other contaminating substances such as acids and pigments.

[0029] During the purification process, the culture broth can undergo the following purification steps to obtain the desired oligosaccharides. 1) Separation of microbial cells from culture broth by microfiltration 2) Separation of proteins from clarified culture broth (= process stream) by ultrafiltration 3) A first nanofiltration step to remove peptides and high molecular weight (HMW) impurities 4) A second nanofiltration step to remove water and salts 5) Activated carbon treatment to remove pigments and other impurities 6) Electrodialysis or diafiltration using nanofiltration membranes for complete removal of contaminating salts 7) Water removal by reverse osmosis Additionally, an additional electrodialysis step can be introduced prior to the activated carbon treatment to reduce the amount of contaminating ions. After purification in aqueous solution, the material can be spray-dried or granulated to provide the desired oligosaccharides in solid form. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows a process scheme of an exemplary embodiment of a method according to the present invention. [Diagram 2] FIG. 2 shows a process scheme of another exemplary embodiment of the method according to the present invention. [Diagram 3] FIG. 3 shows a process scheme of another exemplary embodiment of the method according to the present invention. [Figure 4] FIG. 4 shows a process scheme of another exemplary embodiment of the method according to the present invention. [Diagram 5] FIG. 5 shows a process scheme of another exemplary embodiment of the method according to the present invention. [Figure 6] FIG. 6 shows an HPLC chromatogram of clarified fermentation broth containing 3-fucosyllactose. [Figure 7] FIG. 7 shows an HPLC chromatogram of 3-fucosyllactose after purification from fermentation broth by an exemplary embodiment of the method according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] According to the present invention, the term "purity" refers to chemical purity and defines the extent to which a substance such as 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or any other oligosaccharide of interest (Table 1) is not diluted or mixed with extraneous substances. Thus, chemical purity is a measure of the relationship between a single substance and any by-products / impurities. Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

[0032] Percent Purity = [mass of desired product / total mass of sample] x 100 The purity of the compound in a composition containing the desired oligosaccharide can be determined by any suitable method known to those skilled in the art, for example by high performance liquid chromatography (HPLC). Suitable detectors can be selected from the group consisting of electrochemical detectors, refractive index (RI) detectors, mass spectrometers (MS), diode array detectors (DAD), and nuclear magnetic resonance (NMR) detectors. For example, in HPLC, the purity can be determined by calculating the ratio of the area under the target peak (representing the amount of the desired oligosaccharide) to the sum of the areas under all peaks (representing both the amount of the desired oligosaccharide and compounds different from this substance in the same chromatogram). However, this means that all impurities can be analyzed by the selected HPLC method. Otherwise, a mass balance method is required, i.e., absolute quantification of the desired product. In said technique, a pure substance is used as a standard for the quantification of purity, against which the dry substance obtained from the product (desired product + total impurities) is determined. Said mass balance method can also be used to determine the purity according to the present invention.

[0033] According to the present invention, "culture broth" or "fermentation broth" refers to any liquid after fermentation that contains the 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or any other oligosaccharide of interest (Table 1) to be purified. The terms "culture broth", "fermentation broth" and "culture medium" are used synonymously herein. The culture broth contains the oligosaccharide of interest to be purified as well as biomass (e.g. biological cells and cell debris), medium components, salts, and further contaminating substances such as other acids and pigments. The biological cells contained in the culture broth are biological cells that produce the oligosaccharide of interest intracellularly and secrete this compound into the liquid medium. The biological cells can include or consist of genetically modified biological cells, for example genetically modified E. coli cells. The genetic modification can include or consist of modifications to produce the oligosaccharide of interest, particularly during the growth phase of said biological cells.

[0034] The term "biomass" as used herein refers to the totality of biological cells present in the fermentation broth at the end of the fermentation process. Biomass includes the microbial cells that produced the oligosaccharide of interest, the progeny of this microbial cell that may have lost the ability to produce the oligosaccharide of interest during the fermentation process, as well as any other cells that are unintentionally present in the fermentation broth at the end of the fermentation process. Thus, essentially all of the biological cells present in the fermentation broth at the end of the fermentation process are separated from the fermentation broth such that the clarified fermentation broth, known as the "process stream" (any solution that contains or contains the oligosaccharide of interest to be purified), is substantially or completely free of cells.

[0035] The biomass preferably comprises biological cells producing the oligosaccharide of interest, preferably bacterial cells producing the oligosaccharide of interest, more preferably recombinant bacterial cells producing the oligosaccharide of interest, and most preferably recombinant E. coli cells producing the oligosaccharide of interest.

[0036] The biomass and / or microbial cells can be removed from the fermentation broth by centrifugation and / or filtration. In a suitable centrifugation method for removing biomass from the culture broth, the biomass is obtained as a pellet and the supernatant as a clarified process stream, which is subjected to further processing. In a suitable filtration method for removing biomass from the culture broth, the filtrate is the clarified process stream. The preferred filtration methods for biomass removal are microfiltration and / or ultrafiltration, the latter being capable of removing smaller particles and larger molecules than microfiltration. Microfiltration and ultrafiltration can be operated in a dead-end filtration mode (process stream flows perpendicular to the filter) or in a cross-flow filtration mode (process stream flows parallel to the filter).

[0037] Microfiltration is a physical separation method in which a particle-containing liquid is passed through a medium that includes either a porous material containing tortuous channels to retain the particles (depth filtration) and / or a membrane with a specific pore size that allows particles / molecules smaller than that pore size to pass through (membrane filtration). As used herein, the term "microfiltration" refers to a physical separation method in which biological cells (and cell debris) are removed from a fermentation broth to leave a (clarified) process stream.

[0038] Suitable membranes for biomass removal by microfiltration can be hollow fiber or spiral-wound membranes that may have a pore size of at least 0.2 μm. Alternatively, biomass removal can be achieved by microfiltration using membranes with a MWCO of 100-1000 kDa, preferably 150-500 kDa, to remove biomass, additional cell debris and large proteins. For example, membranes such as TRISEP® DS MVP20 (pore size 0.2 μm) (Microdyn-Nadir GmbH, Wiesbaden, Germany), which are spiral-wound to provide a compact design and better performance, can be used to remove cells from culture broth. Also, spiral-wound membranes with pore sizes of 0.05-0.1 μm, such as TRISEP® DS MP005, a module containing a PES membrane and a nominal pore size of 0.05 μm, can be used for biomass and protein separation.

[0039] In addition, a PES membrane with a MWCO of 150 kDa (5 m 2 Alternatively, a hollow fiber module such as the FS10-FC FUS1582 (Microdyn-Nadir GmbH), which is a hollow fiber filtration module using a fluororesin, may be used.

[0040] In summary, the following possibilities for removing biomass from the fermentation broth are applicable to the present invention: i) Recovery by centrifugation Insoluble components are removed from the culture broth in one step. Advantages: Rapid removal of insoluble components.

[0041] ii) Recovery by microfiltration Insoluble components and large molecules above a certain size are removed from the culture broth in one step. Spiral membrane or hollow fiber cross-flow filters can be used for microfiltration with MWCO ≥ 500 kDa. Advantages: Rapid removal of insoluble components and large soluble molecules above a certain size.

[0042] iii) Recovery by a combination of centrifugation and microfiltration Insoluble components and large molecules above a certain size are removed from the culture broth in two steps. Spiral membrane or hollow fiber cross-flow filters with MWCO ≥ 500 kDa can be used for microfiltration. Advantages: Rapid removal of insoluble components and large molecules above a certain size without clogging of the microfiltration membrane.

[0043] Ultrafiltration is a form of membrane filtration that is not fundamentally different from microfiltration. In ultrafiltration, particles and large soluble molecules are removed by forcing a liquid containing such particles and large soluble molecules through a semipermeable membrane with forces generated by pressure and concentration gradients. Thereby, the particles and large soluble molecules remain in the so-called retentate, while water and low molecular weight (LMW) solutes, such as the neutral and acidic oligosaccharides produced, pass through the membrane into the permeate (filtrate). Membranes for ultrafiltration are defined by their MWCO. The MWCO represents the maximum molecular weight of soluble molecules that can pass through the membrane into the permeate. Any particles, like molecules larger than the MWCO, cannot pass through the membrane and remain in the retentate. Ultrafiltration can be applied in cross-flow mode, where the liquid flow is parallel to the membrane surface, or in dead-end mode, where the liquid flow is perpendicular to the membrane surface.

[0044] The clarified process stream containing the produced oligosaccharides of interest typically contains significant amounts of undesirable impurities, such as, but not limited to, monovalent ions, divalent ions, amino acids, polypeptides, proteins, organic acids, nucleic acids, monosaccharides and / or oligosaccharides.

[0045] Suitable membranes for the removal of most proteins by ultrafiltration have a MWCO of at least 50 kDa, preferably at least 30 kDa, more preferably at least 10 kDa, and can be hollow fiber or spiral wound membranes. For example, membranes such as SPIRA-CEL® DS UP010 (MWCO=10 kDa) or SPIRA-CEL DS UP005 (MWCO=5 kDa) (both sold by Microdyn-Nadir GmbH) are spiral wound to provide a compact design and better performance, and can be used to separate the remaining proteins from the cell-free culture broth.

[0046] Additionally, the ROMICON® HF UF Cartridge PM10 (Koch Membranes Systems, Wilmington, USA; max. 12 m 2 A hollow fiber module such as a PES membrane having an area of ​​100 μm and a MWCO of 10 kDa can alternatively be used.

[0047] Nanofiltration is a membrane filtration process in which the membrane contains nanometer-sized pores (1-10 nm). The pore size of nanofiltration membranes is smaller than that of microfiltration and ultrafiltration membranes, but larger than that 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 a pore density of 1 cm 2 1-10 per 6 It is a hole.

[0048] Nanofiltration is used in purification processes for target oligosaccharides to remove LMW impurities such as peptides and salts and to increase the concentration of oligosaccharides in the clarified process stream or eluate.

[0049] The molecular weight of most HMOs ranges from 400 to 1,200 Da. At least two nanofiltration steps are required to remove HMW impurities (>1200 Da) such as peptides and pigments, as well as LMW impurities (<400 Da) such as salts from the process stream.

[0050] After ultrafiltration, all molecules >5 kDa or >10 kDa (depending on the membrane used) should have been removed from the process stream. To remove impurities below this threshold, such as peptides and pigments, but still larger than the oligosaccharides of interest, a first nanofiltration step can be performed with a MWCO that allows the oligosaccharides to pass through to the permeate but the HMW impurities to remain in the retentate.

[0051] To separate the majority of the desired oligosaccharides from the HMW impurities and increase the yield of the desired oligosaccharides, the process should be run until >70%, preferably >80%, and more preferably >90% of the product passes through the membrane. Additionally, a diafiltration step can be included to increase the yield of oligosaccharides in the clarified process stream.

[0052] After this first nanofiltration step, the only impurities in the process stream containing the target oligosaccharides should be of the same or lower molecular weight as the oligosaccharides and may include monovalent ions, divalent ions, amino acids, organic acids, monosaccharides and / or oligosaccharides.

[0053] Suitable membranes for the first nanofiltration step include polyamide or polypiperazine thin film composite membrane materials that achieve size exclusion in the range of 400-1,200 Da, such as HYDRACoRe70 (MWCO=720 Da) or HYDRACoRe50 (MWCO=1,000 Da) membranes (both from Hydranautics, Oceanside, USA), NADIR® NP010P (MWCO=1,000-1,500 Da) or NADIR® NP030P (MWCO=500-1,000 Da) membranes (Microdyn-Nadir GmbH). Such membranes allow high flux rates. Additional examples of suitable membranes for nanofiltration include Trisep® SBNF (MWCO=2000 Da) cellulose acetate nanofiltration membranes (Microdyn-Nadir GmbH) and GE-series membranes (MWCO=1000 Da) from Suez Water Technologies & Solutions (Ratingen, Germany).

[0054] In additional and / or alternative embodiments, the first nanofiltration step achieves the following parameters: i) The post-filtration recovery of the desired oligosaccharides should be >70%, preferably >80%, and more preferably >90%.

[0055] ii) The concentration of the target oligosaccharides in the process stream should be <50% (w / v), preferably <40% (w / v), more preferably <30% (w / v), preferably <20% (w / v), more preferably <10% (w / v).

[0056] iii) the process should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for nanofiltration). iv) The process should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar.

[0057] In a preferred embodiment of the method, the process stream containing the target oligosaccharides is concentrated and desalted by applying a second nanofiltration step after the first nanofiltration step. The target oligosaccharides can also be concentrated by vacuum evaporation (e.g. using a falling film evaporator or a plate evaporator) or by reverse osmosis. The disadvantage of these two techniques is that the process stream is concentrated but not desalted. Nanofiltration is therefore a preferred method since it allows to achieve concentration and desalting simultaneously, e.g. by using membranes with a size exclusion limit <2 nm.

[0058] The method for purifying the target oligosaccharides comprises 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 (=filtrate or permeate) from the first nanofiltration step is directly subjected to a second nanofiltration step to remove salts and smaller molecules from the clarified process stream.

[0059] A suitable membrane for the first nanofiltration step is FilmtecTM Examples include polyamide or polypiperazine thin film composite membrane materials that achieve size exclusion in the range of 150-300 Da, such as NF270 (Dow Chemical Company, Midland, USA) and Trisep® XN45 or TS40 membranes (Microdyn Nadir GmbH). Such membranes allow high fluxes. Additional examples include Trisep® 4040-XN45-TSF (Microdyn-Nadir GmbH) or GE4040F30 and GH4040F50 membranes (Suez Water Technologies & Solutions).

[0060] Nanofiltration effectively removes significant amounts of salts and LMW impurities from process streams containing the target oligosaccharides prior to electrodialysis. Nanofiltration also effectively removes LMW contaminants after the ultrafiltration step. Removal of such contaminants is beneficial for concentrating and desalting solutions of the target oligosaccharides. By using nanofiltration to concentrate the target oligosaccharides, energy and processing costs are reduced and product quality is improved due to more limited heat exposure.

[0061] The method concentrates a target oligosaccharide from an aqueous solution, the concentration of the target oligosaccharide in the aqueous solution being ≦20%, ≦10%, or ≦5% before concentration. In additional and / or alternative embodiments, concentration by nanofiltration should achieve the following parameters:

[0062] i) the oligosaccharide concentration is >100 g / L, preferably >200 g / L, more preferably >300 g / L, and most preferably >400 g / L; ii) the amount of salt in the purification solution should be <10 wt%, preferably <5%, more preferably <2%; and / or the conductivity should be between 0.5 and 10.0 mS / cm 2 , preferably 1 to 8 mS / cm 2 , and more preferably 1.5 to 4.0 mS / cm 2 It should be.

[0063] iii) the process should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for nanofiltration). iv) The process should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar.

[0064] Dyes in the clarified and / or purified solutions can be removed by treatment with activated charcoal. The advantage of using activated charcoal to remove dyes is that it can remove both charged and uncharged (neutral) dyes.

[0065] Activated carbon (also called activated charcoal) is a form of carbon that has been processed to produce small, low-volume pores that increase the surface area available for adsorption. Typically, just 1 gram of activated carbon can absorb up to 30,000 m of water as measured by gas adsorption, due to its highly microporous structure. 2 It has a surface area of ​​more than

[0066] Colored 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 oligosaccharides produced, the amount of colored substances is rather low and / or in most cases they exhibit hydrophobic behavior. Due to this fact, colored contaminants have a high removal rate from the process stream. Water-soluble substances such as oligosaccharides are weakly bound and can be eluted by rinsing with water, leaving the pigments adsorbed on the surface.

[0067] In additional and / or alternative embodiments, the method for purifying target oligosaccharides further comprises at least one step of treating the clarified process stream or eluate from the previous purification step with activated carbon to remove pigments.

[0068] In a preferred embodiment, the activated carbon treatment comprises: i) after removing water and salts in a second nanofiltration step; and / or ii) after removal of proteins by ultrafiltration or after removal of residual salts by electrodialysis (alternatively a diafiltration step can also be introduced) It should be implemented.

[0069] Suitable activated carbons for removing neutral oligosaccharides, pigments and other contaminants include (but are not limited to) granular activated carbons such as Norit GAC830EN (Cabot Corporation, Boston, USA) and Epibon Y 12x40 spezial (Donau Carbon, Frankfurt, Germany), or powdered activated carbons such as Norit DX1, Norit SA2 (Cabot Corporation) and Carbopal MB 4 (Donau Carbon).

[0070] Electrodialysis is a combination of dialysis and electrolysis that can be used to separate and concentrate ions in a solution based on their selective electromigration through a semipermeable membrane. Industrial applications of electrodialysis date back to the early 1960s, when the process was used to demineralize cheese whey for inclusion in infant formula. Further applications include adjusting the pH of beverages such as wine, grape must (unfermented grape juice), apple juice and orange juice.

[0071] Desalination of brackish water for drinking water production and demineralization of whey for infant food production 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 for ion conduction and connected to a direct current generator. The electrode connected to the positive pole of the direct current generator is the anode, and the electrode connected to the negative pole is the cathode. The electrolyte solution supports the flow of electric current, which occurs as anions and cations migrate to the anode and cathode, respectively. The membranes used in electrodialysis are essentially sheets of porous ion exchange resins bearing negatively or positively charged groups, and are called cationic or anionic membranes, respectively. Ion exchange membranes usually consist of a polystyrene matrix carrying appropriate functional groups (e.g., sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes) cross-linked with divinylbenzene.

[0072] The electrolyte can be, for example, an aqueous solution containing sodium chloride, sodium acetate, sodium propionate and / or sulfamic acid. The electrolyte surrounds the cathode and anode and allows the current to flow through the electrolytic cell. The electrodialysis stack is then assembled between two electrode blocks with the anion and cation membranes parallel, as in a filter press. As a result, the streams undergoing ion depletion and ion concentration are well separated. The two solutions are also called the diluent (which undergoes ion depletion) and the concentrate (which undergoes ion concentration).

[0073] At the heart of electrodialysis is a membrane stack, consisting of several anion and cation exchange membranes placed between two electrodes and separated by spacers. By applying a direct current, anions and cations migrate across the membranes towards the electrodes, producing a (demineralized) diluate stream and a concentrate stream.

[0074] The pore size of the ion exchange membranes used in electrodialysis is small enough to prevent diffusion of product from the diluent stream into the concentrate stream (driven by the high concentration difference between the two streams).

[0075] Electrodialysis is used to remove ions from aqueous solutions while leaving neutral and acidic oligosaccharides in the process stream. An important advantage of electrodialysis is that it allows complete removal of recombinant DNA molecules from solutions containing the oligosaccharides of interest. Furthermore, the amount of salts in the process stream can be significantly reduced by electrodialysis. In fact, sodium chloride can be completely removed from the product stream. This has the advantage that solutions containing the oligosaccharides of interest can be provided free of salts such as sodium chloride, preventing the adverse effects of such salts in the final product, e.g., infant foods.

[0076] After nanofiltration and activated carbon treatment, most of the smaller organic impurities such as salts and organic acids should have been removed from the process stream. An electrodialysis step is performed to ensure effective removal of the remaining salts and small charged organic materials.

[0077] Electrodialysis ensures that the process stream has a stable conductivity of 0.05-1.0 mS / cm 2 , preferably 0.1 to 0.5 mS / cm 2 , and more preferably 0.2 to 0.4 mS / cm 2 Further, electrodialysis can be carried out until the salt concentration is reduced to <10.0 g / L, preferably <5.0 g / L, more preferably <1.0 g / L, and most preferably <0.2 g / L.

[0078] In the case of neutral oligosaccharides, the electrodialysis step should be carried out under acidic or neutral pH conditions, preferably at a pH between 3 and 8, more preferably at a pH between 4 and 7. In the case of acidic oligosaccharides, the electrodialysis step should be carried out under neutral pH conditions, preferably at a pH between 6 and 8, more preferably at a pH between 6.5 and 7.5, since acidic oligosaccharides are unstable under acidic conditions. The pH of the acidic oligosaccharide solution should be controlled during electrodialysis and adjusted with NaOH if necessary.

[0079] Instead of nanofiltration, a reverse osmosis step can also be used to concentrate the target oligosaccharides. Reverse osmosis is a membrane filtration method that concentrates particles larger than 0.1 nm in the retentate of the process stream while removing water. Thus, reverse osmosis concentrates the process stream but does not achieve desalting.

[0080] The method can concentrate target oligosaccharides in an aqueous or organic solvent, where the concentration of the target oligosaccharides is ≦20% (w / v), ≦10% (w / v), or ≦5% (w / v) prior to concentration. In additional and / or alternative embodiments, the concentration step should achieve the following parameters:

[0081] i) Oligosaccharide concentration >300 g / L, preferably >400 g / L, more preferably >500 g / L, most preferably >600 g / L. ii) The process should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for reverse osmosis).

[0082] iii) The process should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar. In additional and / or alternative embodiments of the method, the solution containing neutral and acidic HMOs is concentrated by vacuum evaporation (eg, using a rotary evaporator or plate evaporator) to achieve the following parameters:

[0083] i) Oligosaccharide concentration >300 g / L, preferably >400 g / L, more preferably >500 g / L, most preferably >600 g / L. ii) The process should be carried out at a temperature of <80°C, preferably <50°C, more preferably 20-50°C, even more preferably 30-45°C, most preferably 35-45°C (particularly suitable for vacuum evaporation).

[0084] To remove any possible microbial contamination and endotoxins, the concentrated target oligosaccharides are sterilized by filtration through an ultrafiltration membrane. In a preferred embodiment of the invention, the purified oligosaccharide solution is passed through a filter module of ≦10 kDa, for example a MWCO filter of 5 kDa or 3 kDa. Ultrafiltration membranes suitable for endotoxin removal have a MWCO of at least 10 kDa, preferably at least 5 kDa, and can be spiral-wound or hollow-fiber ultrafiltration membranes. Examples of spiral-wound membranes include SPIRA-CEL® DS UP010 (MWCO=10 kDa) or DS UP005 (MWCO=5 kDa) membranes (Microdyn-Nadir GmbH) and Dairy-Pro® HpHT UF-5K (MWCO=5 kDa) (Koch Membranes Systems). Examples of hollow-fiber modules include ROMICON® HF UF Cartridge PM5 (MWCO=5 kDa) (Koch Membranes Systems) or Microzoa TM series membranes (MWCO=3 or 6) (Pall Cooperation, Port Washington, USA).

[0085] In a preferred embodiment, the purified oligosaccharide solution is spray-dried after sterilization by filtration. The solution is spray-dried using hot air, preferably to remove at least 85 wt%, or more preferably at least 90 wt% of the water. The solution can be spray-dried using any conventional spray drying system, preferably with a fluidized bed dryer attachment.

[0086] The purified solution is spray-dried to achieve a target oligosaccharide concentration of 5 to 60 wt%, preferably 10 to 50 wt%, and more preferably 15 to 45 wt%. The inlet temperature can be maintained within a range of 110 to 150°C, preferably 120 to 140°C, and more preferably 125 to 135°C. The outlet temperature can be maintained within a range of 60 to 80°C, and preferably 65 to 70°C.

[0087] After spray drying, the purified target oligosaccharides can have the following properties: i) in solid granular form; and / or ii) a glass transition temperature of 60 to 90° C., preferably 62 to 88° C., and more preferably 64 to 86° C., as measured by differential scanning calorimetry; and / or iii) a particle size of 5 to 500 μm, preferably 10 to 300 μm, as measured by laser diffraction; and / or iv) an average particle size, as measured by laser diffraction, of 10 to 100 μm, preferably 20 to 90 μm, more preferably 30 to 80 μm, and most preferably 40 to 70 μm; and / or v) in an amorphous state, preferably free of peaks characteristic of crystalline material, as determined by X-ray powder diffraction; and / or vi) A moisture content of ≦10wt%, preferably ≦8wt%, more preferably ≦5wt%.

[0088] The purified oligosaccharides of interest can be used for nutritional purposes, preferably for medical or dairy nutrition (eg cereal products), more preferably for infant nutrition, or in medicine, preferably for the prevention or treatment of gastrointestinal disorders.

[0089] In an embodiment, the oligosaccharide of interest is a neutral oligosaccharide or a sialylated oligosaccharide, preferably a human milk oligosaccharide. The oligosaccharide of interest may be a neutral HMO or a sialylated HMO. In additional and / or alternative embodiments, the oligosaccharide of interest is 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, sialyllactose-N-tetraose. a, sialyllactose-N-tetraose, sialyllactose-N-tetraose c, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose and fucosyl-LST b.

[0090] Aspects of the invention also include the following. Aspect 1. A method for purifying an oligosaccharide of interest from a fermentation broth, the method comprising: - providing a fermentation broth containing the oligosaccharide of interest, biomass, microbial cells, and carbohydrates other than the oligosaccharide of interest; - providing a process stream by removing microbial cells from said fermentation broth; - subjecting said process stream to a first filtration step using a nanofiltration membrane to provide a filtrate containing the desired oligosaccharides; - subjecting the filtrate to a second filtration step using a nanofiltration membrane to obtain a retentate containing the desired oligosaccharides; - removing salts from said process stream using electrodialysis to provide a purified preparation of the desired oligosaccharides. The method includes: 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. A method according to any one of the preceding claims, wherein said at least one filtration step is microfiltration, preferably using a membrane having a molecular weight cut-off of about 500 kDa, more preferably a molecular weight cut-off of about 150 kDa. 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, and more preferably a molecular weight cutoff of 10 kDa. Aspect 5 The method according to any one of aspects 1 to 4, wherein the nanofiltration membrane used in the first filtration step has a molecular weight cutoff of about 700 Daltons to about 3,000 Daltons, preferably about 1,000 to about 2,000 Daltons. Aspect 6. The method according to any one of aspects 1 to 5, wherein the nanofiltration membrane used in the second filtration step has a molecular weight cutoff of 100 daltons to 1,000 daltons, preferably a molecular weight cutoff of about 150 daltons to about 500 daltons, and more preferably a molecular weight cutoff of about 200 to about 300 daltons. Aspect 7 The oligosaccharide of interest is a neutral or sialylated oligosaccharide, preferably a neutral or sialylated HMO, more preferably 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, sialyllactose-N-tetraose a, sialyllactose-N-tetraose, sialyllactose-N-tetraose c, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose and fucosyl-LST b, or any other HMO listed in Table 1. 8. The process stream comprising: i) the amount of salt in the retentate is <10 wt%, preferably <5 wt%, more preferably ≦2 wt%, and / or ii) Conductivity is 0.5 to 10.0 mS / cm 2 , preferably 1 to 8 mS / cm 2 , and more preferably 1.5 to 4.0 mS / cm 2 The method according to any one of aspects 1 to 7, wherein the mixture is subjected to a second filtration step so that the mixture becomes A method according to any of the preceding claims, wherein the process stream is subjected to a concentration step, preferably by reverse osmosis and / or an additional nanofiltration step, preferably by a nanofiltration step using a nanofiltration membrane having a molecular weight cut-off in the range of 200-300 Da. Aspect 10, wherein the concentrating step comprises: by nanofiltration at a temperature of <80°C, preferably <50°C, more preferably between 4°C and 45°C, more preferably between 10°C and 40°C, even more preferably between 15°C and 30°C, most preferably between 15°C and 20°C; and / or by reverse osmosis, at a temperature between 20°C and 50°C, more preferably between 30°C and 45°C, most preferably between 35°C and 45°C; and / or - At pressures of >5bar to <50bar, preferably at pressures of >10bar to <40bar, more preferably at pressures of >15bar to <30bar 10. The method of embodiment 9, wherein the method is carried out. Aspect 11. The method according to aspect 9 or 10, wherein the process stream is subjected to a concentration step to a concentration of the oligosaccharides of interest of ≧100 g / L, preferably ≧150 g / L, more preferably ≧200 g / L. Aspect 12. The method of any of aspects 1 to 11, wherein the process stream is subjected to a dye removal step, preferably by treating the process stream with activated carbon. Aspect 13, the dye removal step comprising: i) before or after the diafiltration step; ii) before or after a concentration step of the process stream; and / or iii) Before or after the electrodialysis step 13. The method of embodiment 12, wherein the method is performed on Aspect 14 The method according to any one of aspects 1 to 13, wherein the electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions. Aspect 15: The process stream, after removal of salts contained therein, i) containing salt in an amount of <1 wt%, preferably <0.5 wt%, more preferably <0.2 wt%; and / or ii) 0.05-1.0 mS / cm 2 , preferably 0.1 to 0.5 mS / cm 2 , and more preferably 0.2 to 0.4 mS / cm 2 having a conductivity of A method according to any one of aspects 1 to 14. 16. The process stream is spray dried, preferably i) at an inlet temperature in the range of 110-150°C, preferably 120-140°C, more preferably 125-135°C (with an outlet temperature in the range of 60-80°C, preferably 65-70°C); and / or ii) The concentration of the target oligosaccharide in the process stream is 5 to 60 wt%, preferably 10 to 50 wt%, more preferably 15 to 45 wt%. 16. The method according to any one of the preceding aspects, wherein the composition is spray dried. Aspect 17. The method of any of the preceding aspects, wherein the purity of the oligosaccharide of interest in the purified preparation is ≧80 wt%, preferably ≧85 wt%, and more preferably ≧90 wt%, based on the dry matter of the purified preparation. The present invention will be described with respect to certain embodiments and with reference to the drawings, but the present invention is not limited thereto, but only by the claims. Moreover, terms such as first, second, etc. in the description and claims are used to distinguish similar elements, and do not necessarily describe an order in time, space, ranking, or any other manner. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the aspects of the invention described herein can be operated in other orders than those described or illustrated herein.

[0091] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. Thus, it should be interpreted as embodying the presence of a specified feature, integer, step or element referred to, but without excluding the presence or addition of one or more other features, integers, steps or elements, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting of only elements A and B. It means that in the context of the present invention, the only relevant elements of the device are A and B.

[0092] Throughout this specification, a reference to "one embodiment" or "an embodiment" means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to one of ordinary skill in the art from this disclosure.

[0093] Similarly, in describing exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof to streamline the disclosure and aid in understanding one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0094] Furthermore, some embodiments described herein include some features (but not other features) included in other embodiments, but a combination of features of different embodiments is meant to be within the scope of the invention as understood by one of ordinary skill in the art to form different embodiments. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0095] Moreover, some of the embodiments are described herein as methods or combinations of method elements that can be implemented by a processor of a computer system or by other means for implementing that functionality. Thus, a processor with the necessary instructions for implementing such a method or method element forms a means for implementing the method or method element. Furthermore, the elements described herein of the apparatus aspects are examples of means for performing the functions implemented by the elements to implement the invention.

[0096] Numerous specific details are set forth in the description and drawings provided herein. However, it should be understood that aspects of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0097] The present invention will now be described by describing in detail some embodiments of the present invention. Other embodiments of the present invention can be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and it is apparent that the present invention is limited only by the provisions of the appended claims. EXAMPLES

[0098] Example 1: Purification of 3-fucosyllactose from bacterial fermentation The HMO 3-fucosyllactose was produced by bacterial fermentation, and the bacteria were removed from the fermentation broth by filtration. To remove bacterial cells, the fermentation broth was filtered by microfiltration using a polyethersulfone membrane with a nominal pore size of 0.05 μm (NADIR® MP005; Microdyn-Nadir, Wiesbaden, Germany) followed by ultrafiltration using a hollow fiber module (ULTRADYN FS-10-FS FUS-1582, 150 kDa MWCO; Microdyn-Nadir, Wiesbaden, Germany).

[0099] The cell-free broth was then treated by diafiltration using a nanofiltration step to remove salts and small molecules to increase the purity of 3-FL. TM The system was equipped with a NF270 nanofiltration module. The inlet pressure was set at 8 bar and the solution was thickened by treating it three times with an equal volume of reverse osmosis water to half 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.

[0100] Ions of similar size to 3-FL were removed from the process stream by electrodialysis using a PCCell P15 system (PCCell GmbH, Heusweiler, Germany). The system was equipped with a PCCell ED 1000A membrane stack containing a CEM:PCSK cation exchange membrane and a CEM:PcAcid60 anion exchange membrane with a size exclusion limit of 60 Da. The conductivity of the starting solution was 8–11 mS / cm. 2 and the conductivity is 0.5mS / cm 2 Electrodialysis was continued until the pH dropped to .

[0101] The 3-FL solution was then treated with activated charcoal powder to remove the pigments. The solution was stirred with Norit DX1 activated charcoal for 2 hours, after which the charcoal was removed by filtration. Another electrodialysis was carried out using the same setup as above. The conductivity of the starting solution was 1.0-1.5 mS / cm. 2 and the conductivity is 0.3mS / cm 2 Electrodialysis was continued until the pH dropped to .

[0102] 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 dropped 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 to 45 wt% dry matter using a Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach, Germany). The highly concentrated 3-FL solution was filter-sterilized by passing it through a 5 kDa Spira-Cell WY UP005 2440 C ultrafiltration membrane (Microdyn-Nadir) to remove endotoxins.

[0103] HPLC chromatograms showing the purification of 3-fucosyllactose from clarified fermentation broth by the present method are shown in Figures 6 and 7. Figure 6 shows an HPLC chromatogram of the clarified fermentation broth, while Figure 7 shows an HPLC chromatogram of the sterile filtered process stream obtained from the clarified fermentation broth resulting in the HPLC chromatogram shown in Figure 6. Comparing these chromatograms, it is clear that the majority of the peaks (each peak representing at least one compound) in the HPLC chromatogram of the clarified fermentation broth are absent from the HPLC chromatogram of the sterile filtered process stream.

[0104] Example 2: Obtaining solid 3-fucosyllactose by spray drying The resulting 3-FL solution was concentrated to 45 wt% by filtration and electrodialysis as described in Example 1 and sterile filtered to remove any bioburden and endotoxin. This highly concentrated and sterile 3-FL solution was then spray dried using a LTC-GMP spray dryer (Nubilosa, Konstanz, Germany). The 45 wt% 3-FL solution was passed through the spray dryer nozzle at 130 °C and 3.5 bar, and the flow rate was adjusted to maintain an exhaust temperature of 66-67 °C. Using these settings, a spray-dried powder with a moisture content of less than 5% was obtained. The moisture content was measured by Karl-Fischer titration.

Claims

1. 1. A method for purifying a target oligosaccharide from a fermentation broth, the method comprising: providing a fermentation broth containing the oligosaccharide of interest, biomass, microbial cells, and carbohydrates other than the oligosaccharide of interest; - providing a process stream by removing microbial cells from said fermentation broth; - subjecting said process stream to a first nanofiltration step using a nanofiltration membrane to remove high molecular weight impurities >1200 Daltons to provide a filtrate containing the desired oligosaccharides; - subjecting the filtrate to a second nanofiltration step using a nanofiltration membrane to remove low molecular weight impurities of <400 Daltons to provide a process stream containing the desired oligosaccharides; - providing a purified preparation of the target oligosaccharide by removing salts using electrodialysis from the process stream obtained after the second nanofiltration step containing the target oligosaccharide; A method comprising: the target oligosaccharide is a neutral human milk oligosaccharide (HMO) or a sialylated HMO; The process does not include an ion exchange step.

2. 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. 3. The method of claim 2, wherein the microbial cells are removed from the fermentation broth by subjecting the fermentation broth to at least one filtration step, the at least one filtration step being microfiltration.

4. 4. The method of claim 3, wherein the process stream resulting from microfiltration is subjected to at least one ultrafiltration step.

5. 5. The method according to any one of claims 1 to 4, wherein the nanofiltration membrane used in the first nanofiltration step has a molecular weight cut-off of between 700 Daltons and 3,000 Daltons.

6. 6. The method of any one of claims 1 to 5, wherein the nanofiltration membrane used in the second nanofiltration step has a molecular weight cut-off of between 100 Daltons and 1,000 Daltons.

7. The target oligosaccharide is 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, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, difucosyl-lacto-N-neohexaose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-sialylpentaose a, lacto-N-sialylpentaose b, lacto-N-sialylpentaose c, fucosyl-lacto-N-sialylpentaose a, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose, sialyllacto-N-tetraose The method according to any one of claims 1 to 6, wherein the HMO is selected from the group consisting of c, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyllactose, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose, and lacto-N-neodifucohexaose I.

8. A process according to any one of claims 1 to 7, wherein the process stream obtained after the second nanofiltration step is subjected to a dye removal step by treating the process stream with activated carbon.

9. The pigment removal step comprises: Before or after the electrodialysis process The method of claim 8, wherein the method is performed on

10. The method according to any one of claims 1 to 9, wherein the electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions.

11. The process stream after electrodialysis is i) containing salt in an amount of <1 wt. %; and / or ii) 0.05-1.0mS / cm ２ having a conductivity of The method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, wherein the purified preparation is spray dried.

13. The method according to any one of claims 1 to 12, wherein the purity of the target oligosaccharide in the purified preparation is ≧80 wt. % based on the dry matter of the purified preparation.

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